Integrated In Silico and In Vitro Investigation of Antimicrobial Activity of Morinda citrifolia Ethanolic Extract

An antimicrobial is an agent that kills microorganisms (microbicide) or stops their growth (bacteriostatic agent). Antimicrobial medicines can be grouped according to the microorganisms they are used to treat.

For example, antibiotics are used against bacteria, and antifungals are used against fungi. They can also be classified according to their function.

Antimicrobial medicines to treat infection are known as antimicrobial chemotherapy, while antimicrobial drugs are used to prevent infection, which known as antimicrobial prophylaxis.

The main classes of antimicrobial agents are disinfectants (non-selective agents, such as bleach), which kill a wide range of microbes on surfaces to prevent the spread of illness, antiseptics which are applied to living tissue and help reduce infection during surgery, and antibiotics which destroy microorganisms within the body.

ANTIFUNGALS

Antifungals are used to kill or prevent further growth of fungi. In medicine, they are used as a treatment for infections such as athlete's foot, ringworm and thrush and work by exploiting differences between mammalian and fungal cells. Unlike bacteria, both fungi and humans are eukaryotes.

ANTIVIRAL

Antiviral drugs are a class of medication used specifically for treating viral infections. Like antibiotics, specific antivirals are used for specific viruses. They should be distinguished from vermicides, which actively deactivate virus particles outside the body.

ANTIPARASITIC:

Antiparasitic are a class of medications which are indicated for the treatment of parasitic diseases, such as those caused by helminthes, amoeba, ectoparasites, parasitic fungi, and protozoa, among others. Antiparasitic target the parasitic agents of the infections by destroying them or inhibiting their growth; they are usually effective against a limited number of parasites within a Particular class

MECHANISM OF ACTION OF ANTI MICROBIALS:

1. Inhibition of Cell Wall Synthesis

• Target: Peptidoglycan layer in bacterial cell walls
• Effect: Weakens the cell wall, leading to cell lysis and death
• Mainly active against: Gram-positive bacteria
  39238058463. β-lactams: Penicillins, Cephalosporins, Glycopeptides: Vancomycin

2. Disruption of Cell Membrane Function

• Target: Cell membrane (phospholipid or sterol components)
• Effect: Destroys membrane integrity → leakage of vital molecules → cell death
  39238058463. Polymyxins (bacteria), Amphotericin B, Nystatin (fungi - target ergosterol)

3. Inhibition of Protein Synthesis

• Target: Bacterial ribosomes (30S or 50S subunits)
• Effect: Blocks protein production → inhibits growth or kills cell
  39238058463. Tetracyclines, Aminoglycosides (30S), Macrolides, Chloramphenicol (50S)

4. Inhibition of Nucleic Acid Synthesis

• Target: Enzymes for DNA or RNA replication
• Effect: Prevents DNA replication or transcription → halts cell division
  39238058463. Fluoroquinolones (inhibit DNA gyrase), Rifampin (inhibits RNA polymerase)

5. Inhibition of Essential Metabolic Pathways

• Target: Enzymes in folic acid synthesis (used for DNA/RNA synthesis)
• Effect: Starves the cell of critical nutrients
  39238058463. Sulfonamide, Trimethoprim

PLANT PROFILE

NONI

Common name: Indian Mulberry, Great Morinda, Cheesefruit,

Scientific name: Morindia citrifolia

Family : Rubiaceae

Geographical  source: Kerala, Karnataka and Tamil Nadu

Scientific classification:

• Kingdom : Plantae
• Order : Gentianales
• Family : Rubiaceae
• Subfamily : Rubioideae
• Genus : Morinda
• Species : Morinda citrifolia

Vernacular names:

English : Indian Mulberry

Tamil : Nuna

Hindi : Bartundi

Telugu : Mogali

Kannada : Tagase maddi

Description:

Morinda citrifolia is a shrub or small tree up to 6 m tall, with grey-brown bark. The twigs are more or less square in cross-section and often fleshy. Stipules are present, very broad and obtuse at the apex, measuring up to 2 cm wide and long. The large glabrous leaves are elliptic to ovate in shape and have 6–9 pairs of lateral veins. The flowers are white and tubular with five lobes, measuring about 15 cm long and across.

The fruits are initially green, transitioning through pale yellow to white or grey, and when ripe they emit a pungent odour. They are irregularly ellipsoid or ovoid.

