|Year : 2020 | Volume
| Issue : 3 | Page : 296-302
Effect of temperature and pressure on antimycobacterial activity of Curcuma caesia extract by supercritical fluid extraction method
Monika Chaturvedi, Reena Rani, Dushyant Sharma, Jaya Parkash Yadav
Department of Genetics, Maharshi Dayanand University, Rohtak, Haryana, India
|Date of Submission||25-Jun-2020|
|Date of Acceptance||01-Jul-2020|
|Date of Web Publication||28-Aug-2020|
Jaya Parkash Yadav
Department of Genetics, Maharshi Dayanand University, Rohtak - 124 001, Haryana
Source of Support: None, Conflict of Interest: None
Background: Supercritical fluid extraction (SFE) is an advanced technique using CO2as a solvent and plant-based drug exploration is a topic of growing interest. Curcuma caesia is a medicinal herb with many medicinal potential. Hence, in the present study, the effect of temperature (40°C–60°C) and pressure (10–20 MPa) on extraction yield and antimycobacterium potential of C. caesia Roxb. dry rhizome powder using supercritical fluid extraction method were evaluated. Methods: The extract of C. caesia by SFE was accomplished using temperature range (40°C–60°C) and pressure range (10–20 MPa). The chemical profile of the extracts were investigated by Gas Chromatography Mass Spectrometry (GCMS) and the antimycobacterial activity of the extracts were analyzed against Mycobacterium smegmatis strains MTCC06 and MTCC994. Compounds found in the extract were further checked by in silico analyses with two protein target 4DRE and 3UCI. Results: Extraction yield ranged from 3.0 to 5.6 g/25g dry substrate, with the highest value being achieved at 50°C and 15 MPa. The results of GCMS analyses revealed the presence of beta-elemene, curzerenone, boldenone, and 2-cyclohexen-1-one, 4-ethynyl-4-hydroxy-3, 5, 5-trimethyl in the extracts. The extract obtained at 50°C temperature and 15 MPa pressure showed the highest zone of inhibition against M. smegmatis strains MTCC06 and MTCC994, that is, 15.6 mm and 13.6 mm, respectively. Active constituents present in the extracts showed good binding energy with 4DRE and 3UCI by in silico analysis. Conclusion: This study identified the effect of temperature and pressure on yield C. caesia extract by SFE method. Furthermore, the effect of different extracts on antimycobacterial potential and docking study validated the antimycobacterial potential.
Keywords: Curcuma caesia, docking, Mycobacterium smegmatis, supercritical fluid extraction
|How to cite this article:|
Chaturvedi M, Rani R, Sharma D, Yadav JP. Effect of temperature and pressure on antimycobacterial activity of Curcuma caesia extract by supercritical fluid extraction method. Int J Mycobacteriol 2020;9:296-302
|How to cite this URL:|
Chaturvedi M, Rani R, Sharma D, Yadav JP. Effect of temperature and pressure on antimycobacterial activity of Curcuma caesia extract by supercritical fluid extraction method. Int J Mycobacteriol [serial online] 2020 [cited 2021 Jan 20];9:296-302. Available from: https://www.ijmyco.org/text.asp?2020/9/3/296/293538
| Introduction|| |
Curcuma caesia Roxb. also known as Kali Haldi belongs to Zingiberaceae family. Various tribal communities have used C. caesia long ago. Traditionally, its rhizomes were used in treating asthma, leukoderma, rheumatic pains, piles, antidiarrheic, bronchitis, and snake and scorpion bite. C. caesia is an aromatic perennial herb with creeping horizontal or tuberous rhizomes having diverse pharmacological activities such as anti-inflammation, anticancerous, antihelminthic, antileprosy, antidiabetic activity, antimutagenic activity, antimycobacterial activity, and antitoxicity against cyclophosphamide. Numerous bioactive metabolites used in pharmacological industries have been reported to present in C. caesia rhizomes such as flavonoids, alkaloids, sesquiterpene, and phenolic.,, Methanol extract of C. caesia was analyzed to have cytotoxicity (IC5090.70 + 8.37 ug/mL) on Ehrlich Ascites carcinoma cell lines.
