INTRODUCTION:

Tuberculosis (TB) is one of the deadliest and chronic infectious, disease resulting from infection with the pathogen Mycobacterium tuberculosis complex (MTC).

Tuberculosis remains one of the top 10 causes of death worldwide and one-third of the world’s population infected with it. In 2019, 1.4 million deaths occurred due to this global burden disease and currently around 10 million people are suffering from TB¹. (World Health Organization (WHO), 2020). This disease affects both developed and developing countries including Sri Lanka².

In 1882, Robert Koch has discovered Mycobacterium tuberculosis (Mtb) which is responsible for TB, thus identifying TB as an infectious disease³. TB is effectively cured by a combination of WHO recommended first-line oral antibiotics including isoniazid (INH), rifampin (RIF), pyrazinamide (PZA) and ethambutol (EMB)^4. In addition, emergence of drug resistance has led to the use of second line drugs for treatment of TB in certain situations. Four agents of Nitroimidazole class including CGI-17341, PA-824, TBA-354 and OPC-67683, have shown the potential anti-TB property⁵.

Medicinal plants are considered as one of the best sources to obtain different types of medicines⁶. Many plants are used in traditional medicine and Ayurveda to treat respiratory diseases including TB⁷. Therapeutic information of some plants is only folkloric based and hence there is need to undertake their scientific rationalization through in vitro and in vivo testing⁸.

Psychotria sarmentosa (Family: Rubiaceae), commonly known as Gonica in Sinhala, is a climbing shrub which is native to tropical Asia and has been used in folk medicine in Sri Lanka. Extracts of different species of the Genus Psychotria have been evaluated for antimycobacterial activity⁹. The literature survey revealed that published scientific information regarding this plant is rare.

Aponogeton crispus (Family: Aponogetonaceae) is an aquatic plant and widely used in Ayrvedic medicine. The plant has medicinal properties like anti-pyretic, anti-inflammatory, hypoglycaemic and anti-nociceptive activities¹⁰. In Ayurveda, A. undulatus is claimed to be effective against tuberculosis¹¹.

Pleurotus ostreatus (American oyster) belongs to the family Pleurotaceae and is considered as one of the most popular edible mushrooms in all over the world. It exhibits antimicrobial, anti-cancer, anti-inflammatory, anti-diabetic, anti-hypercholesterolemia, anti-hypertensive, and hepato-protective activities^12,13. In addition, antimycobacterial activity of ethanolic extract of P.ostreatus has also been recorded¹⁴. Pleurotus cystidiosus (Abalone) belongs to the family Pleurotaceae and has exhibited antimicrobial, antidiabetic, anti-cancer antihypertensive activities and anti-platelet aggregation properties¹². Dichloromethane extract of Pleurotus tuber-regium has exhibited significant anti-TB activity against clinical isolated Mtb¹⁵.

Anti-TB allopathic medications which are prescribed for this disease are associated with numerous side effects. The global TB epidemic has been aggravated by the emergence of disease outbreaks caused by multi drug resistant (MDR)-TB and extensively drug-resistant (XDR)-TB¹⁶. Therefore, there is an urgent need to develop newer anti-TB drugs with unique drug targets.

Even though various biological activities of P. sarmentosa, A. crispus, P. ostreatus and P. cystidiosus have been reported, there were no studies carried out to test anti-TB activity of aqueous extracts of said plants and mushrooms. Hence, this study was aimed to assess the anti-tuberculosis activity of P. sarmentosa, A. crispus and two species of Pleurotus mushrooms.

MATERIAL AND METHODS:

Collection of plant materials:

The leaves of P. sarmentosa were purchased from a supermarket in Rajagiriya, Sri Lanka. A. crispus leaves were collected from a lake located at Kurunegala area in North Western province, Sri Lanka. Fresh P. ostreatus and P. cystidiosus grown using the spawn provided by the Mushroom Cultivation Centre, Export Research Board (Ratmalana, Sri Lanka), were collected from a local farm.

Identification and authentication of plants and mushrooms:

Dried plant specimens of all interested plants were submitted to the National Herbarium located in the premises of the Royal Botanical Gardens, Peradeniya for identification and authentication process. The identification and authentication of P.ostreatus and P. cystidiosus was done by studying the spore print and the shape of the cap (fan-shaped) and the stipe (eccentric).