Chemical constituent:

Morinda citrifolia fruit powder contains carbohydrates and dietary fibre in moderate amounts. These macronutrients reside in the fruit pulp, as M. citrifolia juice has sparse nutrient content. Morinda citrifolia fruit contains diverse phytochemicals, including anthraquinones, lignans, oligo- and polysaccharides, flavonoids, iridoids, such as deacetylasperulosidic acid, scopoletin , fatty acids, catechin, beta-sitosterol, damnacanthal, and alkaloids.

Pharmacological profile:

• Antibacterial
• Antiviral
• Antifungal
• Anthelminthic
• Antitumor
• Analgesic
• Hypotensive
• Anti-inflammatory
• Immune - enhancing effect.                  

MOLECULAR DESIGN:

Molecular design is the process of finding new medicines based on the knowledge of a biological target, it enabled the chemist to predict the structure and then it also allows the medicinal chemist to evaluate the interaction between a compound and its target site before synthesizing a compound so as to increase the ability by reducing the side effects.

Various software used :

• Chem Sketch
• Mol inspiration
• Swiss ADME
• Pro Tox 3.0

MOL INSPIRATION

This software is used to calculate the following properties

• Log P
• Molecular weight
• Number of H-bond donor
• Number of H-bond acceptor
• Number of rotatable bonds

In addiction to “LIPINSKI’S RULE” another rule was proposed VEBER he states that the number of rotatable bonds should be less than 10. This rule is more appropriate for oral drug only. According to the veber’s  rule .

1. The Log P value should not be more than 5
2. The molecular weight of the compound should not more than 500
3. No. of H-bond donor not more than 5
4. No. of rotatable bonds should not be more than 10

3D STRUCTURAL VIEW OF COMPOUNDS:

SCOPOLETIN	LINALOOL
RUTIN	GALLIC ACID
CAFEIC ACID	URSOLIC ACID
OLEANOLIC ACID	β-SITOSTEROL
URSANE	TANNIC ACID
CATECHIN	EPICATECHIN
DEACETYL ASPERULOSIDIC ACID(DAA)	ASPERULOSIDIC ACID(AA)
TIGOGENIN	OLEONANE
QUERCETIN	KAEMPFEROL

Table-1: Properties of Phytoconstituents

Table-2: ADME Properties of Phytoconstituents

Table 3: Toxicity Study of Phytoconstituents

ANTI MICROBIAL ACTIVITY

PREPARATION OF PROTEIN:

The protein target, obtained from the RCSB protein data bank  with the PDB accession. Code 1ULK function as docking receptor. The active site of the receptor was cleared of all sound ligands and water molecules.

CRYSTAL STRUCTURE OF 1ULK

PDB DOI: https://doi.org/10.2210/pdb1ULK/pdb

Classification: SUGAR BINDING PROTEIN

Organism(s): Phytolacca americana

Mutation(s): No

Experimental Data Snapshot

• Method: X-RAY DIFFRACTION
• Resolution: 1.80 Å
• R-Value Free: 0.203 (Depositor)
• R-Value Work: 0.176 (Depositor)
• R-Value Observed: 0.176 (Depositor)

BINDING AFFINITY

MATRIALS AND METHOD

Processing of plant :

The collected plant has identified by Dr.V.Suresh kumar, Assistant Professor, Dept of Botany, Government Arts College, Tiruvannamalai (District), Tamilnadu, India. The plant was washed with tap water 3 times and sterilized by spraying with 70% alcohol.

The purified plant material was shade dried at room temperature to avoid chemical changes and frequently observed for any fungal contamination as the plant material rich in water content. When the plant material was dried entirely, it has subjected to prepare fine powder with help of mixer grinder. The fine material powder is collected and used for extraction of the crude drug in solvent by soxhlet and maceration extraction method.

DRIED FLOWERS

Soxhlet Extraction:

Soxhlet extraction is a method used to remove active compounds from plant material using heat and continuous solvent washing.

Maceration:

Maceration is a method of extracting compounds by soaking plant material in a solvent at room temperature for a long time.

Apparatus:

• Soxhlet extractor
• Round bottom flask
• Condenser
• Heating mantle / water bath, Thimble (filter paper)
• Solvent (ethanol, acetone, etc.)

