The Effect in Vivo and in Silico Citronella Grass Extract (Cymbopogon nardus L.) on the Plasma ACE Inhibitory activity and Antihypertensive effect
Rofiatun Solekha1,4*, Ni Nyoman Tri Puspaningsih3, Putri Ayu Ika Setiyowati4,
Sri Bintang Sahara Mahaputra Kusumanegara5, Fatan Mujahid4, Hery Purnobasuki2*
1Doctoral Program of Mathematics and Natural Science, Faculty of Science and Technology, Airlangga University, Jl. Dr. Ir. H. Soekarno, Mulyorejo, Surabaya 60115, East Java, Indonesia
2Department of Biology, Faculty of Science and Technology, Airlangga University, Surabaya, Jl. Dr. Ir. H. Soekarno, Mulyorejo, Surabaya 60115, East Java, Indonesia.
3Department of Chemistry, Faculty of Science and Technology, Airlangga University, Surabaya, Jl. Dr. Ir. H. Soekarno, Mulyorejo, Surabaya 60115, East Java, Indonesia.
4Department of Biology, Faculty of Science, Technology and Education, Universitas Muhammadiyah Lamongan, East Java, Indonesia.
5Department of Pharmacy, Faculty of Health, Universitas Muhammadiyah Lamongan,
East Java, Indonesia.
*Corresponding Author E-mail: hery-p@fst.unair.ac.id
ABSTRACT:
The mechanism of hypertension is through the formation of angiotensin I into angiotensin II by Angiotensin I Converting Enzyme (ACE) which causes constriction of blood vessels resulting in narrowing of blood vessels. A number of extracts and compounds derived from plants have been proven in vitro as ACE inhibitors including flavonoids. This compound produces the ability to reduce oxidative stress, inhibit angiotensin converting enzyme (ACE) activity, promote vascular endothelial relaxation, and regulate cell signaling and gene expression by lowering Heat Shock Protein 70(HSP 70). The purpose of this study was to determine the effectiveness of the optimal dose of Cymbopogon nardus (L.) Citronella grass extract in its activity as a hypertension reducer and the effectiveness of the compound for inhibiting HSP-70 as an antihypertensive. The study employed bioinformatics modeling in its effectiveness in inhibiting HSP-70 in silica and in vitro using Cymbopogon nardus (L.) Citronella grass extract with various doses of 25, 50, and 100mg/kg BW in BALB/C mice. Na-CMC was used as a positive control and lead acetate was used as a negative control. Modeling with in silico method was used to observe the inhibition of compounds from Citronella grass stems against heat shock protein 70(HSP-70). The in vitro method with the maceration method was used in its extraction. The HPLC method was used for testing ACE inhibitors. The results of this study were treated with Na-CMC suspension (66.3±1.2%), acetic acid (65.7±0.7%), a dose of 25mg/kg BW (80.9±1.3%), a dose of 50 mg/kg BW was 88.2±1.7 and a dose of 100mg/kg BW (93.9±2.5%). In conclusion, HSP-70 can be used as an indicator of in silico inhibition of hypertension and is effective in reducing hypertension in vitro.
KEYWORDS: Ace inhibitor, Anti-hypertension, Citronella grass, In silico, HSP-70.
INTRODUCTION:
Hypertension is an increase in a person’s blood pressure that is higher than normal and can result in morbidity and mortality1.
Hypertension has been a serious problem until now. WHO (World Health Organization) states that hypertension affects 22% of the world’s population, and reaches 36% of the incidence in Southeast Asia. Hypertension is also a cause of death with 23.7% of the total 1.7 million deaths in Indonesia in 20162. The mechanism of hypertension is through the formation of angiotensin I into angiotensin II by Angiotensin I Converting Enzyme (ACE) which causes constriction of blood vessels and results in narrowing of blood vessels. ACE inhibitor drugs are the first class of hypertension treatment including Captopril, Lisinopril, Enalapril, and Ramipril. Prolonged use of these drugs can cause side effects such as dizziness, cough and angioneurotic edema. Prolonged use of these drugs can cause side effects such as dizziness, cough and angioneuritic edema, prolonged use of hypertension drugs will also cause complications of hypertension which causes 30% of human deaths3, this triggers most research to target bioactive compounds from nature. Some examples are peptides, anthocyanins, flavanols, and triterpenes.
