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Year : 2019  |  Volume : 9  |  Issue : 1  |  Page : 48-52

The potency of Pinus merkusii extract nanoparticles as anti Mycobacterium tuberculosis: An in vitro study

1 Department of Pharmacology, Faculty of Veterinary Medicine, Airlangga University, Surabaya, Indonesia
2 Department of Pharmacy Biology, Faculty of Pharmacy, Hang Tuah University, Surabaya, Indonesia
3 Department of Conservative Dentistry, Faculty of Dentistry, Airlangga University, Surabaya, Indonesia
4 Study Program of Environmental Health, Polytechnic of Health, Surabaya, Indonesia
5 Faculty of Health, Muhammadiyah University, Gresik, Indonesia

Date of Web Publication30-Apr-2019

Correspondence Address:
Sri Agus Sudjarwo
Department of Pharmacology, Faculty of Veterinary Medicine, Airlangga University, Surabaya
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijnpnd.ijnpnd_57_18

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Objective: Herbal nanoparticles have gained interest in nanomedicine, and development of new therapeutic with improved bioavailability, increased sensitivity and specificity, and reduced toxicity. The aim of this study was to evaluate the antimycobacterial activity of the Pinus merkusii extract nanoparticle in vitro. Materials and Methods: Ethanolic extract of P. merkusii was set by maceration method. Tripolyphosphate (TPP) was used to make P. merkusii nanoparticles by ionotropic gelation method. The size and morphology of the P. merkusii nanoparticle was analyzed using scanning electron microscope (SEM). The broth microdilution and micro diffusion methods were used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of P. merkusii nanoparticle on strain Mycobacterium tuberculosis H37Rv. Results: The SEM micrographs of the nanoparticle extract of P. merkusii showed that they were approximately uniform spheres with rough surface morphology and a solid dense cubical or rectangular structure. The size of P. merkusii nanoparticle ranged from10 to 800 nm; most were 500 nm. Using the broth microdilution and micro diffusion susceptibility method, P. merkusii nanoparticle was found to have the antimycobacterial effects with a MIC value of 1000 µg/ml and MBCs value of 2000 µg/ml for M. tuberculosis H37Rv. Conclusion: P. merkusii extract nanoparticle has the lead compounds that may be developed further into antimycobacterial drugs.

Keywords: Minimal bactericidal concentration, minimal inhibitory concentration, M. tuberculosis H37Rv, P. merkusii nanoparticle

How to cite this article:
Sudjarwo SA, Wardani G, Eraiko K, Koerniasari, Ernawati. The potency of Pinus merkusii extract nanoparticles as anti Mycobacterium tuberculosis: An in vitro study. Int J Nutr Pharmacol Neurol Dis 2019;9:48-52

How to cite this URL:
Sudjarwo SA, Wardani G, Eraiko K, Koerniasari, Ernawati. The potency of Pinus merkusii extract nanoparticles as anti Mycobacterium tuberculosis: An in vitro study. Int J Nutr Pharmacol Neurol Dis [serial online] 2019 [cited 2022 Aug 8];9:48-52. Available from:

   Introduction Top

Tuberculosis (TB) is the disease caused by Mycobacterium tuberculosis, which in 99% of cases was reported in developing countries. Over eight million new cases of infection are diagnosed every year. The disease causes more than two million deaths per year. The global incidence rate for TB is growing each year by approximately 1.1% and the number of cases by about 2.4%. Resistance to anti-TB drugs continues to be recognized as a clinical problem. As a result, Multi Drug Resistant (MDR) and Extensively drug-resistant (XDR) tuberculosis are now becoming a major threat to health worldwide, accounting for almost 3% of all newly reported cases of TB.[1] Due to increased drug-resistant strains of bacteria such as M. tuberculosis and methicillin resistant Staphylococcus aureus there has been renewed interest in natural products as potential sources of novel antibiotics. [2],[3]

The use of natural products as medicines is well known in rural areas of many developing countries. The natural products claim that their medicine is cheaper, more effective and has least side effects as compared to synthetic medicines.[4],[5] Medicinal plant products have long been used as anti bacterial in traditional medicines for the treatment of many diseases such as tuberculosis.[6],[7] The anti M. tuberculosis medicinal plant products have become subject to scientific investigations worldwide; their active components have a potential to provide an alternative to conventional medicine in the treatment of M. tuberculosis. In this context, the development of medicinal plant product-based drug candidates as anti M. tuberculosis has gained momentum in research studies directed toward design and discovery of drugs.[5],[6] Piliostigma thonningii, Curtisia dentata, Combretum zeyheri, Artemisia nilagirica and Murraya koenigii are among the medicinal plants claimed to possess potential antimycobacterial agent.[5],[6],[7]