Preparation of aqueous extracts:

Freshly collected leaves of P. sarmentosa, A. crispus and the whole mushrooms of P. ostreatus and P. cystidiosus were washed under slow running tap water to remove humus and other debris on the plant part surfaces and then were rinsed 3-4 times with distilled water followed by washing in hydrogen peroxide (6%) for 15 minutes. The leaves and mushrooms were dried until a constant weight under the ambient condition. The dried materials were pulverized into particles. A weight of 120g of the powdered plant materials was allowed to heat at a moderate temperature in 1.9 L of distilled water and the final volume was reduced to 240ml according to Ayurvedic protocol. Filtrate was obtained by filtering through a muslin cloth. Each filtrate was transferred to a sterile glass container and stored in a refrigerator at 4ºC until subjected to freeze-drying process. The freeze-dried extracts were collected into a clean, dry, well-closed container and were stored in a desiccator.

Preparation of Middlebrook 7H11 media:

A weight of 10.25g of Middlebrook 7H11 agar base was dissolved in 450ml distilled water containing 2.5ml glycerol. The mixture was heated to boiling to dissolve the medium completely. Prepared media were sterilized by autoclaving at 15 lbs pressure, 121⁰C for 10 minutes. Media were cooled to 50⁰C and aseptically added contents of 1 vial (50ml) of Middlebrook OADC (Oleic acid, bovine albumin, sodium chloride, dextrose and catalase) growth supplement and mixed thoroughly without letting bubbles to form.

Preparation of serial dilutions of inoculum suspension:

Fresh growth of the standard strain of Mtb H₃₇Rv grown on L-J medium was used as the source of inoculum. A loopful (3mm disposable loop) of the inoculum was scraped from the L-J medium and the culture was emulsified on the wall of a sterile McFarland bottle with 1 ml sterile distilled water and six 5mm glass beads. The suspension was vortexed for 20-30 seconds until homogenized. A volume of 4ml of sterile distilled water was added and mixed thoroughly. Then the suspension was rested for 5 minutes. The supernatant was decanted to another sterile McFarland bottle. Turbidity of Mtb H₃₇Rv suspension was visually adjusted by comparing it to the reference McFarland standard 1.0 which contained approximately 10⁸ colony forming units (CFU)/ml (NEAT), by addition of sterile distilled water. Serial dilutions of 10^-2(10⁶ CFU/ml) and 10^-4 (10⁴ CFU/ml) of the inoculum suspensions were prepared by diluting sequentially 100.0µl of the standard suspension in a sterile bijou bottle containing 900.0µl of sterile distilled water.

Indirect agar proportion method for drug susceptibility test:

Drug susceptibility test was conducted using the standard indirect agar proportion method. Anti-TB drugs; INH, RIF, streptomycin and EMB were tested against standard strain of H₃₇Rv Mtb. Stock solutions of standard drugs were prepared. Each standard drug solution was filter sterilized using 0.2 μm filter and stored at -20⁰C. Stock solutions were thawed and mixed with Middlebrook 7H11 medium with OADC supplement under sterile conditions in order to get the required drug concentration in the final agar mixture as represented in Table 1.

Media with standard drugs and the drug- free control media were then transferred separately into the sterile culture plates (20ml) under sterile conditions. Inoculation and incubation were carried out in a bio-safety level 3 (BSL3) laboratory. The culture plates were inoculated with 0.01ml of freshly prepared two ten-fold dilutions (10^-2 and 10^-4) of McFarland 1 suspension of standard Mtb strain H₃₇Rv suspensions. Plates were sealed with parafilm to avoid contamination and incubated at 37°C in the presence of 5-10% CO₂ incubator. Results were recorded after four weeks. The assay was done in duplicate. The numbers of colony forming units, growing on the medium containing the drug were compared with the numbers on the control plate. Inhibition percentage of each test sample was calculated using following formula.

[No. of colonies on control medium – No. colonies on test medium]

Percentage of = --------------------------------------------------------- X 100

inhibition No. of colonies on control medium

Table 1. Stock concentrations and final concentrations of standard anti-TB drugs

Standard anti-TB drugs	Concentration of stock solution (µg/ml)	Concentration of working solution (µg/ml)
Isoniazid	1000	0.2
Rifampicin	100	1
Ethambutol	100	5
Streptomycin	1000	2

Determination of anti-tuberculosis activity of P. sarmentosa, A. crispus, P. ostreatus and P. cystidiosus:

A weight of 20.00mg of freeze-dried aqueous extract of each plant and mushroom were dissolved in sterile distilled water (4.00ml) separately. The solutions were filter sterilized using 0.2μm filter and the sterile filtrates were obtained at a concentration of 5.00mg/ml. Sterile crude extracts were in cooperated in to freshly prepared Middlebrook 7H11 agar medium containing the OADC enrichment separately and the concentrations of final agar media were 1mg/ml. The media with each crude extract were then transferred in 20ml amounts into sterile culture plates separately under sterile conditions using the pour plate method. A volume of 0.01ml of freshly prepared dilution (10^-2 and 10^-4) of the inoculum was placed in plates containing crude extracts. This assay was done in triplicate and inoculation and incubation were carried out in a bio-safety level 3 (BSL3) laboratory.

