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Chemical composition of fixed oil and in vitro antimicrobial activity of Andrographis paniculata root
⁎Corresponding authors. shaziz2408@yahoo.com (Shahin Aziz), sharif@acce.iu.ac.bd (Sharif Md. Al-Reza)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
The present study estimates the antimicrobial potential of root extract of Andrographis paniculata which are extensively used in traditional medicine against clinical pathogens. The in vitro antibacterial activities were done with ten pathogen bacterial strains. Among the three extracts, the methanol extract exhibited the highest zone of inhibition against Salmonella typhimurium (45 mm), while ethanol and ethyl acetate extract revealed strong in vitro antibacterial against Shigella flagella (42 mm) and E. faecalis (38 mm), respectively at 200 µg/ml concentration. Antifungal activities of ethanol and methanol extract were done against four reference fungal strains. The ethanol and methanol extract confirmed the highest antifungal activity against Trichophyton mentagrophytes (38 mm) and Candida albicans (38 mm), respectively. The constituents of the fixed oil of petroleum ether root extract were evaluated by Gas Chromatography-Mass Spectrophotometer. Eleven composites have recognized, methyl palmitate (56.732%) was the major constituent. The investigation come to an end that the phytochemical constituents of methanol and ethanol root extract have capacity to kill both drug resistant bacteria and fungal.
Keywords
Andrographis paniculata
Antimicrobial activity
Fixed Oil
Gas Chromatography–Mass Spectrophotometer
1 Introduction
Natural products are strong and potential sources of drugs as they are very safe and free from any toxicity (Deora, 2017). Synthetic drugs are often treated chemicals so sometimes our body has difficulty to recognize it and causes toxicity and unhealthy side effect by extreme uses of different synthetic drugs. Nowadays, from the medicinal herbs or partial chemical synthesis maximum drugs are developed (Sukanya et al., 2009). In ancient times medicinal plants are widely used around the whole world as a great source of antimicrobial agents as plants are rich in phytochemical constituents (Ravikumar et al., 2010; Pandy et al., 2012). Andrographis paniculata (Burm. F) Ness belongs to the Acanthaceae family which is one of the most valuable medicinal plant and commonly used for the design of natural medicine of various diseases like as diarrhea, eczema, dysentery, chickenpox, fever, common cold, pneumonia and hepatitis (Akbar, 2011; Jarukamjorn and Nemoto, 2008). Andrographis paniculata is widely used from the ancient periods in India, Bangladesh, Hongkong, Indonesia, Pakistan and Thailand (Kabir et al., 2014). In south Asian countries it is known as “Kings of bitters” or “Kalmegh”. It has been used for various therapeutic values such as anti-cancer, anti-inflammatory, anti-HIV, anti-diabetic, anti-allergic, anti-oxidant, anti-microbial and reliefs in striking cholera, influenza, bronchitis, swellings, itches and gonorrhea (Richard et al., 2017; Harjotaruno et al., 2008; Sheeja et al., 2006).
Formerly different parts of Andrographis paniculata have been considered for the isolation of pharmacologically active compounds but no orderly study has been done for comparative study of in vitro anti-microbial activities of root against various pathogenic microorganisms and fatty acid existing in the root of this plant. For the discovery of pure compounds of natural herbs, extraction must be needed. Such as solvent extraction, distillation method, pressing and sublimation have been done on different conditions. Among all processes solvent extraction is very popular because of its different beneficial properties; needs minimum energy, worthy grade extract, control the outcome destruction and instability, the process is very ordinary and easy to separate and collection of the resultant and also can reuse the solvent. Ethyl Acetate, ethanol and methanol are highly recommended for phytochemical as they have great properties to the entrance. (Qing-Wen et al., 2018). In view of the enormous application of all the parts of Andrographis paniculata in Ayurveda and Siddha, in vitro microbiological study and fatty acid composition of root were analyzed by Gas Chromatography-Mass Spectrophotometer.
2 Materials and methods
2.1 Chemicals
This experiment employed only analytical or laboratory-grade chemicals and solvents. The solvents ethyl acetate (purity 99.5%), ethanol (purity 99.8%) and methanol (purity 99.8%) were procured from E. Merck (Germany).
