Nitric oxide (NO) free radical scavenging activity and Superoxide (O2-) radical scavenging activity
The nitric oxide free radical scavenging assay estimation was done by method described by Parul et al., 2013. Among the plant extracts undertaken the Hibiscus sabdariffa showed percentage inhibition of 68% (IC50 value 317.74 µg/mL) followed by the plant extract of Azadirachta indica inhibition of 62% and IC50 value (320.7473 µg/mL), and Moringa oleifera inhibition of 48.50% (IC50 value 350.618 µg/mL), (Table-1). While the ascorbic acid (positive control) showed inhibition of 73.50% (IC50 value 302.222 µg/mL) (Figure-1 and Table-1). Based on the free radical scavenging activity the IC50 value of Hibiscus sabdariffa (317.74 µg/mL) were observed closed to IC50 of positive control (302.222 µg/mL). The need of the hour is to harness and explore novel plant sources having appreciable bioactivity against NO radical accumulation and the accompanying inflammatory reaction. In the present work, Azadirachta indica and Moringa oleifera display higher NO free radical scavenging activity as compared to Hibiscus sabdariffa. One of the main enzymes involved in NO production is nitric oxide synthase (NOS) (Ondua et al., 2019). NO scavenging activity of the three plants was evaluated spectrophotometrically at 595 nm. The variations in optical density give a fair assessment of the NO scavenging potency of diverse plant extracts. The phytochemical profile of these plant extracts exhibited the presence of flavanoids and phenolic compounds which are important phytobioactives known for their appreciable radical scavenging potential (Kaurinovic and Vastag, 2019). All the three plants analyzed in the present investigations demonstrate ample NO free radical eradication ability and thus exemplify their intrinsic medicinal potential. Superoxide radicals play a key role in signal transmission at the cellular level (Luis et al., 2018). However, their production beyond a certain threshold in cells has proven to be counterproductive (Basu and Hazra, 2006). This ultimately produces a deleterious effect on biomolecules like proteins, lipids and nucleotides (Engwa, 2018). The most potent superoxide inhibitor proved to be Moringa oleifera followed closely by Azadirachta indica. These plants were traditionally incorporated in folk medications and hence it becomes imperative to prepare a comprehensive biochemical and antioxidant profile so as to harness their antioxidative stress release potential. These plants also displayed higher radical scavenging activity than the positive control used in these experiments. This further underscores the superoxide radical neutralization effect of these medicinal plants capable enough to prevent cell damage. A wide variety of superoxide scavenging phytoconstituents have been identified and characterized which include mangiferin, naringin, quercitin, myticetin among others (Septiana et al., 2010). The elucidation of optimum concentration of these natural superoxide deradicalizers is a key aspect in their use as efficient therapeutic agents.
LC-MS analysis of active Fractions of Moringa oleifera, Azadirachta indica, and Hibiscus sabdariffa
While doing the LC-MS analysis of the isolated molecule from chloroform fraction of Moringa oleifera, we found a peak of 274 m/z in the mass spectrum (Figure 2). Which was assumed to be a fragmentation of beta-sitosterol 414 m/z. Hence, the primary conformation of the isolated molecule was beta sitosterol (414 m/z). For further conformation we have done 1H-NMR and 13C-NMR.
1H‐NMR and 13C‐NMR spectra were done using CDCl3 as solvent on Bruker Advance II 400 NMR spectrometer at the department of Pharmacy at Guru Jambeshwar University Hisar, Haryana. The 1H-NMR spectrum of compound varied between δ 0.5 to 1 ppm. This spectrum shown fifteen protons on carbon C18, C21, C26, C27 and C29 position in the compound structure (Supplementary Figure -1 and Table-1). proton signal between δ 2.6-4.2 ppm at C4, C7, C25 and 1.2 to 2.5 ppm 47 proton present at different-different carbon position. Chemical shift of δ 5.3 corresponds to oleifinic proton. 13CNMR spectrum of Compound (1) shows signal at C3 β-hydroxyl group 17.064 and 11.0 for angular methyl carbon atoms for C19 and C18 respectively. The C5, C6, C22 and C23 appeared to be alkene carbons. The value at 17.064 ppm corresponds to angular carbon atom (C19). Spectra show twenty-nine carbon signal including six methyls, nine methylenes, eleven methane and three quaternary carbons (Supplementary Figure-2 and Table-2).