After inoculation, the plates were sealed with parafilm and incubated at 37°C in the presence of 5-10% CO₂ incubator. Results were recorded after 4 weeks. The numbers of CFU, growing on the medium containing each crude extract were compared with the number on the control plate. Inhibition percentage of each test sample was calculated using the following formula.

[No. of colonies on control medium – No. colonies on test medium]

Percentage of = --------------------------------------------------------- X 100

inhibition No. of colonies on control medium

RESULTS:

Indirect agar proportion method for drug susceptibility test:

The inhibition percentages of anti-TB drugs at 10^-2 dilution inoculum of H₃₇Rv Mtb strain and the inhibition percentages of anti-TB drugs at 10^-4 dilution inoculum of H₃₇Rv Mtb strain are shown in Table 2 and Table 3 respectively. Mean inhibition percentages of standard anti-TB drugs inoculated with two ten-fold dilutions (10^-2 and 10^-4) of H₃₇Rv Mtb strain are shown in Table 4.

Table 2: Inhibition percentages of anti-tuberculosis drugs at 10^-2dilution inoculum of H₃₇Rv Mtb

Test sample	Concentration of drug (µg/ml)	Mean CFU	Standard error of mean (SEM)	Inhibition percentage %
Control	-	>300	0	-
Isoniazid	0.2	0	0	100
Rifampicin	1	0	0	100
Ethambutol	5	0	0	100
Streptomycin	2	0	0	100

Table 3: Inhibition percentages of anti-tuberculosis drugs at 10^-4 dilution inoculum of H₃₇Rv Mtb

Test sample	Concentration of drug (µg/ml)	Mean CFU	SEM	Inhibition percentage %
Control	-	>300	0	-
Isoniazid	0.2	0	0	100
Rifampicin	1	0	0	100
Ethambutol	5	0	0	100
Streptomycin	2	0	0	100

Table 4: Mean inhibition percentages of standard anti-Tuberculosis drugs

Test sample	Concentration of drug (µg/ml)	Mean inhibition percentage %	SEM
Isoniazid	0.2	100	0
Rifampicin	1	100	0
Ethambutol	5	100	0
Streptomycin	2	100	0

The sensitivity to detect susceptibility to INH, RIF, streptomycin and EMB were 100%. As per the results, there was no growth of standard strain colonies observed at both ten-fold dilutions of the inoculum in the presence of standard drugs.

Determination of anti-tuberculosis activity of P. sarmentosa, A. crispus, P. ostreatus and P. cystidiosus:

In the present study, we examined the in vitro antimycobacterial activity of P. sarmentosa, A. crispus, P. cystidiosus and P. ostreatus against a slow growing Mtb strain of H₃₇Rv which was susceptible to the first-line TB drugs. Inhibition percentages of P. sarmentosa, A. crispus, P. cystidiosus and P. ostreatus at 10^-2 and 10^-4 dilution inoculum of H₃₇Rv Mtb are indicated in Table 5 and Table 6 respectively. The inhibitory effect of each extract was calculated by the mean reduction of the number of colonies on the extract containing medium compared to control based on measures of central tendency as the standard strain of H₃₇Rv Mtb was tested in triplicate.

Table 5: Inhibition percentage of P. sarmentosa, A. crispus, P. cystidiosus and P. ostreatus extracts at 10^-2dilution inoculum of H₃₇Rv Mtb

Test sample	Mean CFU	SEM	Percentage inhibition %
Control	>300	0	-
P. sarmentosa (Gonika)	110.33	67.9	66.6
A. crispus (Kekatiya)	152	120.8	49.3
P. cystidiosus (Abalone)	>300	0	0
P. ostreatus (Oyster)	>300	0	0

Table 6: Inhibition percentage of P. sarmentosa, A. crispus, P. cystidiosus and P. ostreatus extracts at 10^-4dilution inoculum of H₃₇Rv Mtb

Test sample	Mean CFU	SEM	Percentage inhibition %
Control	41.8	28.5	-
P.sarmentosa (Gonika)	10.33	3.4	75.4
A.crispus (Kekatiya)	24	17.1	42.6
P.cystidiosus (Abalone)	8.75	0.86	79.1
P. ostreatus (Oyster)	5.5	1.2	86.8

The inhibitory effect of each extract was calculated by the number of colonies on the extract containing medium compared to extract-free control medium. The mean percentage inhibition exerted by P. sarmentosa, A. crispus, P. ostreatus and P. cystidiosus were 71.0 %, 46.0 %, 43.4 % and 39.5 % respectively. Accordingly, the highest mean percentage inhibition was exhibited by P. sarmentosa (71.0 %).