2.2 Plant materials
Fresh Andrographis paniculata roots were obtained in April 2018 from Jahangirnagar University in Dhaka, Bangladesh, and identified by a taxonomist at the Bangladesh National Herbarium in Dhaka, where a voucher specimen (No. 45932) was submitted. The dried plant material was then ground into a fine powder and stored in an airtight container at ambient temperature for future uses. The moisture content of the powder sample was 2.8%.
2.3 Preparation of extracts
For five days at room temperature, 100 g of powdered sample was submerged in appropriate solvents with increasing polarity commencing with ethyl acetate, ethanol, and methanol in an air-tight separating funnel with intermittent shaking and stirring. A rotary evaporator was used to concentrate the various extract under reduced pressure. Finally, the extracts were kept at 4℃ for subsequent analysis. % of yield = (W1 × 100)/W2
W1 = weight of the extract residue obtained after solvent removal
W2 = weight of the plant powder
2.4 Bioactivity screening
2.4.1 Microbial strains
The antibacterial properties of the plant sample were tested against four gram-positive bacteria, including Bacillus cereus, Bacillus thuringiensis, Staphylococcus aureus, and Enterobacter faecalis, with six gram-negative bacteria, including Shigella flagella, Salmonella typhimurium, Klebsiella pneumonia, Escherichia coli, Pseudomonas arerogenosa, and Vibro parahaemolytices. The antifungal test included Aspergillus niger, Trichophyton mentagrophytes, Candida albicans, and Candida tropical. The Institute of Food Science and Technology (IFST), BCSIR, Dhaka, Bangladesh, provided all microorganisms as pure cultures.
2.4.2 Antibacterial assay
The agar well diffusion method was used to investigate the possible antibacterial activity of root extracts (Valgas et al., 2007). The sterile petri plates were filled with 25 ml Muller Hinton agar, which was allowed to solidify. A sterile borer was used to punch 6 mm diameter wells in the medium before streaking the plates with bacterial culture. The bacterial cultures were inoculated after the agar had been set by distributing them in petri plates using sterilized cotton swabs. Then, directly onto the surface of Muller Hinton agar containing bacterial lawn, 0.1 ml of plant extract was put. The inoculation plates were kept at 37 °C overnight to allow for bacterial development, and the diameter of the zone of inhibition was measured in millimeters.
2.4.3 Antifungal assay
100 µl of the ethanol and methanol root extracts were put into the wells in the plates. 100 µl of 10% DMSO was used to make a negative control well. Plates were placed in a laminar flow for 30 min to allow for pre-diffusion of the extract, then incubated at room temperature for three days. After the incubation period, the inhibition zone was evaluated. All of the experiments were carried out in triplicate. Each sample was tested in three different ways.
2.5 Extraction of fatty acids and preparation of methyl ester (FAMEs)
The root of Andrographis paniculata was collected and cleaned individually under running tap water to remove dust, debris, and other soil particles. Then, the root was dried at room temperature and made powder by a Fritsch mortar grinder from Germany. The natural fatty acids were extracted separately from the plant's root powder (100 g) using petroleum ether (b.p 40 °C-60 °C) for 72 h in a Soxhlet device. In a rotary evaporator, the extracts were concentrated under reduced pressure. The extracts were filtered through Whatman No. 1 filter paper before being vacuum distilled to eliminate the solvent completely. In a refrigerator, the extracts were stored in a nitrogen atmosphere. The fatty acids in the extracts were first converted to fatty acid methyl esters (FAMEs), and then GC–MS was examined using the method described by Griffin (Griffin, 1960).
2.6 Gas Chromatograph-Mass spectrum analysis
On an Agilent 7890A system with a mass Spectrophotometer detector and split less injection system, GC–MS analysis of petroleum ether extract from the root was performed. An HP-5MS capillary column (30 m × 0.25 mm: film thickness: 25 m) was installed in the GC. The following was the temperature schedule: initial oven temperature was 70 °C, then increased at 10 °C/min to 150 °C for 5 min, then 12 °C/min to 200 °C for 15 min, and finally 12 °C/min to 220 °C for 15 min. Helium was employed as the carrier gas, with a flow rate of 0.6 ml/min and a pressure of 17.69 psi. The samples were dissolved in methanol, and a 1 µl aliquot was automatically injected. MS had been set to scan mode. It was electron ionization that caused the ionization. The mass range was chosen to be between 50 and 550 m/z. MS spectra of isolated components were identified for fatty acid composition using NIST libraries.