While doing the LC-MS analysis of the chloroform fraction of Azadirachta indica, we found a peak of 425.19 m/z. Which was assumed to be a fragmentation of Azadiradionolide m/z 480.574g/mol. The Moringa oleifera compound i.e. beta sitosterol, similar to cholesterol was also found in fungi, animal and plants. Beta- sitosterol belong to phytosterol which play role in heart disease, antioxidant, diabetes. These are mainly found in the mixture of stigmasterol. The difference between β- sitosterol and stigmasterol is single bond and double bond respectively. Study done by Pierre et al., (2015) found β-sitosterol from Odontonema strictum. Hence, the primary conformation of the isolated molecule was Azadiradionolide, which was 425.19 m/z (Figure-3) (274 m/z). The 1H NMR spectrum of compound varied between δ 0.5 to 1.75 showed the 15 proton at C18 and C19 methyl group carbon (supplementary file Figure-3 and Table-3). In δ 2.30 ppm shown 7 protons at C6, C9, C11 and C12, δ 3.72 shown 3 protons, δ 3.30-7.55 shown 8 protons at C17, C23, C15, C2, C1 and C21 (supplementary file Figure-4 and Table-4).
The LC-MS analysis of the chloroform fraction of Hibiscus sabdariffa, we found a peak of 274.25 m/z. Which was assumed to be a fragmentation of Hibiscitrin m/z 496.377g/mol (Figure-4). As the molecule was peak of Hibiscitrin after fragmentation is 274.25 m/z (Figure-4). By 1HNMR analysis δ 1.01-1.47 ppm shown 12 protons at C26, C27, C28 and C31 and δ 2.3- 2.8 shown 4 protons at C23, C24 and C25 (supplementary file Figure-5 and Table-5). At δ 3.68-7.28 ppm shown also shown 4 protons at C13, C6 and C23. Methylene group shown at C28 (Figure-6 and Table-6).
Angiotensin converting Enzyme Inhibitory Assay
The six plants extract under this study subjected to ACE inhibitory activity. The percentage inhibition of Azadirachta indica, Hibiscus sabdariffa, Moringa oleifera, Punica granatum, and Allium sativum were showed 74.00±0.7937 %, 73.47±1.434 %, and 71.80±2.650 %, (Figure-5). The lowest IC50 activity were showed by Azadirachta indica (255.991 µg/ml) followed by Hibiscus sabdariffa (267.722 µg/ml), and Moringa oleifera (294.397 µg/ml) (Table-2). Among them based on the IC50 the following plants extracts (Azadirachta indica, Hibiscus sabdariffa, and Moringa oleifera) were used for fractionation. The Azadirachta indica fraction i.e. n- butanol, ethyl acetate and chloroform showed the percentage inhibition and IC50 value were 42.45±1.050% (IC50 value 508.5438 µg/mL), 53.25±1.750 % (IC50 373.0502 value µg/mL), and 68.45±0.550 % (IC50 value 264.4097 µg/mL) respectively. The Hibiscus sabdariffa fraction i.e. n- butanol, ethyl acetate and chloroform showed the percentage inhibition and IC50 value were 28.67±1.167 % (IC50 value 550.3377 µg/mL), 47.17±1.424 % (IC50 value 340.875 µg/mL), and 62.63±1.660 % (IC50 value 304.246 µg/mL) respectively. The Moringa oleifera fraction i.e. n- butanol, ethyl acetate and chloroform showed the percentage inhibition and IC50 value were 32.00±0.981 % (IC50 value 461.9108 µg/mL), 43.57±0.263 % (IC50 value 338.2692 µg/mL), and 58.50±0.458 % (IC50 value 296. 7497 µg/mL) respectively. The percentage inhibition and IC50 of positive control (captopril) was 81.13±0.753% (IC50 value 203.2158 µg/mL) (Figure-5). Among those fractions the chloroform fractions showed best inhibition activity. Tutor JT and Chichioco-Hernandez CL, 2018 worked on plant extract and ethyl acetate fraction of Eleusine indica and calculated percentage inhibition is 68.84% and 51.51% respectively. In our study n-butanol, ethyl acetate, chloroform fraction and extract of A. indica shows 30.8%, 45.5%, 63.9% and 67.0% inhibition respectively. The positive control captopril shows 76.0% inhibition for ACE. Deo et al., 2016 and Khan et al., 2019 reported that the 50% inhibition of enzyme by plant extract and fraction consider is active.
