INTRODUCTION:

Plants contain the rich source of natural products like vitamins, minerals and other immune- modulators used for human welfare specially to cure disease caused by pathogenic microorganisms. Clerodendrum infortunatum Linn is one of them. Considerable utilisation and progress have been achieved regarding its biological activities¹. Clerodendrum is a very large and diverse genus and till now five hundred and eighty species of the genus have been identified and are widely distributed in Asia, Australia, Africa and America. The whole parts of the plants are used in microbial infection. Leaf extract is used as antimicrobial, anthelmintic, analgesic, anticonvulsant, antidiabetic agent and also to increase haemoglobin level in the blood. Clerodolone, clerodone, clerodol are different isolated compounds obtained from this plant.² A sterol, now designated as clerosterol obtained from the leaves is used as an antitumor agent. Percentage of haemoglobin increased due to leaf extract. Piles patient treated with leaf extract gives considerable relief. Sugar percentage reduced in diabetic patient. Slightly warm leaves applied with edible oil on the painful part of human body gives considerable relief within twelve hours³.

Morphology:

Clerodendrum infortunatum Linn. (Family: Verbanaceae, Kingdom: Plantae, Genus: Clerodendron), is a flowering shrub or small tree, and is so named because of its rather ugly leaf. The stem is erect, 0.5–4 m (1.6–13.1 ft) high, with no branches and produce circular leaves with 15 cm (5.9 in) diameter. Leaves are simple, opposite; both surfaces sparsely villous-pubescent, elliptic, broadly elliptic, ovate or elongate ovate, 3.5–20 cm (1.4–7.9 in) wide, 6–25 cm (2.4–9.8 in) long, dentate, inflorescence in terminal, peduncled, few-flowered cyme; flowers white with purplish pink or dull-purple throat, pubescent. Fruit berry, globose, turned bluish-black or black when ripe, enclosed in the red accrescent fruiting-calyx. The stem is hollow and the leaves are 15–20 cm (5.9–7.9 in) long, borne in whorls of four on very short petioles. The inflorescence is huge, consisting of many tubular snow white flowers in a terminal cluster up to 0.6 m (2 ft) long. The tubes of the flowers are about 10 cm (3.9 in) long and droop downward, and the expanded corollas are about 5 cm (2.0 in) across. The fruits are attractive dark metallic blue drupes, about 1 cm (0.4 in) in diameter. Fruit usually with four dry nutlets and the seeds may be with or without endosperm. It flowers from April to August.^5,6 The microscopic characters of the root involve stratified Cork, phelloderm, stone cell layer, cambium, large lignified vessels, xylem fibre etc⁷. (Figure no:1)

Figure No :1 Clerodendrum infortunatum

Phytochemical Aspects:

The leaves of the plant were reported to contain saponin, alkyl sterols, some enzymes, and 2,-(3, 4-dehydroxyphenyl) ethanol1-O-α-2 rhamnopyranosyl (1→3)-β-D-(4-O-caffeoyl) glycolpyranoside (acetoside)^8,9. It was also found that the leaves contain a fixed oil which consists of glycerides of linoleic, oleic, stearic and lignoceric acid¹⁰.

Drug discovery and development^11,12:

Drug discovery and development is an intense, interdisciplinary and endeavour, when a compound is under taken for discovery. It involves synthesis, characterisation, screening and evaluation for therapeutic efficiency. A number of researches have been performed to identify and isolate biologically active compounds from different species of Clerodendrum. Research report revealed that steroids, terpenoids and flavonoids are major among them. The aim of the present study was to evaluate, the antimicrobial activity of natural extract and also perform molecular docking studies of the chemical constituents clerodin, cleroflavone and hispidulin. This study explains the evidence-based information regarding the different pharmacological activities with a correlation of chemical constituents available with this plant so that it may serve as a reference for further studies. Some of the important chemical compounds encountered in Clerodendrum are depicted in Figure No:2,3,4.

Clerodin (figure No:2)

Cleroflavone (figure No:3)

Hispidulin (figure no:4)

Chemical structures of some important constituents of Clerodendrum. infortunatum Linn.