A number of extracts and compounds derived from plants have been shown in vitro to be ACE inhibitors4. This beneficial effect generally stems from the presence of flavonoid molecules whose complex chemical derivatives can reach into the active center of ACE5. Flavonoids are one of the secondary metabolites found in plants which can inhibit many oxidation reactions Flavonoids have the ability as antioxidants because they are able to transfer free radical compounds6. These compounds produce the ability to reduce oxidative stress, inhibit the activity of angiotensin converting enzyme (ACE), increase vascular endothelial relaxation, and regulate cell signaling and gene expression7.
Citronella grass (Cymbopogon nardus) contains active phenolic compounds which act as antioxidants8 Ethanolical extract of lemongrass (C. citratus) leaves contained alkaloids, saponins, flavonoids, phenols, and steroids9. The compounds found in citronella could inhibit the HSP-70 protein through modeling protein inhibition by compounds found in citronella using a bioinformatics approach10. In silico methods are particularly interesting because they can be easily integrated into the early stage of the drug discovery process using only the “virtual” structure of the compounds11. Moreover, they are less time consuming and cheaper than wet experiments so that large numbers of compounds can be evaluated. There are several kinds of in silico models which focus on targets at different levels including addressing the whole body, or specific organs, or certain biological processes, or focused biochemical mechanism such as binding to a receptor.
In comparison with the models presented above, in silico models for organ-specific effects are generally focused on pharmaceuticals since data availability is most abundant for drug-like compounds. Among them, hepatotoxicity has been frequently investigated12, and nowadays increasing interest is also placed on cardiotoxicity and nephrotoxicity13. In previous studies, in silico and in vitro were used for anti-cancer activity 4H-chromenes with C4-active methine groups14.
There are several studies that examine the potential of rhizomes as a natural blood pressure lowering drug, but there is no research that specifically discusses the genus Cymbopogon like lemongrass (Cymbopogon nardus L.). The respondent's blood pressure after 2 weeks of being given ginger in the intervention group showed a mean systolic of 152.55mmHg, which was decreased15. In this study, in silico and in vitro observations were carried out to validate and provide the effectiveness of citronella in reducing hypertension. One of the parameters, in silico, is the presence of compounds bound to HSP-70 protein as binding ligands and receptors in antihypertensives16. In vitro parameter was a decrease in Angiotensin Converting Enzyme (ACE) inhibitor from Citronella grass extract with various doses of 25, 50, and 100 mg/kg BW. In this study, the researchers used hippuryl-L-histydyl-Lleucine (HHL) for comparison. The purpose of this study was to determine the potential of the compound in silico as well as the optimal dose of the use of citronella extract in its activity to decrease in hypertension In vitro.
MATERIALS AND METHODS:
Materials:
Sample preparation:
The PuChem database (https://pubchem.ncbi.nlm.nih.gov/) was used in this study for the preparation of chemical compounds samples from the results of GCMS analysis of lemongrass stems. Thirty-four compounds were successfully obtained from the database with PubChem Compound ID (CID) information which consisted of CID 7503, CID 460, CID 10329, CID 332, CID 7041, CID 1715136, CID 1146, CID 6432308, CID 92138, CID 92231, CID 95997 CID 12301996, CID 226486, CID 518516, CID 3084311, CID 521216, CID 5974, CID 592628, CID 9983, CID 26397, CID 527256, CID 75303, CID 594234, CID 554084, CID 12366, CID 565584, CID 606866, CID 548034, CID 543959, CID 15256789, CID 5363269, CID 1234769878, & CID 8791. Other information was 3D structure with file structure data format (sdf). The process of energy minimization and conversion of sdf files to protein databank format (pdb) was carried out on all samples of compounds through OpeBabel 2.4.1 software. Minimization of energy in the compound aimed to increase the flexibility of the molecule through positive bond energy. The 3D structure of the target protein, Hsp70 (RCSB ID: 4IO8) was obtained from the RCSB PDB database (https://www.rcsb.org/), protein sterilization was carried out using PyMol 2.5 version software through the removal of water molecules and contaminant ligands17.