ACE is a significant enzyme playing a critical role in regulation of blood pressure. Various plant constituents have the ability to inhibit the action of ACE. The active site of ACE is influenced by the phytochemicals present in neem plant. Flavonoids, terpenoids and phenolic compounds have the ability to forge chemical bonds with the amino acids present in close proximity with the active centre of ACE. These new chemical interactins tend to produce distortions in the catalytic centre of ACE thus causing inhibition. Since ACE is a metalloenzyme having zinc atom at its active site, a majority of hydrogen bonds and chemical bridges involve not just conserved amino acids of the active site but also zinc atom (Yusuf et al., 2000). In this study the ACE inhibiting compound is identified as azadiradionolide. It is a terpenoid comprising multiple rings. The tetracyclic nature of azadiradionolide is responsible for high ACE inhibitory activity of neem extract. The presence of carbonyl, lactones and methyl groups in azadiradionolide is responsible for enhancing the potency of neem plant extract against ACE. These chemical groups may be acting as causative chelating agents for the zinc atom located at the nerve centre of ACE. The in silico studies reveal that azadiradionolide due to its unique structure is able to gain access into the active site of ACE, establish chemical interactions simultaneously with amino acids and zinc atom and causing alterations in the 3-D structure of catalytic protein thus proving to be a successful ACE inhibitor.
In silico study of antihypertensive bioactive compounds against Renin angiotensin components
The in silico study is based on the lock and key theory for drug design. This is used for check the biological activity of the natural and synthetic compound and it is time consuming, reasonably and accurate method. The docking of the ligand and enzyme was done by autodock vina software and visualize the hydrogen bonding by chimera and PyMOL. The hydrogen bonding of enzyme and ligand is the important aspect for ligand and enzyme complex formation. In the present study natural isolated compounds targeted the ACE enzyme which has a role in blood pressure regulation. The Angiotensin-converting enzyme converts angiotensin I to angiotensin II a potent peptide hormone exerts its effect predominantly via AT1R (Donoghue et al., 2000). The bioactive molecule reduces the action of angiotensin converting enzyme and control the systolic blood pressure. The fractions of the selected plants were subjected to LCMS and 1H-NMR and 13C-NMR and following compounds were analyzed. Beta- sitosterol was determined in chloroform fraction of Moringa oleifera, Azadiradionolide was determined in chloroform fraction of Azadirachta indica and Hibiscitrin found in chloroform fraction of Hibiscus sabdariffa. All three compounds were docked with one of the major cascade renin angiotensin component i.e. angiotensin converting enzyme. The compounds i.e Hibiscitrin, beta-sitosterol, and azadiradionolide were showed very low binding energy i.e. -12.3kcal/mol, -11.2kcal/mol and -11.3kcal/mol binding energy with ACE crystal structure respectively. The standard drugs are used in this study is captopril and enalpril which shown -5.6kcal/mol and -8.1kcal/mol binding energy with ACE. The Hibiscitrin compound was interacting with six amino acid residues i.e. Asn-263, Gly-254, Thr-358, His-331, Tyr-498, Lys-489 (Table-3, Table-4). This Hibiscitrin compound shown hydrophobic interaction with ten amino acid residues i.e. His 491, Ala322, Gln 259, Asp255, Thr 144, Glu 262, Glu 431, Ser 357, Phe 435, Phe 505. The beta-sitosterol shown hydrophilic interaction with Asn 145, Gly 254 and Thr 144 and hydrophobic interaction with Thr 352, Asp 255, Asp 340, Leu 139, His 331, Gln 355, and Asp 354 amino acids residues. The compound isolated from neem i.e. azadiradionolide has shown hydrophilic interaction with Asn 145, Asn 263, Thr 144 and hydrophobic interaction Asp 354, Gly 254, Ala 148, Asp 255, Leu 147, Leu 139, Asp 140, His 331, Gln 355, and Thr 352 amino acid residues. It can be observed that compounds Hibiscitrin a greater number of hydrogen bond interactions with various amino acid residues of the Angiotensin converting enzyme residues. We believe that these hydrogen bond interactions play an important part in the inhibition of the Angiotensin converting enzyme residues, and ultimately better anti-inflammatory activity of the compounds Hibiscitrin (Figure-6). An overlay of the docked pose of Azadiradionolide and Beta-sitosterol also exhibited that these compounds are superimposable with each other (Figure-7, Figure-8.). This indicates that the active site of the Angiotensin converting enzyme residues can accommodate these compounds, which synergizes the Angiotensin converting enzyme residues inhibitory activity of the compounds Azadiradionolide and Beta-sitosterol. All these three compounds have shown better binding affinity in comparison to standard drugs (captopril and enalpril). Further these compounds are used for toxicity of the molecule by ADMETSAR software. The toxicity of the compound is the main reason of drug failure in the drug development field (Table-5).