Nowadays, herbal medicines are gaining much importance as they are less toxic than chemical based drugs. Extraction of secondary metabolites from plant for the preparation of herbal medicine is increasing. Supercritical fluid extraction (SFE) method, which employs fluids in their supercritical states for the extraction of solid samples, is a powerful technique for separation of natural compounds from plants. In recent years, many investigations have been made on apparent industrial applications of the SFE, which offer some benefits over the conventional techniques, mainly in food, chemical, pharmaceutical, and oil industries. The SFE method has advantage over conventional methods of extraction due to less use of toxic solvent, extraction of heat-labile metabolite, and contamination-free product.,
Therefore, the objective of the present study is the experimental study of SFE of C. caesia in a bench-top unit to study the effect of pressure and temperature on the extraction yield. The chemical constituents of C. caesia extract were also analyzed by gas chromatography–mass spectrometry (GCMS) and their antimycobacterial activity was checked against Mycobacterium smegmatis. Moreover, the docking study was also conducted to explore the possibility of metabolite as a future drug against Mycobacterium tuberculosis.
| Methods|| |
Curcuma caesiacollection and preparation
C. caesia Roxb. was collected and identified from ICAR-Indian Institute of Spices Research Kozhikode, Kerala, with Accession No: 1154 (Voucher no: 266608) and grown in the herbal garden of Maharshi Dayanand University, Rohtak, Haryana. Harvesting of rhizome was done in November 2018. Rhizomes of C. caesia were washed with distilled water, shade-dried for 1 week, and then grinded into fine powder. The moisture content of the rhizome was calculated by hot air oven drying method.
Supercritical fluid extraction
A laboratory scale SFE unit (Speed™ SFE Prime of Applied Separations, Allentown, PA, USA) was used to perform the SFE assays to obtain the extracts from C. caesia by loading 10 g of substrate in a 25 ml of extraction vessel. Glass wool was cast off in the extraction vessel at both the ends to avoid entrainment of the sample. All the SFE extractions were performed in a constant extraction time, i.e., 60 min in static mode. The influence of pressure and temperature was estimated on the yield of C. caesia extract with temperature ranges from 40°C to 60°C and pressure from 10 MPa to 20 MPa. The mass of extract was evaluated by collecting in a preweighed clean and dry glass vial. Extraction yield (Y) of C. caesia was indicated as grams extract per 25 g of dry substrate (g/25 g d.s.). The packed bed supercritical extraction procedure was carried out.
Experimental design and statistical analysis
The effect of independent variables (temperature and pressure) on yield (response variable) of C. caesia extract was evaluated by central composite rotatable design of response surface methodology. The factors and their levels are shown in [Table 1]. A total of 11 runs were carried out, which include 4 factorial points, 4 axial points, and 3 center points with a value of α = 1.41. It was created on a two-factor factorial design (n = 2), by two levels (coded values −1 and +1). Coded temperature (A1) in degree centigrade and coded pressure (A2) in MPa calculated by Equation 1 and 2 respectively.
|Table 1: Independent variable with level of central composite rotatable design matrix|
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The experiment data were based on the second-order model Eq. (3), which expressed the yield (Y) (response variable) as a function of temperature (A 1) and pressure (A 2) (independent variable). Experiments were conducted in a randomized order to reduce the effect of unexpected variability in the observed response due to unnecessary factors.
Y = X0 + X1A1 + X2A2 + X12A1A2 + X11A12 + X22A22(3)
where X0 is a constant; X1 and X2 are linear coefficients, and X12 is a cross-product coefficient. Quadratic coefficients are symbolized by X11 and X22. Three-dimensional (3D) surface response plots were generated and goodness of fit was assessed by analysis of variance (ANOVA). Using Design-Expert Software, version 12 (Stat-Ease, Inc., Minneapolis, MN, USA), the coefficients of response surface equation were assessed.