Procedure:

• Dry and powder the plant material.
• Place the powdered sample inside a thimble.
• Insert the thimble into the Soxhlet extractor.
• Add solvent into the round bottom flask.
• Attach condenser on top.
• Heat the system.
• Solvent evaporates → condenses → drips into thimble.
• When chamber fills, siphon tube returns extract to flask.
• This cycle repeats for 6–8 hours.
• After extraction, evaporate solvent to obtain crude extract.

Advantages

• Efficient extraction
• Uses less solvent
• Continuous extraction

Disadvantages

• Time consuming
• Not suitable for heat-sensitive compounds

Maceration

Apparatus:

• Conical flask / beaker
• Stopper or aluminum foil
• Stirring rod
• Filter paper
• Funnel

Procedure

• Dry and powder plant material.
• Place powder in a flask.
• Add suitable solvent (enough to cover sample).
• Close the flask.
• Keep for 24–72 hours at room temperature.                                   
• Shake occasionally.
• Filter the solution.                                                        
• Evaporate solvent to get extract.

Advantages

• Simple method
• No heating required
• Suitable for heat-sensitive compounds

Disadvantages

• Takes longer time
• Less efficient than Soxhlet

                                Maceration process                                                     Soxhlet method

CONFIRMATION TESTS FOR CRUDE EXTRACTS

METHOD

• In-vitro Anti microbial activity of plant extract

Plant extracts possess significant antimicrobial activity due to compounds like alkaloids, flavonoids, tannins and essential oil, Phenols offering natural alternative to synthetic drugs by inhibiting various bacteria and fungi, targeting pathogens like (BACILLUS, STAPHYLOCOCCUS).

Preparation of nutrient broth medium:

PROCEDURE:

• Weigh above all ingredients and make up 500 ml with distilled water.
• Mix well and constant stirring until the power dissolves completely.
• Autoclave the medium at 121 ? C for 15 minutes under 15psi pressure.
• Cool to about 45?C.
• Pour into sterile petri dishes (20ml per plate).
• Spread the microbial suspension evenly over the surface of the agar plate using a sterile swab to create a lawn of growth.
• Incubate at 37 ?C for 48hours.

COMMON METHODS:

1. Disc diffusion method (standard-ciprofloxacin, sample-crude drug).
2. Agar well diffusion method (make small well in the agar & fill with the (standard & sample).

• Measure inhibition zones around plant extract discs and well.
• Larger zones indicate greater antimicrobial potency.

BACILLUS

                                    STANDARD                                                                SAMPLE

STAPHYLOCOCCUS

STANDARD                                                        SAMPLE

SERIAL DILUTION:

BLANK:

Add 1.5 g of Beef extract

↓

Add 2.5 g of Peptone

↓

Add 2.5 g of Sodium chloride

↓

Make up for 500ml with distilled water

↓

Autoclave the medium at 121?C for 15 minutes under psi pressure

↓

Cool to about 45-50 ?C

↓

Incubate at 37 ?C for 48 hours

↓

Measure absorbance at 254nm

CONTROL:

Add 10ml of nutrient broth

↓

Add 0.5ml of Micro-organism (Bacillus, Staphylococcus)

↓

Incubate at 37 ? C for 48hours

↓

Measure the absorbance at 254nm

STANDARD:

Add 10 ml of nutrient broth solution

↓

Add 0.5 ml of micro-organism (bacillus, staphylococcus)

↓

Add 0.5ml of 50 mcg Ciprofloxacin solution

↓

Incubate at 37 ?C for 48 hours

↓

Measure the absorbance at 254nm

TEST:

TEST FOR

100 mcg/ml, 200 mcg/ml, 300 mcg/ml, 400 mcg/ml, 500 mcg/ml, 600 mcg/ml, 700 mcg/ml, 800 mcg/ml, 900 mcg/ml, 1000 mcg/ml.

Add 10ml of nutrient broth solution

↓

Add0.5ml of micro-organism (Bacillus & Staphylococcus)

↓

Add 0.5ml of plant extract

↓

Incubate at 37 ?C for 48 hours

↓

Measure the absorbance at 254nm

RESULT

CALCULATION FORMULA:

% of inhibition =

(Absorbance of control - Absorbance of  test) ×100

                 (Absorbance of control)

OBSERVATION

• Ciprofloxacin at 50µg/ml showed 80-85% inhibition, validation of an assay.
• The plant extract showed dose-dependent inhibition, reaching 80-92% at 1000µg/ml
• This suggests that the extract contains active phytochemicals capable of inhibiting cell wall synthesis, potentially beneficial for managing micro-organism.

GRAPH