Molecular Dynamic Simulation Study
MD simulation study has been also executed to investigate the conformational stability of Beta-sitosterol, Hibiscitrin and Azadiradionolide in active site of Angiotensin converting enzyme in water at 300 K. To examine the structural stability of Angiotensin converting enzyme and complex with beta-sitosterol, hibiscitrin and Azadiradionolide. We monitored the time evolution plot of all atom RMSD, Rg, RMSF and SASA. In the fourth parameter the angiotensin converting enzyme with beta sitosterol complex has shown a reduced SASA from 310 nm2 to 285 nm2 up to 5 ns. The solvent accessibility surface area for the of angiotensin converting with beta sitosterol complex indicated a more compact size at the end of the simulation period. SASA. In this case of hibiscitrin (depicted in green), was reduced from 310 nm2 to 290 nm2 and in the case of azadiradionolide reduced from 310 nm2 to 280 nm2. This indicates that beta sitosterol complex will provide more compactness than the hibiscitrin and azadiradionolide, and aromatic ring has less exposed the later on complex. We have detected that complex of protein and compounds shows relatively higher SASA value than wild type protein this could be explain by the presence of relatively larger hydrophobic packed core region in native protein as compare to protein compound complex. In one such similar studies these parameters (RMSD, Rg, SASA and RMSF) were studied by Fang et al., (2019) for ACE that support that support our study.
Radius of gyration
Another important parameter, radius of gyration (Rg) is used to determine the dynamic adaptability of A CE in water and three different compounds which namely, beta sitosterol, hibiscitrin and azadiradionolide. A time evolution Rg plot of backbone atoms (Figure-9 A) shows that the native conformation of ACE is retained in water Rg trajectory in water (Black) quickly achieves the stable equilibrium in few nanoseconds. The average Rg values of 2.37±0.01 nm is maintained throughout the simulation period. In the presence of beta sitosterol (depicted in red), 298 K temperature, the system is decreased in the fluctuation of trajectory is suggested that initial compaction in structure with comparison to water (depict in black) an average value is 2.31 ± 0.02. while the previous condition is remaining same and only change the compound instead of Beta sitosterol to used hibiscitrin (depicted in green). Now, the system is gradually increase the fluctuation throughout the simulation relatively less stable than Beta sitosterol, and the average Rg values of 2.34 ± 0.04 which indicates a less stable structure and system is expanded. In the presence of Azadiradionolide, 298 K temperature, the Angiotensin converting enzyme is more stabilized than other three compounds. In Rg trajectory of Azadiradionolide (Blue line) depicted that quickly achieves stability at 5 ns and system remain stable and the average Rg values of 2.28 ± 0.01. nrg for native protein is comparatively more than protein compound complex. The Rg results shows that native protein was more compact after the binding of compound.
Root means square distance (RMSD)
RMSD are calculated as a function of time, with respect to the initial conformation and its shown in Figure-9 B. Visual inspection of this plot show that Angiotensin converting enzyme in water (black) is unstable during the 0-10 ns time interval and after 10-30 ns system have attained the equilibrium is maintained till the end of simulation of 30 ns. Addition of in the presence of Beta sitosterol (depicted in red), 298 K temperature to the system resulted in structural perturbation is slightly come into picture at time 0–10 ns which is relatively more than water and the simulation end up with an increase in RMSD of ~0.012 nm as compare to native condition. The remaining condition is remain intact only compound is change Hibiscitrin (depicted in green) the morphology of structure is change more than Beta sitosterol and the system remain unstable during the entire time evolution of simulation. The presence of azadiradionolide (depicted in blue) at 298 K temperature, the ACE in presence of azadiradionolide the system is unstable during the 0-10 ns time interval and after 10-30 ns system. While the behavior of morphology is in between the Beta sitosterol and Hibiscitrin.
