Formulation And Evaluation of Herbal Antimicrobial Granules Using Bacopa Monnieri (Brahmi)

The exposure and rapid spread of antimicrobial resistance (AMR) pose one of the greatest challenges to global public health in the twenty-first century. Decades of extensive antibiotic use—both appropriate and inappropriate—have applied intense evolutionary pressure on pathogenic bacteria, leading to the selection and proliferation of resistant strains. The World Health Organization (WHO) has identified AMR as a major threat to progress in modern medicine, the effective treatment of infections, undermining surgical procedures, and increasing healthcare costs. In response, researchers worldwide are seeking new antimicrobial agents, novel therapeutic strategies, and alternative approaches—among which medicinal plants and herbal formulations have re-emerged as promising reservoirs of bioactive molecules. Herbal medicines have a long history of use in traditional healing systems around the world. In India, Ayurveda and other ancient systems have categorized hundreds of plants with antimicrobial, anti-inflammatory, antioxidant, and immune-modulating activities. Although many ancient texts emphasize the use of whole plant powders, decoctions, and oils, modern pharmaceutical development requires standardized, stable, and patient-friendly dosage forms. This need has driven interest in converting traditional herbal preparations into granules, tablets, capsules, and other advanced delivery systems, thereby combining the benefits of phytotherapy with the rigors of contemporary quality control and formulation science. Among the vast repertoire of Ayurvedic herbs, Bacopa monnieri (commonly known as Brahmi) has attracted considerable attention. Traditionally, Brahmi is celebrated for its nootropic properties—enhancing memory, learning, and cognitive function—and is extensively studied for its neuroprotective, antioxidant, and anti-anxiety effects. The principal active constituents in Brahmi are triterpenoid saponins known as bacosides (primarily bacoside A and B), along with alkaloids (e.g., brahmin), flavonoids, and other polyphenolic compounds. These phytochemicals contribute not only to its cognitive benefits but also to a range of activities including anti-inflammatory, anti-ulcer, hepatoprotective, and indeed antimicrobial effects. The antimicrobial potential of Bacopa monnieri is attributed to its diverse secondary metabolites. Saponins, for instance, can disrupt microbial cell membranes through interaction with sterols, increasing membrane permeability and causing leakage of cellular contents. Flavonoids and polyphenols act as enzyme inhibitors, chelators of metal ions required for microbial growth, and free-radical scavengers that interfere with pathogen survival. Alkaloids often intercalate into microbial DNA or inhibit specific enzymes critical for replication.

Several in vitro studies have demonstrated inhibitory effects of Brahmi extracts against a variety of pathogens:

1) Gram-positive bacteria: Staphylococcus aureus, Bacillus subtilis

2) Gram-negative bacteria: Escherichia coli, Pseudomonas aeruginosa

3) Fungi: Candida albicans, Aspergillus niger

These investigations have employed extraction solvents ranging from water to organic mixtures (ethanol, methanol, diethyl ether, ethyl acetate), revealing that the choice of solvent markedly influences the spectrum and potency of antimicrobial activity. For example, organic extracts often yield higher zones of inhibition in agar diffusion assays, likely reflecting greater solubilization of nonpolar bioactives.

 Rationale for Granule Formulation

While many studies report antimicrobial activity in crude extracts, few translate these findings into standardized formulations suitable for preclinical or clinical evaluation. Powders—even when finely milled—can exhibit poor flow, content uniformity challenges, and variable dissolution profiles. Granulation, especially wet granulation, is a time-tested technique in pharmaceutics that improves powder flow, enhances compressibility, and yields granules with more uniform particle size. These granules can be further compressed into tablets or filled into capsules, facilitating precise dosing, ease of administration, and patient compliance.

Key advantages of formulating herbal extracts into granules include:

1. Improved Flow and Compressibility: Granules possess better flow behavior (lower angle of repose) and higher bulk density, reducing manufacturing variability and improving tablet hardness consistency.

2. Controlled Disintegration and Dissolution : By selecting appropriate binders and disintegrants, the release profile of active constituents can be modulated, potentially optimizing bioavailability.

3. Enhanced Stability: Granules packaged in moisture-protective containers exhibit reduced degradation of sensitive phytochemicals compared to loose powders.

4. Standardization: Granule formulations facilitate uniform distribution of active extract throughout the batch, improving reproducibility across production lots.

Objectives and Scope of the Study

The principal aim of this research is to develop and characterize a wet-granulated herbal formulation containing Bacopa monnieri extract with demonstrable antimicrobial activity. To achieve this, the following objectives are delineated:

1. Extraction and Phytochemical Profiling

Shade-dry fresh Brahmi leaves and prepare a coarse powder. Perform solvent extraction (e.g., ethanol) to concentrate bacosides and related metabolites. Conduct qualitative phytochemical tests to confirm presence of saponins, flavonoids, tannins, and alkaloids.

2. Granule Formulation

Optimize a wet granulation procedure, selecting suitable binders (e.g., starch paste, polyvinylpyrrolidone), diluents (lactose, microcrystalline cellulose), and disintegrants (sodium starch glycolate).Evaluate granule characteristics: moisture content, particle size distribution, flow properties (angle of repose, bulk and tapped density, Carr’s index, Hausner ratio).

3. Antimicrobial Efficacy Testing

Prepare standardized granule extracts and perform agar well diffusion assays against representative pathogens (E. coli, S. aureus).Compare zones of inhibition with a reference standard antibiotic (e.g., Norfloxacin).Determine minimum inhibitory concentration (MIC) via broth dilution, if resources permit.