Gas chromatography–mass spectrometry analysis
The extracts obtained were further analyzed for chemical profile by BRUKER SCION 436-GC SQ GCMS instrument equipped with RESTEK Rtx®-5 (Crossbond® 5% diphenyl/95% dimethylpolysiloxane) with 30 m length, 0.25 μm df, and 0.25 mm ID column. Helium as a carrier gas was used at a flow rate of 1 ml/min in split mode and was used for the separation of phytochemicals. GCMS protocol was used following the method of Chaturvedi et al.
The antimycobacterium activity of all SFE extracts of C. caesia was analyzed using agar well diffusion assay against M. smegmatis strains, i.e., MTCC06 and MTCC994. Both the strains of M. smegmatis obtained from the National Jalma Institute of Leprosy and other Mycobacterial Diseases, Agra, Uttar Pradesh, India. The rifampicin disk was used as a standard against M. smegmatis with a concentration of 10 μg.
Molecular docking analysis was performed using MGL docking tool through AutoDock 4.2 software from the Scripps Research Institute of USA for the prediction of interaction between metabolite present in SFE extract and potential drug targets of mycobacterium, i.e., enoyl-acyl reductase enzyme InhA (PDB ID: 4DRE) and gyrase type IIA topoisomerase (PDB ID: 3UCI) of Mycobacterium tuberculosis. The generated results of docking were analyzed and visualized by Discovery Studio. Lipinski's rule of five helps in preliminary analysis of molecule that it can be used as drug or not. Before docking analysis, compounds were analyzed by SCFBIO-IIT-Delhi (http://www.scfbio-iitd.res.in/software/drugdesign/lipinski.jsp) online software for Lipinski's rule of five. The ADMET properties of all ligands was analyzed using an online server, i.e., Admet SAR (http://lmmd.ecust.edu.cn/admetsar2/about).
| Results|| |
In packed bed SFE the solid particle size has a great effect on the yield because small particle size provides larger surface area and lower internal diffusion resistance. If the particle size is below 0.71 mm, its effect can be neglected as according to Del Valle et al. The average particle size of C. caesia dry sieved powder was 0.64 mm. Moisture contents were calculated by hot air oven drying method, which is found to be 8.0% ± 0.5%. Plant materials were characterized with bed porosity of 0.38 + 0.004 and solid density of 791.6 + 3.2 kg/m3 and apparent density of 189.4 + 1.6 kg/m3.
Experimental extraction yield and predicted yield using supercritical CO2 from dry rhizome powder of C. caesia are depicted in [Table 2]. The yield was 2.5 times higher than the lowest yield as it ranges from 3.0 g/25 g to 5.6 g/25 g d.s. The statistical indicators obtained by the analysis of the variance applied to the selected second-order model Eq. (3) [Supplementary Table 1]. P < 0.05 indicated that model terms were significant.
|Table 2: Experimental and predicted extraction yield as a function of T and P|
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The second-order model Eq. (4) characterizes the effects of independent variable on response variable, i.e., pressure and temperature on yield in the particular experimental section. The equation in terms of coded factors can be used to make predictions about the response for given levels of each factor.
Y = 5.43 + 0.10359 (A1) – 0.0268 (A2)– 0.1500A1A2 – 1A12 – 1.5A22(4)
The ANOVA was used to specify which terms are statistically important and that have a positive or a negative effect on yield [Supplementary Table 2].