Furthermore, figure 1 shows the appearance of Mtb colonies on P. sarmentosa extract containing plate and the control plate after inoculating 10^-2 dilution inoculum of H₃₇Rv Mtb. This Figure further supports the effectiveness of P. sarmentosa on the inhibition of H₃₇Rv Mtb.

Figure 1: Number of colonies recorded after 4 weeks at 10^-2 dilution inoculum of H37Rv Mtb. A: Control plate; B: P. sarmentosa extract incorporated plate.

DISCUSSION:

According to the results of the present study, the highest anti-mycobacterial activity was shown by P. sarmentosa, followed by A.crispus, P. ostreatus and P. cystidiosus. Thus, by having an inhibitory activity greater than 50%, P. sarmentosa has a potential to be used in the treatment of TB. To the best of our knowledge, the in vitro anti-mycobacterial activity of P. sarmentosa, A. crispus and P. cystidiosus.is reported for the first time in the current study.

In the present study, the highest anti-mycobacterial activity was shown by P. sarmentosa. Acetone extract of Psychotria zombamontana and P. capensis had been evaluated against different mycobacterial species including M. tuberculosis H₃₇Rv in a previous study. The minimum inhibitory concentration (MIC) values of the above extracts have demonstrated reasonable antimycobacterial activity against all tested strains and comparable with the positive control; RIF¹⁷. Ethanolic extract of P. vellosiana had exhibited anti-mycobacterial activity against M. tuberculosis H₃₇Rv¹⁸. In the present study, aqueous extract of P. sarmentosa was evaluated against M. tuberculosis H₃₇Rv. Anti-micobacterial activity of ten Psychotria species including P. pubigera, P. ruelliifolia, P. suterela, P. stachyoides, P. capitata, P. glaziovii, P. leiocarpa, P. nuda, P. pubigera, P. racemosa, P. vellosiana have been evaluated in a previous study¹⁹. All ten species have demonstrated anti-mycobacterial activity against Mycobacterium bovis¹⁹. In the present study, anti-micobacterial activity of P. sarmentosa was evaluated against M. tuberculosis H₃₇Rv.

Anti-mycobacterial activity of P. vellosiana is related to the presence of alkaloids¹⁸. Further, Junior et al., 2019 has demonstrated the anti-TB activity of a mixture of triterpenes and two alkaloids which were isolated from methanol extract of P. nuda against Mycobacterium tuberculosis H₃₇Rv²⁰. Presence of various bioactive phytochemicals such as tannins, polyphenols and flavonoids has been observed in P. sarmentosa²¹. Therefore, the antimycobacterial activity of P. sarmentosa observed in this study could be attributed to the presence of various bioactive principles which were investigated in P. sarmentosa. However, in the present study, the active compounds present in the crude extract of P. sarmentosa have not been investigated. Therefore, further studies are recommended to isolate and characterize compounds from P. sarmentosa aiming at developing a new class of drugs against tuberculosis.

Aponogeton crispus has emerged as the next candidate of interest with an inhibition percentage of 46.0 % among the other extracts. Published scientific information regarding this plant is rare and effectiveness as an anti-mycobacterial agent is also not reported. In Ayurveda, Aponogeton undulatus is claimed to be effective against tuberculosis¹¹. However, this is the first report of anti-TB activity of A. crispus.

Dichloromethane extract of Pleurotus tuber-regium has exhibited significant anti-TB activity against clinical isolated M. tuberculosis¹⁵. Ethanolic extract of Pleurotus ostreatus had demonstrated significant activity against H₃₇Ra Mtb, 2 clinical isolates and one Mycobacterium other than tuberculosis¹⁴. In the present study, anti-mycobacterial activity of aqueous extract of P. ostreatus was evaluated against M. tuberculosis H₃₇Rv.