Molecular Docking and 3D Visualization:
The ability of inhibitor activity by query compounds from lemongrass stems on Hsp70 in this study was predicted through molecular docking simulations. Molecular docking was used to measure the specific activity ability of the ligand and the pattern of molecular interactions through the value of binding affinity (Lipinski et al., 2001). This study used PyRx 0.9.9 version software to identify the ability of inhibitor activity on Hsp70 and activator on AR by compounds from lemongrass stems through molecular docking simulations with grid position Center (Å) X: 11,444 Y: -3.991 Z: 15,181 Dimension (Å) X: 36,592 Y: 38.011 Z: 35,415, Center (Å) X:6.891 Y: 30,858 Z:11,548 Dimension (Å) X:34, 254 Y:29, 278 Z:29, 846, and Center (Å) X: 19,813 Y: 8,863 Z: 38,209 Dimension (Å) X: 39, 358 Y: 37,212 Z: 53, 400. Visualization of the 3D structure of the ligand-protein complex was carried out using PyMol 2.5 version software and the structure was displayed with cartoons, transparent surfaces, and selected staining18.
Chemical Interaction Analysis:
Identification of molecular interactions of compounds from lemongrass stems with all target proteins in this study were identified through the Discovery Studio 2016 version of the software. Types of chemical bond interactions such as Van der Waals, hydrogen, hydrophobic, electrostatic, and pi were present in molecular complexes. The interaction formed was a weak bond that played a role in triggering the activity of the target protein19.
Molecular Dynamic Simulation:
The validation of the docking results was carried out through molecular dynamic (MD) simulation which aimed to identify the flexibility of ligand binding in the protein domain. The flexibility of the bond was shown by the RMSF plot on the CABS-flex 2.0ver server (http://biocomp.chem.uw.edu.pl/CABSflex2).
Parameters used for MD simulation were RNG seed, temperature, side-chain, C-alpha, rigidity, and trajectory. A stable binding interaction on the ligand-protein complex should had an RMSF value < 4Å.
Bioactivity Prediction:
The compound from the lemongrass stem extract with the strongest binding activity on Hsp70 was tested with the SwissADME server (http://www.swissadme.ch/) for the prediction of adsorption and distribution via Canonical SMILE. The prediction aimed to identify physicochemical properties, bioavailability score, and prediction as a drug-like molecule using various methods such as Lipinski, Ghose, Egan, and Muegge. Validation of inhibitory properties on specific compounds was further identified through the Molinspiration Chemoinformatics server (https://www.molinspiration.com/cgi-bin/properties), and predictions with positive results as inhibitors on query compounds were shown through more positive scores on probability scores.
Preparation of 0.5% Na-CMC suspension:
Na CMC was weighed 500mg then it was mixed with 100ml of warm sterile distilled water and stirred to mix evenly.
Preparation extract of C. nardus and suspension:
A total of 500grams of citronella stems were cleaned and then cut into small pieces, then dried using an oven at 40oC for 2 hours. The dried lemongrass stalks were then mashed using a blender. The stem powder was then macerated using 70% ethanol for 3 days. The maceration results were filtered using filter paper and then the extract was separated from the solvent by heating it using a water bath at 82oC to make suspension, then extract c. nardus was dissolved with 0.5% Na-CMC. The suspension concentration was 0.5%, 1%, and 2%.
Preparation of Lead acetate suspension:
A total of 10mg of lead acetate was mixed with 20ml of sterile distilled water and then stirred so that the lead acetate dissolved in distilled water.
Preparation of mice:
Balb c mice were acclimatized for a week. Mice were 8 weeks old and obtained from the Veterinary and Pharma Center, Surabaya, East java. They were housed under standard laboratory conditions (temperature 28-30°C, 12-h/12-h light/dark cycle) and given food and water ad libitum.