It has been demonstrated that the medicinal properties of pinus plant are due to its phytochemicals, including alkaloids, polyphenols, flavonoids, lignans, triterpenes, sterols, glycosides, triterpenoids, and saponins.[8],[9] Recent research activities have shown that Pinus plant is an important source of pycnogenol that contains proanthocyanidins (procyanidins).[10],[11],[12] Proanthocyanidins are potent free radical scavengers; they exhibit vasodilatory, antiallergic, antiinflammatory, cardioprotective, immune-stimulating, antiviral, estrogenic, and antibacterial properties.[8],[9],[13]

In recent years, synthesis of nanoparticles has drawn the interestof nanoscience and nanobiotechnology.[14],[15]The plants seem to be the best suited for large-scale biosynthesis of nanoparticles. Pinus plant nanoparticles have drawn the attention of researchers because of their suitable applications in the fields of material science, and medicine. The objective of the present study was to evaluate the antimycobacterial activity of the nanoparticle extract of P. merkusii in vitro. P. merkusii nanoparticles have a potential as antimycobacterial of new pharmacological and therapeutic drug with improved biodistribution and increased specificity and sensitivity and reduced pharmacological toxicity

   Materials and Methods Top

Preparation of Ethanol Extract of Pinus merkusii

Plant material and extract of P. merkusii leaf were collected from Surabaya, Indonesia. P. merkusii leaves were cleaned under the running tap water and then chopped into pieces. They were dried under shade at ambient temperature for five days, and the air dried P. merkusii were then ground to powder for extraction. The powdered P. merkusii (1 kg) was macerated with ethanol (5 L) for a week at 37°C9. The supernatant was then collected and filtered through Whatman No. 1 filter paper in a Buchner funnel under vacuum. The filtrate was concentrated by evaporation with a vacuum rotary evaporator at 45°C. The extract was dried at reduced pressure, stored at 0−4°C, and used for the experimentation.

Preparation of Pinus merkusii Extract Nanoparticle

P. merkusii extract of nanoparticles was prepared by ionic gelation of chitosan and sodium tripolyphosphate (TPP) anions. One gram P. merkusii extract was dissolved in 50 ml tween 80 solution and added 100 ml chitosan in glacial acetic acid (0.25% v/v). 350 mL of 0.84% w/v TPP was added under magnetic stirring at room temperature in drops using 10 ml syringe needle into P. merkusii extract and chitosan solution. The pH of the solution was adjusted by adding 0.1M NaOH solution to the Chitosan P. merkusii complex and stirred for two hours on a magnetic stirrer. Chitosan-P. merkusii complex was centrifuged at 12,000 rpm for another 30 min and decanted.[16],[17] The supernatant was kept and dried in an oven at 40C overnight for onward usage.

Electron microscopy analysis

After the preparation of the synthesized P. merkusii nanoparticles, the characterization of the nanoparticle was examined by scanning electron microscope (SEM).[16] The particle size and morphology of synthesized nano materials were determined using a field emission scanning electron microscope (FE-SEM, 15 kV, model 54160, Hitachi, Japan).

Culture and preparation of Mycobacterium tuberculosis

M. tuberculosis strains H37Rv were obtained from the Institute of Tropical Disease, Airlangga University, Surabaya, Indonesia. M. tuberculosis was cultured at 37°C in Middlebrook 7H9 broth (Becton Dickinson, Sparks, MD) supplemented with 0.2% glycerol (Sigma Chemical Co.,St. Louis, MO) and 10% OADC (oleic acid-albumin-dextrose-catalase; Becton Dickinson) until logarithmic growth was reached. Each culture was mixed with a sufficient volume of sterile supplemented Middlebrook 7H9 broth to achieve a turbidity equivalent to that of McFarland’s No. 1 standard. To obtain the test inoculum, this suspension was further diluted 1:50 with the same culture medium to approximately 6 × 106 colony-forming units (CFU)/mL immediately before use.[18],[19].