Evaluation of biological activity:

Primarily, evaluated the anti bacterial activities of Clerodendrum infortunatum (Verbenaceae) leaves with two different solvents viz ethyl acetate and acetone against gram negative E. coli bacterial strains. The in-vitro screening for antimicrobial activity was carried out by using well-plate techniques. The test compounds were compared with standard Ciprofloxacin. Solvents ethyl acetate and acetone were kept as a control.

Molecular docking studies:

In the field of molecular modelling, docking is a method which predicts the orientation of one molecule to a second when bound to each other to form a stable complex. Preferred orientation helps to predict the strength of association or binding affinity between two molecules. The associations with biological molecules such as proteins, nucleic acids, carbohydrates and lipids play an important role in signal transduction. The orientation of the interacting molecules decides the signal transduction i.e. agonism or antagonism. So docking is an useful tool for predicting both the strength and type of signal produced¹³. Flavonoids and terpenoids can inhibit the enoyl ACP reductase that present in the Escherichia coli is an essential bacterial enzyme. Fabl, the enoyl acyl carrier protein reductase of the Escherichia coli species is one of the attractive targets in E. coli associated diseases.By considering the above observations, an attempt is made here to molecular docking studies of flavonoids and terpinoids present in Clerodendrum infortunatum with enoyl ACP reductase (PDB ID: 1C14) by using in silico studies by molinspiration online tool and evaluated for their antimicrobial activity^{14 - 18}.

MATERIALS AND METHODS:

IN-SILICO METHODS

Softwares and databases used

· AutoDock 4.2 combines

· AutodockTools1.5.4

· Python Molecule viewer1.5.4

· Vision 1.5.4

· Python 2.5

· Accelrys discovery studio viewer

· Pre ADMET software

· Molinspiration server

· RCSB protein data bank

· Online SMILES translator

All the in-silico experiments are carried out at Sanjo College of Pharmaceutical Studies, Vellapara, Palakkad. The leads selected were substituted with various substituents and they were optimized for the pharmacokinetic parameters by evaluating. The in-vivo absorption capabilities of the designed molecules were assessed by the means of Lipinski’s rule of five using molinspiration server.

Docking studies for the lead molecules:

After the lead has been optimized, the protein was subjected to docking studies using AutoDock 4.2 for evaluating the binding interactions.

Enzyme and ligand preparation:

The X-ray crystal structure of the enzyme enoyl ACP reductase (FabI) of E. coli (PDB entry: 1C14) was obtained from Protein Data Bank¹⁹

Selection of Enoyl-[acyl-carrier-protein] reductase from PDB:

E coli enoyl reductase-NAD+- triclosan complex (1C14) Escherichia coli PDB accession code is 1C14 Resolution is 2 Chains A, B Sequence Length 262.

Anti-bacterial studies^20-23:

Apparatus and chemicals required:

Sterile swab : Hi Media Non-absorbent cotton: Rama Raju Surgical cotton Ltd. Conical flask: Borosil Test tubes: Borosil Petri dishes: SD Fine – Chem Ltd. Micropipettes: VARI pipettes (Hi – Tab Lab) Autoclave: Universal Autoclave Laminar air Flow unit: CLEAN AIR Instruments Inc. Micro tips: Tarsons The antibacterial screening was carried out in the Pharmaceutical Microbiology laboratory, Sanjo College of Pharmaceutical Studies, Vellapara, Palakkad.

Procedure for antibacterial activity:

The sterilized (autoclave at 1200 c for 30 minutes) nutrient agar medium (40-500 c) was inoculated with the suspension of microorganism and mixture was transferred to sterile petridishes and allowed to solidify. Ciprofloxacin disc of conc. 1µg placed kept as standard and ethyl acetate and acetone extract of Clerodendrum infortunatum were poured. The plates were kept in refrigerator for 30 minutes to allow the diffusion of sample to the surrounding agar medium. The plates were incubated at 37±20° c for 24 hours and observed for antibacterial activity. The diameter of zone of inhibition were measured and compared with that of standard. The values were tabulated.

