Department of Biosciences, Veer Narmad South Gujarat University, India

^*Corresponding author: Patel H, Department of Biosciences, Veer Narmad South Gujarat University, Surat, Gujarat, India, Email: hinalbiotech@gmail.com

Copyright: © Patel H, et al. 2020. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Article Information: Submission: 07/02/2020; Accepted: 16/03/2020; Published: 20/03/2020

Hypertension is one of the most common health problems that carry a high risk of stroke, myocardial infection and renal disease. ACE is one of the important enzymes that responsible for hypertension. Ocimum sanctum plant is very important for their therapeutic potential. There are so many phytochemicals are present in Ocimum sanctum among which flavonoids are most important for their health benefits. In present study prepared flavonoid extract from Ocimum sanctum leaves using methanol and ethyl acetate solvent. Isolate flavonoids from prepared solvent extract using preparative HPTLC method and identified it with the help of HRLC-MS/Q-TOF technique. Molecular docking study was conducted using iGEMDOCK V.2 windows software to find out efficiency of isolated flavonoids with ACE along with standard drug. This information can be further utilized for design of potential therapeutic drug from plant flavonoid in regulation of hypertension.

Hypertension is the most common serious persistent health problem that carries a high risk of arteriosclerosis, stroke, myocardial infarction and end stage renal disease [1]. Angiotensin converting enzyme inhibition is one of the Morden therapeutic approaches to treat hypertension [2]. ACE catalyses the degradation of bradykinin [3]. ACE also converts angiotensin I into potent vasoconstrictor angiotensin II [4]. Therefore to generate antihypertensive effect, inhibition of ACE is required. Captopril, enalapril like synthetic drugs are widely used to treat hypertension but they show some major side effects like renalin fatigue, angioneurotic edema etc. so there is a requirement of new Natural based ACE inhibitors that greatly beneficial to hypertensive patients.

Natural has many useful plants and herbs for human beings. The attention paid by health authorities to use of herbal medicine has increased considerably to meet its health needs. Medicinal plants are rich in secondary metabolites like alkaloids, flavonoids, glycosides, tannins and essential oil which are good source of drugs in pharmaceutical industries [5,6]. Among the plants having the medicinal value, the plants of genus Ocimum belongs to the family labiacea are very important for their therapeutic potential [7]. Ocimum sanctum has been used for thousands of the year in Ayurveda for its healing and other medicinal properties. In Hindu tradition, Ocimum sanctum get important symbol. Ocimum sanctum is a herbal remedy for many common ailments like healing power , fever, common cold, sore throat, anti-inflammatory action, antibacterial etc.

Secondary metabolite like flavonoids – A product of secondary metabolism produced by plant in higher amount and distributed widely in plant kingdom. Flavonoids represent a one of the important class of phytochemicals. They belong to group of polyphenols that are present in fruits, vegetables, medicinal plants and its products. Interest in isolation and structure elucidation of many natural flavonoids get increasing because of its ecology and health benefits of bioactive flavonoids. They are renowned for their number of health [8]. Phenolic compounds such as ferulic acid and tannic acid and flavonoids such as quercetin, anthocyanins, flavones and flavonols have shown to exhibit a capacity to inhibit different zinc metalloproteinases, including ACE [9,10].

Molecular docking is a very important tool useful in the structure based rational drug designing. Using iGEMDOCK software of molecular docking, enzyme interact with ligands like smaller molecules such as inhibitors or drug candidate to form stable complex.

The main object of this study was to extract flavonoids from Ocimum sanctum leaves extract using solvent maceration method and get isolated using preparative HPTLC and identified by HRLCMS/QTOF technique. Then to perform molecular docking of that identified flavonoids (inhibitors) with ACE, to determined its binding efficacy.

Dried leaves powdered of Ocimum sanctum plant (50 gm) extracted with direct methanol (95%) by maceration method for 24 h in shaking condition.

The extract was filtered and evaporated in vacuum condition. The filtrate obtained was resuspended in ethyl acetate and extract it successively to yield ethyl acetate fraction following the method [11] with some modification.

High performance thin layer chromatography was carried out after successful development of TLC plate by using toluene: ethyl acetate solvent system. HPTLC was carried out using CAMAG HPTLC system for prepared flavonoid extract. Prior to sample application, activate HPTLC plate (silica gel 60 F254, Merck) at 100 oC for 30 min.