Minimum Inhibitory Concentration determination by Resazurin Microtiter Plate Assay (REMA) method

REMA was performed with minor modifications.[20],[21]The resazurin microtiter assay (REMA) plate method was performed in 7H9-S medium containing Middlebrook broth, 0.1% Casitone, and 0.5% glycerol and supplemented with oleic acid, albumin, dextrose, and catalase (Becton-Dickinson). Briefly, 100 µL of Middlebrook 7H9 broth was dispensed into each well of the microtitre plate. Serial five-fold dilutions of P. merkusii extract nanoparticle (0; 125; 250; 500; 1000; and 2000 µg/ml) and standard antimycobacterial drugs were made in the plate. M. tuberculosis strains H37Rv suspension (100 µL) containing approximately 6 × 106 CFU/mL was added to all the wells. Sterility control and growth control were also included. The plate was wrapped in aluminum foil and incubated at 37°C for seven days. After completion of the incubation period, 30 µL resazurin solution (100 µg/mL) was added to each well and plate was again wrapped in aluminum foil and incubated overnight. The plate was then observed for change in color. The color changed from blue to pink or colorless indicating growth of the bacteria. The lowest concentration of nano particle extract that prevented color change from blue to pink was taken as the upper limit for Minimum Inhibitory Concentration (MIC) range; the highest nanoparticle extract concentration that showed a change in color from blue to pink was considered the lower limit. All evaluations were carried out in quadruplicate.

Minimum Bactericidal Concentration Determination Using the Paper Disc Method

Screening of P. merkusii nanoparticles and its solvents for antimycobacterial activity against M. tuberculosis strain H37Rv was done using the paper disc method.[22],[23] For the treatment, each of 0; 125; 250; 500; 1000; 2000 µg/ml chitosan nanoparticles solutions were slowly absorbed into the sterilized paper disc (diameter: 8 mm, Watman, England) and adhered to the surface of the plate on which M. tuberculosis strains H37Rv at a concentration of 106 CFU/ml had been inoculated in Middlebrook 7H9 broth. Sterilized distilled water was used as a control. After culturing for 24 hours in an incubator at 37°C. Antibacterial activity was defined as the diameter (mm) of the clear inhibitory zone formed around the discs. MBC was defined as the lowest concentration that induced the clear inhibitory zone formed around the discs.

   Results Top

Scanning Electron Microscopic (SEM) Studies of Pinus Merkusii Extract Nanoparticle

The SEM micrograph details of the P. merkusii extract and P. merkusii extract nanoparticle are represented in Figure 1. The SEM micrograph obtained for the P. merkusii extract revealed that the texture is plain without pores having smooth, compact and homogeneous even surface structure with no gross effects, while the SEM micrograph of P. merkusii nanoparticles ([Figure 1]) revealed the rough surface morphology with solid dense cubical or rectangular structure and not aggregated. The spheres had mean diameter of around 500 nm. The nanoparticles dry powder consists of individual nanoparticles, which touch each other, but retain their original size and shape. The size variation may be related to different conditions of sample preparation for SEM.
Figure 1 Scanning electron microscopic (SEM) of P. merkusii extract (A) and P. merkusii extract nanoparticles (B).

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The MIC of the Pinus merkusii extract nanoparticle against Mycobacterium tuberculosis on Resazurin Microtiter Plate Assay (REMA) method

The MIC of the P. merkusii nanoparticles were determined for their antimycobacterial activity using resazurin as an indicator of M. tuberculosis viability in 96-well microplates. The investigation showed that P. merkusii nanoparticles were active against M. Tuberculosis. In this study, MIC of chitosan nanoparticles against M. tuberculosis strains H37Rv was 1000 µg/mL ([Figure 2]).
Figure 2 MIC of P. merkusii extract nanoparticles against Mycobacterium tuberculosis H37Rv on Resazurin Microtiter Plate Assay (REMA) method was 1000 μg/ml (Black arrows). Serial five-fold dilutions of P. merkusii extract nanoparticles at dose 0 μg/ml; 125 μg/ml; 250 μg/ml; 500 μg/ml; 1000 μg/ml; and 2000 μg/ml.

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The MBC of the Pinus merkusii nanoparticles against Mycobacterium tuberculosis on the paper disc method

The antimycobacterial activity of P. merkusii extract nanoparticle against M. tuberculosis strains H37Rv was done using the paper disc method. MBC was defined as the lowest concentration that induced the clear inhibitory zone formed around the discs. The MBC P. merkusii extract nanoparticle against M. tuberculosis strains H37Rv was 2000 µg/mL, which induced the clear inhibitory zone formed around the discs was 7.3 ± 0.3 mm ([Figure 3]).
Figure 3 MBC of P. merkusii extract nanoparticles against Mycobacterium tuberculosis H37Rv on the paper disc method was 2000 μg/ml (Black arrows).