Mice acclimatization:
Acclimatization was carried out for 7 days with cage conditions at a temperature of 28-30oC. The process of acclimatization aimed to keep the animals alive during the research.
Experimental design:
Animals were divided randomly into five equal groups (n=6) as follows: the negative control group were given a subcutaneous injection of 0.1ml Na-CMC 0.5% within 40 days; positive control group were given a subcutaneous injection of 0.1ml lead acetate with a dosage of 7mg/kg BW within 5 days. The treatment group was given a subcutaneous injection of 0.1ml lead acetate with a 7mg/kg body weight dosage within 5 days. Then, the subcutaneous injections of 0.1ml with various dosages were given in each treatment group. The first treatment group was 25 mg/kg BW, the second treatment was 50mg/kg BW, and the third treatment was 100mg/kg BW within 35 days. After the end of the procedure, all mice were killed using chloroform20.
ACE inhibitor analysis:
The substrate HHL was dissolved (5mM) in 0.1M sodium-borate buffer (pH 8.3) containing 0.3 M NaCl. The assay was performed by mixing 100 lL of substrate solution with 25 lL of inhibitor solution (or borate buffer for control). After 10 min of incubation at 37 C, 10lL of ACE solution (100mU/mL) were added and the sample was further incubated at 37C for 30 min with continuous agitation at 450rpm. The reaction was stopped by adding 100 lL of 1 M HCl and the solution was filtered through a 0.45-lm nylon syringe filter before being analyzed by reversed-phase HPLC. The HPLC analysis was performed on a C18 column (150 3.0mm i.d.), particle size 5 lm with a Varian chromatographic system and analytes were detected at the wavelength of k = 228 nm. The column was eluted at a flow rate of 0.5mL min1 with a two solvents system: (A) 0.05% TFA in water and (B) 0.05% TFA in acetonitrile. The gradient consisted 5–60% B in 10 min, maintained for 2 min at 60% B, then returned to 5% B for 1 min. This was followed by isocratic elution for 4 min at 5% B.(Sharifi et al., 2013).
ACE inhibition (%) =
[1-(AUC inhibition/AUC control)] x 100
AUC inhibitor = AUC of the HA peak of Inhibitor
AUC control = AUC of HA peak of control sample without inhibit
RESULT:
Figure 1. Spathulenol Chemical Element
The compound with the most negative binding affinity value was predicted to trigger stronger activity on Hsp70 than other compounds. Visualization of the Spathulenol_Hsp70 molecular complex was carried out through rigid and transparent surfaces, cartoons, sticks structures with colored selection (Figure 2).
Figure 2: Structural visualization from the docking simulation Spathulenol_Hsp70.
Hsp70 activation was carried out by acetylation at the active site with lysine residues by the native ligand HOPX to produce a biological response to Hsp70. Spathulenol compounds were predicted to act as inhibitors of Hsp70 protein activity because they inhibited the binding site of the native ligand on Hsp70.
Figure 3: The ligand-protein interaction Spathulenol_Hsp70.
Data on Table 1 showed that the ACE inhibitor activity in each treatment had different values. The significant difference was according to the treatment. The results showed in the treatment with Na-CMC suspension (66.3 ± 1.2%), acetic acid was (65.7 ± 0.7%), the dose of 25 mg/kg BW was (80.9 ± 1.3%), the dose of 50 mg/kg BW was 88.2 ± 1.7 and a dose of 100 mg/kg BW (93.9 ± 2.5%).
Tabel 1: ACE inhibitor activity in several treatments
|
No. |
Treatment |
Precent of Inhibition (%) a |
|
1 |
Na-CMC |
66.3 ± 1.2 |
|
2 |
Acetic acid |
65.7 ± 0.7 |
|
3 |
25 mg/kg |
80.9 ± 1.3 |
|
4 |
50 mg/kg |
88.2 ± 1.7 |
|
5 |
100 mg/kg |
93.9 ± 2.5 |
a Values are means ± SD of three measurements.