Impregnated 200 μl of prepared extract on HPTLC plate (20×20) as a single band of 180 mm length using CAMAG automatic TLC sampler III (CAMAG, Switzerland).The plate was then air dried and kept for development in the twin trough chromatographic chamber containing 200 ml standardized solvent system, toluene: ethyl acetate (9.3:0.7).

As the above procedure, 5 HPTLC plates were impregnated with 200 μl of same sample same as performed for the above plate. Examine under UV chamber at 366 nm wavelength, after the successful development of plates.

The plate which was developed was further proceeded for isolation of flavonoid constitution. The developed plate was then marked with graphite tip from 1 to 10 cm with mm marking scale along the solvent system movement. Put the plate under UV chamber to mark flavonoid band on the scale. By using sharp scalpel, scratched the selected area along with silica and collected in Eppendorf tube and eluted flavonoid constituent from silica gel with ethyl acetate and pooled the content.

The pooled content was evaporated at room temperature to form concentrated and store in small glass bottle for further analysis.

Identification of extracted flavonoid from desired plant solvent extract was analysed by LC/Q-TOF Mass Spectrometer, Agilent technologies, Bombay India. The HR-LCMS analysis of plant material was analysed by using UHPLC-PDA detector Mass spectrometer. Sample was separated on SB-C18 column(2.1×50 mm, 1.8 particle size). The solvents used were Water: Methanol. MS detection was performed in MS /Q-TOF Mass spectrometer.

Identified flavonoids through HR-LCMS get derived from PubChem in 3D structure along with standard drug, captopril. Convert their structure in mol. Format using software OPENBABEL. Three dimensional experimentally determined X-ray crystal structure of Angiotensin converting Enzyme (ACE) were taken from protein data bank(PDB)(www.rcsb.org/) having PDB code108A. Once the ligand and protein have been prepared, download the file in iGEMDOCK software. Docking was performed using iGEMDOCK software. At the end of each run, compare the binding of ligand and protein by binding energy i.e. the lowest energy shows highest binding affinity.

Cardioprotective potential of flavonoids gained more interest in research for other potential properties [12]. Flavonoids or their derivatives are widely used as pharmaceutical agents for their Vasoprotective properties [13]. Flavonoids have been extracted from the leaves of Ocimum sanctum using methanol and ethyl acetate as a solvent and prepared concentrated extract in ethyl acetate. Thin layer chromatography process confirmed the presence of flavonoids by observing the florescent band. Extraction method of phytochemicals is one of the most important procedure in pharmaceuticals use of plant species having medicinal importance [14]. Now a day in botany, HPTLC become very useful technique for pattern generation, separation and further isolation. The crude flavonoid extracts revealed brown, one bright florescent band along with light blue band. The clear bright blue florescent band was considered to be possible flavonoids and was selected for isolation (Figure 1).

HRLC-MS analysis lead to the identification of flavonoids from leaves extract of Ocimum sanctum which showed the presence of four flavonoids are tubulated (Table 1). Identified flavonoids were Daidzin, Apigenin, and Rutin and Quercitrin. Retention time, molecular formula, m/z value were evaluated in (Table 1). Nature, molecular formula and structure of flavonoids compounds were analyzed by using mass spectrometer at different time. In these, appearances of peaks are due to large compounds splits into small ones at various m/z ratios and these HRLC-MS spectra are fingerprint of that compounds and it identified with data library [15]. The cleaned and optimized structure of identified flavonoids was derived from PubChem. 3D structure of ACE was derived from PDB. All the procedure has been performed using iGEMDOCK V.2 windows software. Flexible docking has been done between ACE and identified flavonoids. And also between ACE and standard drug captopril. Low energy with high binding affinity of flavonoids with ACE is given in (Table 2). Best conformation with highest binding affinity with low energy have been chosen for the protein-ligand interaction study and shown in Figure 2-6.

In most of the studies, it have been shown that plant extracts rich in phytochemicals found to be very effective in angiotensin converting enzyme (ACE) inhibition. It can be conclude that, Ocimum sanctum contain flavonoids, that may important for the ACE inhibition. The above stated procedure is say to be an efficient and simple workout for the isolation of compounds from plant extract. Present molecular modeling studies demonstrated that isolated flavonoids have as same binding efficiency as standard drug. There for these compounds may be an effective therapeutic candidate for control of ACE. Further studies with structural modification are required to develop potential therapeutic entities for the treatment of hypertension and related disorder.