The correlation between predicted responses with experimental responses has been shown in [Figure 1]a. The graph shows that the experimental values are closely matched with the predicted values. It means that the second-order model provides a statistically significant relation between the response variable and the independent variables. The data were analyzed by difference in fits (DFFITS) plots (the effect on the predicted value of each point is calculated for the reliability evaluation of the model) and Cook's distance method (measure of how the regression changes if the case has been removed) to confirm the adequacy of model or absence of outlier in the experimental data. The Cook's distance plot [Figure 1]b and DFFITS plots [Figure 1]c analysis revealed that model show no unpredicted errors. [Figure 2] shows the 3D graphical response of Eq. (4). The extraction yields increased with increasing the temperature and pressure upto a certain limit and then decline. The highest yield was 5.6 g/25 g d.s. at 50°C temperature and 15 MPa pressure.
|Figure 1: Diagnostic plot of model (a) predicted versus actual (b) graphical plots Cook's distance (c) difference in fits plot normal probability versus residual|
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|Figure 2: Three-dimensional response surface graph for extraction yield (Y, g/25 g dry substrate) as a function of temperature (T, °C) and pressure (P, MPa)|
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Gas chromatography–mass spectrometry profile
The major metabolites present in different SFE extracts was identified by GCMS [Table 3]. Metabolites, beta-elemene and curzerenone, were present in almost every extracts with variation in the area percentage value of peak. GCMS chromatogram of extract obtained at 50°C and 15 MPa condition showed the highest area of percentage of beta elemene and curzerenone [Supplementary Figure 1]. At this condition, the peak area of beta-elemene and curzerenone was 16.38% and 18.097%, respectively. The peak area of boldenone and 2-cyclohexen-1-one, 4-ethynyl-4-hydroxy-3, 5, 5-trimethyl was 15.646% and 10.063%, respectively.
Antimycobacterium study results showed that the extracts at 50°C and 15 MPa condition were most efficient with respect to all other extracts. The zone of inhibition of various extracts obtained by SFE against M. smegmatis strains MTCC06 and MTCC994 were analyzed at concentration of 50 mg/ml [Supplementary Figure 2]. The extract at 50°C and 15 MPa condition showed the maximum activity with zone of inhibition 15.6 mm and 13.6 mm against MTCC06 and MTCC994, respectively.
Molecular docking studies were done with four metabolites, namely, beta-elemene, curzerenone, boldenone, and 2-cyclohexen-1-one, 4-ethynyl-4-hydroxy-3, 5, 5-trimethyl with 4DRE and 3UCI. Enoyl-acyl reductase enzyme (InhA) is necessary for cell wall synthesis as it synthesized mycolic acid a vital component of mycobacterium cell wall and gyrase type IIA that helps in reducing topological strain in DNA helix during replication. ADMET properties and Lipinski's rule of five for all the four metabolites have been shown in [Supplementary Table 3]. All the four metabolites followed the Lipinski's rule of five. The estimation of the ADMET properties plays a significant role in the early phase of drug formulation process. Caco-2 cell permeability, blood–brain barrier penetration, human intestinal absorption, and Ames test properties were calculated. After drug likeness properties, the docking study was done. The docking study revealed that boldenone showed the highest binding energy against both the receptor 4DRE and 3UCI. The binding energy and inhibition constant of the four ligands along with standard ethionamide drug are shown in [Table 4].
|Table 4: The binding energy, inhibition constant, hydrogen bond, and hydrophobic interaction|
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The 3D interaction of 4DRE and 3UCI with ligands have been shown in [Figure 3] and [Figure 4] respectively. The 3D interaction shows the bond length, type of bond, and amino acid involved in the interaction. The most efficient binding was shown by the 3UCI protein with boldenone, that is, −8.45 Kcal/mol with three hydrogen bond and nine hydrophobic interactions.