Root means square fluctuation (RMSF)
To capture the dynamics progression of Angiotensin converting or flexibility of structure in water and three different complex with compounds of the Beta-sitosterol (depicted in red), Hibiscitrin (depicted in green) and are Azadiradionolide (depicted in blue) are depicted in Figure-10 A. RMS fluctuations showed that the most flexible residues 291–301, 338-354 are located in loop regions and 324–347 are located in the loop regions, which connect the Beta-sheets to alpha-helices. RMS fluctuation is more in the of Angiotensin converting +Azadiradionolide (depicted in blue) complex compared to the protein +beta sitosterol (depicted in red) complex. Comparatively, Angiotensin converting +Hibiscitrin (depicted in green) showed high fluctuations at 53–65, 66–144, 145–192 positions, these amino acids mostly constituting the loop region, alpha helices played no role in Azadiradionolide (depicted in blue) binding.
Solvent accessible surface area (SASA)
The SASA for Angiotensin converting + water (in depicted black colour) was 190nm2 at the beginning of the simulation and retained the area throughout the simulation period shown in the Figure 10 B Which is quite stable, apart from this addition of in the presence of Beta sitosterol (depicted in red), 298 K temperature to the system the solvent accessible surface area (SASA) for Angiotensin converting + beta sitosterol was observed to be 310 nm2 at the start of simulation; as dynamics proceeded, it continuously decreased to 285 nm2. The Angiotensin converting + beta sitosterol complex has shown a reduced SASA from 310 nm2 to 285 nm2 up to 5 ns.
The solvent accessibility surface area for the of Angiotensin converting + beta sitosterol complex indicated a more compact size at the end of the simulation period. SASA. In this case of Hibiscitrin (depicted in green), was reduced from 310 nm2 to 290 nm2 and in the case of azadiradionolide reduced from 310 nm2 to 280 nm2. This indicate that beta sitosterol complex is provide more compactness than the Hibiscitrin and azadiradionolide, and aromatic ring has less exposed the later on complex. We have detected that complex of protein and or compounds shows relatively higher SASA value than wild type protein this could be explain by the presence of relatively larger hydrophobic packed core region in native protein as compare to protein compound complex.
Cell toxicity assay
To determine the optimum concentration of the fractionated compounds for biological assays the cell toxicity was determined by MTT assay. The chloroform fraction containing predominantly beta-sitosterol, Hibiscitrin and azadiradionolide compounds were subjected to MTT assay on BHK-21 cell lines. The results revealed that with the treatment of bioactive fraction bearing hibiscitrin (HS) compound showed 99.34% viability at 970ug/ml followed by beta sitosterol (BS) with 99.13% viability at 970ug/ml and azadiradionolide (AZ) with 98.67% viability at 970ug/ml. Thus, results indicated that these compounds were least toxic to the cells, further suggesting these compounds are biocompatible and can be utilized as a drug (Figure-11). The Nemudzivhadi and Masoko (2014) showed the 90% cell viability of bioactive compound from Ricinus communis. In another study by Raiola et al., (2016) found the 80% cell viability on HEK- 293 cell lines by the extract of tomatoes which is lower from the present study. In support to our findings Arevalo et al., (2018) found 50% cell viability by the extract of M. oleifera and A. indica on MDCK cell lines.
Summary
The present study suggested the consideration of medicinal plants of Himachal Pradesh for targeting the role in hypertension. The isolated and characterized bioactive molecule from Moringa oleifera, Hibiscus sabdariffa and Azadirachta indica were found to be beta-sitosterol, hibiscitrin and azadiradionolide respectively. The most potent molecule to inhibit the ACE of these was hibiscitrin. So this compound could play a significant role in inhibiting the conversion of angiotensin I to angiotensin II to control the systolic blood pressure. The current study delivers a new perspective for the drug development against systolic blood pressure regulation and also opens new horizons for considering alternate highly potent drug target for hypertension.