Figure 4. Chromatogram of hippuryl-histidyl-leucine of sample of dose 100 mg/BB as a product of ACE inhibitors detected by HPLC
We can see the correlation between dose and chromatography (figure 4) of the GCMS detector which shows high levels of HHL In the detector picture it can be seen that the pik on HHL shows the highest pik compared to the others. This is correlated with the effective dose to the concentration level according to the chromatographic image.
DISCUSSION:
Medicinal plants can play vital role in the development of new drugs22. The compound from lemongrass stem extract with the most negative binding affinity value for each of the highest target proteins was Spathulenol (-7.9 kcal/mol) (Figure 1). Spathulenol as one of the immunomodulatory compounds23 and as antioxidant, anti-inflammatory, antiproliferative and antimycobacterial activities of the essential oil of Psidium guineense Sw24. MD simulations were carried out to validate the docking results in this study. MD aimed to identify the stability of the protein-ligand molecular complex with reference to the RMSF value25.
From these results, it can be analyzed that the higher the activity of ACE inhibitors, the higher the effective dose in treating hypertension. Flavonoids26, flavanols, flavanols, anthocyanins, isoflavones, flavones 27 and other phenolic compounds have proved to be effective in decreasing the ACE activity. This is in line with previous research which mentioned that citronella contained flavonoids, tannins, saponins and terpenoids. A dose of 100 mg/kg BW of citronella extract is an effective dose for increasing ACE inhibitors in reducing hypertension. In addition, antihypertensive activity is reported in traditional medicine for similar species of the above-mentioned plants including Crataegus oxyacantha28, Onopordon leptolepis, and Onopordon carmanicum29. Total phenolics and flavonoids contents among the selected species of Syzygium cumini leaves which is well known for diabetes treatment30.Research on essential oil from Blepharocalyx salicifolius showed an increase in ACE inhibitors and ATPase possible due to the presence of high concentrations of spathulenol31. S. pallasii essential oil was found to exhibit a dose-dependent ACE inhibitory activity with an IC50 value of 0.33 mg/ml32. In silico evaluation of ACE inhibitory activity of the individual components showed that spathulenol exhibited the best binding affinity with ACE, and the lowest binding energy of 7.5 kcal/mol33. ACE inhibitors are enzymes that play a role in blocking ACE so that ACE in the blood is not high, so ACE inhibitors are often used as a standard in the analysis of hypertension 34. In the food area, studies have been mainly focused on the identification of food components, principally peptides, able to inhibit ACE activity with the aim to control hypertension and then to prevent cardiovascular diseases through diet. In fact, nutrition has been reported as one of the main factors influencing blood pressure35. Figure 4 indicated the dose of 100 mg/BW experienced a high increase in HHL as a higher inhibitory response than other doses of citronella extract. It can be assumed that the most effective dose in reducing hypertension is a dose of 100 mg/BB. Certain conditions, such as narrowed arteries in your heart (coronary artery disease) or high blood pressure, gradually leave your heart too weak or stiff to fill and pump efficiently. Herbal drugs like digitalis, squill, and stropanthus commonly find the treatment of heart diseases36. This assumption needs further confirmation with further research in the form of clinical trials in accordance with ethical clearance to determine the safety, pharmacokinetic and pharmacodynamic aspects of the drug so that dose-related side effects can be assessed.
CONCLUSION:
Citronella stem extract has the potential to inhibit HSP-70 which causes hypertension with a bioinformatics approach in the form of in silico. This correlates with the in vitro method that citronella extract is effective in reducing hypertension at a dose of 100 mg/BW.
CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
ACKNOWLEDGMENTS:
The authors would like to thank Airlangga University and Universitas Muhammadiyah Lamongan for their kind support during molecular and all other lab studies.
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Received on 25.10.2022 Modified on 30.12.2022
Accepted on 07.03.2023 © RJPT All right reserved
Research J. Pharm. and Tech 2023; 16(10):4487-4492.
DOI: 10.52711/0974-360X.2023.00731