3 Results
3.1 Oil extraction yield
A. paniculata root showed different yield (%) for the different solvents; 6.4% for ethyl acetate, 8.9% for ethanol and 12.6% for methanol.
3.2 In vitro antibacterial activity assay
The in vitro antibacterial activity of various solvent extracts of A. paniculata root at different concentrations (10, 40, 80, 120, 160, 200 µg/ml) was qualitatively measured by zones of inhibition against employed bacteria. Consequences showed that all the crude extracts have potential against all the bacterial strains screened (Figs. 1–4). Methanol extract was more effective compared with the ethanol and ethyl acetate extract. Methanol extract was exposed the greatest amount of bacterial activity against the maximum number of pathogen bacteria. Maximum activity of methanolic extract was observed against Salmonella typhimurium (45 mm), while ethanol and ethyl acetate extract revealed strong in vitro antibacterial activity against Shigella flagella (42 mm) and E. faecalis (38 mm), respectively at 200 µg/ml concentration.
Zones of inhibitions (mm) showing antibacterial activity of ethyl acetate extract of Andrographis paniculata root.
Zones of inhibitions (mm) showing antibacterial activity of ethanol extract of Andrographis paniculata root.
Zones of inhibitions (mm) showing antibacterial activity of methanol extract of Andrographis paniculata root.
Antibacterial Zones of inhibitions of Root Extract (A) E.A extract with different con. against V. parahaemolytices (B) Ethanol extract with different con. against S. aureus (C) Methanol extract with different conc. against P. arerogenosa (D) MBC of E.A extract against V. parahaemolytices (E) MBC of Ethanol extract against E. coil (F) MBC of Methanol extract against V. parahaemolytices.
Furthermore, MIC and MBC values (Table 1) of the root extracts were studied and compared with the standard by the well diffusion method. Ethyl acetate extract and ethanol extract lowest MIC value was 2.5 µg/ml against S. flagella and E. coli, respectively, whereas methanol root extract stood 2.8 µg/ml against P. arerogenosa. A. paniculata root extract disclosed possibly antibacterial action opposed to the microorganism with MBC from 165 µg/ml to 195 µg/ml. The highest MBC value was 195 µg/ml for Vibro parahaemolytices. The lowest MBC value was 165 µg/ml for Bacillus cereus and Salmonella typhimurium. The MIC and MBC evaluation of vigorous solvent extract in contradiction of strains presenting auspicious outcomes of antibacterial perspective of the A. paniculata.
SI No
Name of Bacteria
Ethyl acetate extract
Ethanol extract
Methanol extract
MIC (µg/ml)
MBC (µg/ml)
MIC (µg/ml)
MBC (µg/ml)
MIC (µg/ml)
MBC (µg/ml)
1
Bacillus cereus
3
165
4
170
3.5
175
2
Bacillus thuringiensis
3
170
3.5
180
4
180
3
Staphylococcus aureus
3.5
185
3
175
3.5
185
4
Enterobacter faecalis
3
180
3
185
4.5
170
5
Shigella flagella
2.5
175
3
190
4
165
6
Salmonella typhimurium
3
165
3.5
194
3.5
185
7
Klebsiella pneumonia
3
170
3.5
187
3.6
175
8
Escherichia coli
3.5
175
2.5
180
3
170
9
P. arerogenosa
3.5
185
3
187
2.8
180
10
Vibro parahaemolytices
4
185
4.5
195
3.5
190
3.3 In vitro antifungal activity assay
Additionally, the results of fungal bioassays of root ethanol and methanol extract were also determined (Fig. 5 and Table 2). This assay exposed that the ethanol root extract showed the maximum amount of antifungal activity (38 mm) and also methanol root extract showed the same value against Trichophton mentagrophytes and Candida albicans, respectively. The lowest antifungal activity was disclosed (32 mm) by ethanol root extract against Aspergillus niger. This assay discovered that A. paniculata had a strong antifungal perspective too.
Antifungal ZIB (A) Ethanol Root Extract against Trichophyton mentagrophytes (B) Methanol extract against Candida tropicals.
SI No.