Method: Agar well-plate Method

Test organism: E. coli

Standard drug: Ciprofloxacin (1µg/ml)

Control: Ethyl acetate and acetone

Culture medium: Nutrient agar medium

RESULTS AND DISCUSSION:

Docking studies:

The results of docking of Enoyl-[acyl-carrier-protein] reductase (1C14.pdb) with chemical constituents clerodin, cleroflavone and hispidulin are reported below. The best docked structure should have the binding energy higher than that of the standard. The binding sites were represented in the snap shots and the binding energy was compared with the standard ligand, ciprofloxacin. (-5.45K cal/mol). The chemical constituent cleroflavone showed highest binding energy -6.03 kcal/mol. Clerodin and hispidulin have good binding energies -5.06 and -5.04Kcal/mol respectively. The results are mentioned in the table(table No:1) followed by the snapshots (figure No:5,6,7,8).

Table No:1 Binding energies of clerodin, cleroflavone and hispudulin with enoyl ACPreductase (1C14.pdb

Sl. No	(Chemical constituents)	Binding energy (∆G =Kcal/mol)
01	Clero flavone	-6.03
02	Clerodin	-5.06
03	Hispidulin	-5.04
04	Ciprofloxacin (STD)	-5.45

(Figure No:5) Snapshots and binding interactions of ciprofloxacin with enoyl ACP reductase (Binding Energy= -5.45)

(Figure No:6) Snapshots and binding interactions of Cleroflavone with enoyl ACP reductase (Binding Energy= -6.03)

(Figure No:7) Snapshots and binding interactions of Hispidulin with enoyl ACP reductase (Binding Energy= -5.04)

Antibacterial activity:

The zone of inhibition of ethyl acetate and acetone are shown in figure No:7 and their diameters were compared with that of the standard ciprofloxacin mentioned in Table No: 2

S.No	Compound with code	Zone of inhibition
01	Ethyl acetate extract (EAS)	1.1cm
02	Acetone extract (ACS)	0.5cm
03	Ciprofloxacin (STD)	2.3cm

Diameter of zones of inhibition (mm) of ethyl acetate and acetone extracts of Clerodendrum infortunatum against microorganism E.Coli.

Among these, ethyl acetate extract of Clerodendrum infortunatum showed highest zone of inhibition (1.1 cm) compared with standard ciprofloxacin (2.3 cm). Acetone extract of this leaves also have moderate antibacterial activity against E.Coli.

(Figure No: 8) The inhibition zone of ethyl acetate and acetone extracts of Clerodendrum infortunatum against microorganism E.Coli.

CONCLUSION:

Computer aided drug design helps to minimise the tedious drug discovery process over the traditional method. In silico ADME study and drug likeness score of the ligands observed helped to predict a better pharmacokinetic activity and oral bioavailability of the designed leads. The binding energies obtained from docking study of enoyl ACP reductase confirms that the lead compound inhibit the enzymes present in Escherichia coli. The screening of synthesized compounds for antibacterial study revealed that ethyl acetate extract show good antibacterial activity. Thus the present study depicts that the utilization of computer aided drug design is an efficient tool in predicting the effectiveness of a series of compounds under study and thus can result in the design of potent antibacterial agent. Although CADD is an efficient tool in determining the efficiency of chemical moieties, the data obtained from the docking studies in the present study comply with the result obtained from antimicrobial studies conducted. From this study, we concluded that the antibacterial potency of ethyl acetate and acetone extracts may be due to the presence of the chemical constituents Clerodin, Hispidulin and cleroflavone in Clerodendrum infortunatum. Isolation of these constituents leads to discover novel medicinal compounds to inhibit Escherichia coli. With this point of view the present article aims at focusing the attention of research scholars on the unexplored and untouched areas related with Clerodendrum infortunatum Linn.

ACKNOWLEDGEMENT:

The authors are thankful to Management, Principal and Staff, Sanjo College of Pharmaceutical Studies, Vellapara, for providing necessary facilities and support to carry out this work.

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Received on 29.12.2019 Modified on 19.01.2020

Asian J. Research Chem. 2020; 13(2):128-132.

DOI: 10.5958/0974-4150.2020.00026.7