|Figure 3: The three-dimensional interaction of ligands with 4DRE receptors (a) beta-elemene (b) curzerenone (c) boldenone (d) 2-cyclohexen-1-one, 4-ethynyl-4-hydroxy-3, 5, 5-trimethyl (e) ethionamide (green line hydrogen bond, purple line hydrophobic interaction)|
Click here to view
|Figure 4: The three-dimensional interaction of ligands with 3UCI receptors (a) beta-elemene (b) curzerenone (c) boldenone (d) 2-cyclohexen-1-one, 4-ethynyl-4-hydroxy-3, 5, 5-trimethyl (e) Ethionamide (green line hydrogen bond, purple line hydrophobic interaction)|
Click here to view
| Discussion|| |
This study describes the correction between different temperatures and pressures on extraction yield of extract using SFE method. Furthermore, apart from yield, the chemical profile of different extracts was changed with respect to their antimycobacterium potential. In the present study, the highest yield was obtained at 50°C temperature and 15 MPa pressure. Brunner suggested that the pressure increases the CO2 density which further enhances the solvent power by decreasing the distance between CO2 molecules. Extraction yield in Smyrnium cordifolium Boiss leaves is directly proportional to temperature (40°C–60°C) whatsoever the pressure (10–30 MPa). Many studies suggested that indirect relational between extraction yield and pressure is due to diffusion coefficient, which is inversely proportional to pressure. Wang et al. suggested that at high-pressure levels, repulsive interactions occur between solvent and solute which may decrease the extraction yield of Cyperus rotundus Linn.
Extract obtained at 50°C and 15 MPa showed the highest one of inhibition with M. smegmatis. This may be due to the presence of four metabolites, namely, beta-elemene, curzerenone, boldenone, and 2-cyclohexen-1-one, 4-ethynyl-4-hydroxy-3, 5, 5-trimethyl. Many studies suggested that sesquiterpene has antimycobacterium activity for example β-elemene have the capacity to alter the expression of dprE1 gene needed for cell wall synthesis and clgR genes regulate cell membrane structure of mycobacterium. The essential oil of ginger mainly composed of monoterpenes and sesquiterpenes exhibited inhibitory activity against Mycobacterium tuberculosis. Antimycobacterium activity of Salvia tomentosa, Origanum minutiflorum, O. syriacum, and Thymus revolutus is due to the presence of caryophyllene, germacrene D, and β-elemene in their essential oil.,,, Hence, the presence of sesquiterpene in extract is responsible for antimycobacterium activity. In contrast, steroids are also effective in reducing tuberculosis mortality, including pulmonary tuberculosis. Steroids were given in combination with antituberculosis drugs showed the decrease in the mortality rate in patient suffering from tuberculosis of central nervous system. Testosterone and estradiol derivatives have potential as antimycobacterium activity with IC50 at 10.6 μM. Corticosteroids are also recognized as having beneficial effect on persistence of tuberculosis patients. In the present study, the antimycobacterium activity of C. caesia seems to be due to the presence of β-elemene (sesquiterpenoid), curzerenone (monoterpenes), and boldenone (steroid).
In silico analysis is an attractive method used by researchers to recognize the interactions among drug and protein. In silico analysis is advantageous for the synthesis of a better drug for specific pathogen. The two bacterial proteins, viz., 4DRE and 3UCI, were used for the in silico analysis with four metabolites present in the extract obtained at 50°C and 15 MPa. All four metabolites show good binding energy, but boldenone which is a steroid shows the highest negative binging energy. Docking results validate that antimycobacterium activity is due to these four metabolites.
| Conclusion|| |
This study investigated the effects of pressure and temperature on the extraction yield of C. caesia rhizome powder from SFE. A second-order regression model was used to predict the experimental results. The maximum yield of 5.6 (g/25g d.s) was found under 50°C and 15 MPa with the highest zone of inhibition against antimycobacterium activity. GCMS analysis shows four metabolites in prominent in almost all extracts. Docking analysis of these four metabolites with two proteins of mycobacterium also validate that these metabolites help in antimycobacterium activity. It is concluded that the extract obtained at 50°C and 15 MPa condition showed the presence of four major metabolites that may contribute for antimycobacterium activity of C. caesia.
The author would like to thank the University Grant Commission (UGC), New Delhi, for financial support by UGC SAP [F. 20/2012(SAP II)] project fund and also ICAR-Indian Institute of Spices Research, Kozhikode, Kerala, for providing the samples.
Financial support and sponsorship
This study was financially supported by the UGC, New Delhi, by award number UGC SAP [F. 20/2012(SAP II)] project fund.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]