In this study, dried plant materials and mushrooms were pulverized into particles. When the particle size is reduced, the surface area in contact with the solvent is increased. This in turn leads to efficient extraction. Moreover, moderate temperature was supplied by heating during the extraction process. Moderate temperature aids in the optimal extraction process while high temperature could reduce the activity of the compounds extracted into the solvent. Bioactive compounds such as phenolic compounds, enzymes etc. are thermal sensitive. Some compounds degrade in the presence of high temperature²². Therefore, it is recommended to evaluate the anti-TB activity of suspensions of freeze-dried and powdered Pleurotus mushrooms which were not exposed to high temperature. Jayasuriya et al., 2015 and 2020, have reported the hypoglycemic activity of P. ostreatus and P cystidiosus and anti-inflammatory activity of P. ostreatus using suspensions of freeze-dried and powdered mushrooms^12,13.

Freeze-dried aqueous extracts of P. sarmentosa, A. crispus and Pleurotus mushrooms were used to prepare the final known concentrations. By freeze-drying, the plant extracts properly preserve the medicinal qualities of the extracts and therefore using freeze-dried extracts is better when compared with other methods of drying. The principle involved in freeze-drying is called sublimation²³.

Anti-mycobacterial activity exhibited by aqueous extracts of Pleurotus mushrooms were less when compared with the anti-mycobacterial activity exhibited by P. sarmentosa in the present study. The compounds present in the Pleurotus mushroom extracts, which are responsible for anti-mycobacterial activity. may be extracted in less amount to the aqueous solvent. Hence, low polar compounds which are more likely to dissolve in organic solvents (less polar solvent than water) may not have been extracted into the aqueous extract. Those compounds could exert anti-mycobacterial activity. Anti-inflammatory activity of P. ostreatus was exhibited by the acetone extract of P. ostraetus¹³. Furthermore, an oxidized ergosterol active against anthracnose causing Colletotrichum gloeosporioides have been isolated from the acetone extract of P. cystidiosus and characterized²⁴. Moreover, dichloromethane and ethanolic extracts of other Pleurotus species have exhibited significant anti-micobacterial activity^14,15. Therefore, it is recommended to evaluate the anti-TB activity of different solvent extracts of two Pleurotus species extracted with organic solvents with different polarities. The concentrations of the extracts of Pleurotus used in the agar plate may be insufficient to show the inhibitory effect on mycobacteria. In addition, the soil composition and other environmental factors could affect the concentration of active compounds present in a plant extract.

The H₃₇Rv Mtb was inhibited effectively by WHO recommended first-line oral therapeutic drugs including INH, RIF, EMB and by streptomycin which is usually administrated as a parental drug for TB treatment. However, sometimes, these drugs are ineffective in the presence of drug resistance strains²⁵. Drug susceptibility testing were carried out to identify whether any particular drug is resistant to the tested TB strains. The anti TB activity of standard drugs were compared with anti TB activity of P. sarmentosa and A. crispus plants and Pleurotus mushrooms in the present study. Therefore, the susceptibility tests for standard drugs were carried out to determine the sensitivity of the H₃₇Rv strain to standard drugs. The major disadvantage of the agar based drug susceptibility test is that it is time- consuming since the results can be observed only after the visible appearance of the growth of the slow- growing colonies.

Recent studies have shown that there is a possibility of using plant extracts in combination of two or more as anti-mycobacterial agents and effective against different strains of Mtb²⁶. Similarly, some studies have been carried out to evaluate the synergism between antimicrobial drugs and plant extracts²⁷. It is recommended to conduct studies with different plant materials together in search of synergistic anti-TB activity.

This study provides novel information about the anti-TB activity of P. sarmentosa, A crispus, P. ostreatus and P.cystidiosus. Further studies to investigate anti-TB bioactive constituents from these plants and mushrooms are important to prove the possible application in TB therapy.

CONCLUSION:

In the current study, P. sarmentosa A. crispus and P. ostreatus and P.cystidiosus were screened for their anti-tuberculosis activity. Accordingly, the highest mean percentage inhibition was shown by P. sarmentosa (71.0 %). The mean percentage inhibition exerted by A. crispus, P. ostreatus and P. cystidiosus were 46.0%, 43.4% and 39.5% respectively against H₃₇Rv Mtb. Freeze-dried aqueous extract from leaves of P. sarmentosa has exhibited higher activity against the tested standard mycobacterial strain H37Rv and has a potential to be used as an anti-TB drug component.

ACKNOWLEDGEMENT:

The authors would like to thank National Institute of Fundamental Studies, Hantana Road, Kandy for providing laboratory facilities and University of Sri Jayewardenepura is gratefully acknowledged for providing financial assistance (Grant no. ASP/01/RE/MED/2015/43).

CONFLICT OF INTEREST:

Authors declare no conflict of interest.

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Received on 08.03.2021 Modified on 18.07.2021

Research J. Pharm.and Tech 2022; 15(3):954-960.

DOI: 10.52711/0974-360X.2022.00159