Fungal Strain
Zone of Inhibition (mm)
Ethanol (100 µg/ml)
Methanol (100 µg/ml)
1
Aspergillus niger
32 ± 0.2
34 ± 0.3
2
Trichophton mentagrophytes
38 ± 0.5
37 ± 0.7
3
Candida albicans
35 ± 0.7
38 ± 0.5
4
Candida tropicals
33 ± 0.3
36 ± 0.5
3.4 Investigation of fatty acid by GC–MS
The constituents of the fatty acids from the root of Andrographis paniculata from petroleum ether extract confirmed the presence of 11 compounds, comprising the vigorous type of fatty acids that have been represented in Table 3. The fatty acids composition of root defended the herb’s acceptability in the ayurvedic remedies. They are associated with extended biological signing mechanisms. The highest component was methyl palmitate (56.732%) with a retention time of 11.59 min which is a saturated fatty acid. Most of the saturated fats have very important properties in heart disease. They also have been revealed to shield the glandular organ from liquor and drugs. Lack of them the immunity system of our body also reduced against predators or microorganisms. Unsaturated fatty acids are prime resources of power and strength from nutriment (Peichev et al., 2000).
SI No.
Retention time (min)
Name of the compound
Types of fatty acid
Concentration (%)
1
5.62
Methyl Caproate/Methyl Hexanoate
Saturated
0.961
2
8.70
Methyl Laurate
Saturated
0.259
3
9.96
Methyl Myristate/ Methyl Tetradecanoate
Saturated
1.151
4
10.71
Methyl Pentadecanoate
Saturated
0.547
5
11.59
Methyl Palmitate
Saturated
56.732
6
12.56
Methyl Heptadecanoate
Saturated
0.897
7
13.65
Methyl Stearate
Saturated
9.978
8
13.82
Trans-9-Elaidic acid methyl ester
Unsaturated
5.968
9
13.96
Cis-9-Oleic acid Methyl ester
Unsaturated
8.875
10
14.57
Methyl Linoleate
Unsaturated
12.192
11
16.07
Methyl Eicosanoate
Unsaturated
2.442
4 Discussion
Globally transferrable diseases are the foremost reason for illness and temporariness. At this time, the enduring conflict contrary to bacteria and fungus wins through the confidence of growing, resistance. Nevertheless, development in health field outcomes in further sufferers being in serious and resistant repressed conditions, hence producing a continuous necessity for new antibiotics and antifungal drugs. Consequently, now is the great stage to find out novel microbicidal drugs (Mahesh and Satish, 2008). Natural medicines are a greater significant subject of interest in therapeutic industries for the preparation of important remedies (Sule et al., 2010). At extreme methanolic extract, concentration disclosed the highest bacterial inhibitory activity compared to other extracts. This result is a similar statement stated by several scientists (Parekh and Chand, 2010; Al-Bayati, 2008; Kaushik and Goyal, 2011, Shalini and Narayanan, 2015). There is very few work has been done on the antimicrobial activity of A. paniculata root, this study was done with ten microorganisms and interestingly the growth of all the bacteria has inhibited whereas researchers previously did by four bacteria (Shalini and Narayanan, 2015). The composition of fatty acid is subject to changing in topographical and environmental circumstances. Their hydrophobic behaviors, good collaboration with phospholipid bilayer characteristics might show the anti-microorganism properties. (Carvalho de Lima et al. 2016).
5 Conclusion
From the primordial periods, A. paniculata has been used in traditional treatments as a chemotherapeutical agent. It has the least side effects and worldwide field for numerous infections in the pharmacological markets with extract or isolated composites. The research was accomplished to open a new era in improving traditional medicine with modern microbiology. The encouraging outcome of this investigation may bring about the root of A. paniculata would be a prospective source of antibiotics. This in vitro study of A. paniculata is an absolutely new finding in Bangladesh. This is not to claim that the assays' results are conclusive; they may be the first stage in a considerable process that will ultimately lead the new researchers to the selection of more valuable substances in the plant.
Acknowledgment
We are grateful to IFST, BCSIR, Dhaka for giving us the opportunity to do the antimicrobial assay of the crude extract of plant materials. We are also thankful to the Director, BCSIR Laboratories, Dhaka, for providing the necessary facilities to carry out this research work.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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