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   Discussion Top

Tuberculosis is a chronic disease caused by M. tuberculosis. The emergence of antibiotic-resistant strains of this species underscores the need for novel effective drugs against resistant mycobacteria as first-line antituberculosis medications.[1] The uses of natural product as traditional medicines are accepted, particularly in developing countries.[2] This led us to the investigation of the effects of P. merkusii nanoparticles on antimycobacterial activity.

We made P. merkusii extract that was encapsulated into chitosan nanoparticle using sodium tripolyphosphate on ionotropic gelation method, which has more advantages over Pinus merkusii extract. Due to this modification, we can improve biodistribution and increase specificity and sensitivity, and reduce pharmacological toxicity.[13],[15],[17] The topography, surfaces, structures, morphologies, and composition of the P. merkusii extract nanoparticle were studied using Scanning Electron Microscopic (SEM). The SEM micrograph obtained for P. merkusii revealed that the texture is plain without pores having smooth, compact and homogeneous even surface structure with no gross effects. While the SEM micrograph of P. merkusii nanoparticles revealed the rough surface morphology. The SEM picture of P. merkusii nanoparticles demonstrated good dispersion of the nanoparticles which were entangled one on the other with a larger exposed surface making the material very suitable for adsorption. In this study, we successfully synthesized and optimized P. merkusii extract nanoparticle. On comparing the SEM micrograph details of P. merkusii extract nanoparticle with P. merkusii extract, it was observed that the P. merkusii extract nanoparticle have a relatively rough surface with the uneven structure which exhibited highly amorphous feature.

Particle size has an important role in obtaining optimal in vitro efficacy. Particle size also has a crucial impact on the in vivo fate of a drug delivery system. Decreasing the particle size could increase the dissolution and thus increase the bioavailability of poorly water-soluble molecules[15],[17] In our study, P. merkusii extract nanoparticle have mean diameters around 500 nm. The smaller size particles have efficient interfacial interaction with the cell membrane compared to larger size particles due to the endocytosis of small size particles. Small size particles improve the efficacy of the particle-based oral drug delivery systems. Also, the use of small particle size can increase the bioavailability and prolong the blood half-life of drugs and increase thier efficacy.

Investigation of MIC and MBC plays an important role during the process of screening, prioritizing, and optimizing a chemical series during early antibacterial drug discovery. Minimum Inhibitory Concentration was determined using Resazurin Microtiter Plate Assay (REMA) method. Many researchers have used the Resazurin Microtiter Plate Assay (REMA) method to screen test substances for antimycobacterial activity against M. Tuberculosis.[19] Resazurin, an oxidation-reduction indicator, has been used to assess viability and bacterial contamination and to test for antimicrobial activity. Results obtained using the REMA assay is faster and less expensive. Bearing in mind considerations of rapidity, low technology requirements and low cost, microplate assays that use Resazurin type compounds have the potential of becoming the methods of choice for drug susceptibility testing of M. tuberculosis in places where TB is a major problem.[21] The MBC of P. merkusii extract nanoparticle against M. tuberculosis was conducted on the paper disc method. In this study, MIC of P. merkusii extract nanoparticle against M. tuberculosis strains H37Rv was 1000 µg/mL, while MBC of P. merkusii nanoparticles against M. tuberculosis strains H37Rv was 2000 µg/mL. This suggests that P. merkusii has potent activity as antibacterial and this has been confirmed experimentally.These results are in agreement with reports in the literature that have documented the antibacterial activity of Pinus plant against a large number of gram-positive and gram-negative bacteria. Some researchers have also shown that P. merkusii generally has stronger effects for gram-positive bacteria (e.g. Listeria monocytogenes, Bacillus megaterium, B. cereus, Staphylococcus aureus, Lactobacillus plantarum, L. brevis, L. bulgaris, etc.) and for gram-negative bacteria (e.g. E. coli, Pseudomonas fluorescens,  Salmonella More Details typhymurium, Vibrio parahaemolyticus, etc.).[24],[25],[26]

   Conclusion Top

The findings from this research can thus be concluded: P. merkusii extract nanoparticles have a relatively rougher surface with an uneven structure that exhibit highly amorphous feature with promising anti-tuberculosis activity by preliminary in vitro techniques. Therefore, P. merkusii potential as a source of compounds that may be developed further into anti-mycobacterial drugs.


This study was supported by the Ministry of Research, Technology and Higher Education of the Republic of Indonesia. Grants No: 004/SP2H/LT/DRPM/IV/2017, February 17, 2017

Financial support and sponsorship


Conflict of interest

Authors have no conflict of interest.

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