4. Optional Characterization

Assess granular disintegration time, friability, and drug content uniformity. Conduct preliminary stability studies under ambient and accelerated conditions.

5. Data Analysis and Interpretation

Analyze physicochemical and microbiological data using appropriate statistical tools. Discuss implications for herbal dosage form development and potential clinical applications.

 Significance of the Study

This research fills a critical gap by bridging empirical antimicrobial findings with practical pharmaceutical formulation of a widely used Ayurvedic herb. By demonstrating that Brahmi extract retains activity when converted into granules—and by establishing robust physicochemical and microbiological evaluation protocols—this work lays the foundation for further scale-up, in vivo pharmacodynamic studies, and eventual clinical translation. Given the urgent need for novel antimicrobial agents, especially from natural sources, standardized herbal granule formulations could emerge as adjunctive or alternative therapies in the fight against drug-resistant infections. Moreover, successful formulation strategies developed here can be adapted to other medicinal plants, advancing the integration of phytomedicine into mainstream healthcare.

Drug Profile

a) Brahmi

Brahmi (commonly referred to as Bacopa monnieri) is a well-known medicinal herb in Ayurvedic medicine. It has a broad range of pharmacological activities, including antimicrobial, antioxidant, neuroprotective, and anti-inflammatory effects. The antimicrobial activity of Brahmi is attributed to several phytochemical constituents.

Fig Brahmi

 Chemical constituent

Bacosides (especially Bacoside A and B):  Exhibit bactericidal and fungicidal activity.

Brahmin: An alkaloid with potential antibacterial activity.

Herpestine: Another alkaloid contributing to antimicrobial effects.

Flavonoids (e.g., luteolin, apigenin): Possess broad-spectrum antimicrobial activity.

Triterpenoid & saponins: Show potential antifungal and antibacterial actions.

Sterols (e.g., β-sitosterol):  Have been reported to exert mild antibacterial activity.

Uses:

1. Cognitive Enhancer / Memory Booster Bacosides (especially Bacoside A) Enhances memory, concentration, learning capacity, and attention span. Modulates cholinergic system, reduces oxidative stress in the brain.

2. Antioxidant Activity Scavenges free radicals, protects against neurodegeneration and aging.

3. Antimicrobial Activity  Bacoside A, Brahmin, Herpestine, flavonoids  Inhibits growth of various bacteria and fungi.

4. Cardioprotective Lowers blood pressure, supports healthy cardiac function. Antioxidant activity, reduction of lipid peroxidation.

 5. Anxiolytic and Antidepressant Reduces anxiety and symptoms of mild to moderate depression. Modulates serotonin and dopamine pathways.

6. Anti-inflammatory Helps reduce inflammation in chronic conditions. Inhibits COX enzymes, reduces inflammatory cytokines.

b) Neem:

Neem, scientifically known as Azadirachta indica, is a tree native to the Indian subcontinent. Various parts of the neem tree have been used in traditional medicine for centuries due to their therapeutic properties.

Figure 03: Neem

Chemical constituent

a) Azadirachtin: A prominent compound known for its insecticidal properties.

b) Nimbin: A bitter compound with antifungal and antibacterial properties.

c) Nimbidin: Another compound with anti-inflammatory and antipyretic properties.

d) Quercetin: A flavonoid with antioxidant and anti-inflammatory effects.

Uses:

?Antimicrobial Properties: Neem extracts are used for their antibacterial, antiviral, and antifungal properties.

?Skin Care: Neem is used in various forms (oils, creams) for treating skin conditions like acne, eczema, and psoriasis.

?Oral Health: Neem twigs are traditionally used as toothbrushes (neem sticks) due to their antimicrobial effects.

?Insect Repellent: Neem oil is used as a natural insect repellent.

?Hypoglycemic Effects: Neem extracts have been shown to lower blood glucose levels in animal studies. Compounds like nimbidin and quercetin are believed to enhance insulin sensitivity and improve glucose metabolism.

?Antioxidant Properties: Diabetes is associated with oxidative stress, which can lead to complications. Neem's antioxidant properties help in reducing oxidative damage, potentially mitigating the effects of diabetes.

?Anti-Inflammatory Effects: Chronic inflammation is a contributing factor in diabetes and its complications. Neem’s anti-inflammatory properties can help in managing inflammation associated with diabetes.

METHODOLOGY (MATERIAL & METHODS)

1. Material

1)The following materials/chemicals were used in the work as follow

The materials/chemicals used in this research work is mentioned in table 1

Table 01: Chemicals Used in The Research Work

2)The equipment used in the present work is as follows:

The equipment used in the present work is shown in table 2

Table 02: Equipment Used in The Research Work

3) List of Drug in the present work

Table 03: The Drug Used in Present Research Work

1. Methodology

1)Collection of plant material

Brahmi are collected from the botanical garden and Neem leaves are collected from our locality, Nandgaon, Murbad. These leaves were dried for a period of one week to remove excess moisture. Then the dried leaves  were powdered using a suitable grinder and they were sieved by using mesh no. 20.

2)  Herbal extraction method

Reflux condensation method

1)Take a 20g of herbal powder and 200 ml of methanol (80%) solvent in a RBF and  connect the flask to a reflux condenser and place it on a water bath..

2)Heat the mixture at 60–80°C for 2–3 hours, allowing continuous boiling and condensation without loss of solvent.

3)After extraction, cool the mixture and filter it using filter paper.

4)Dry the extract in a hot-air oven at <40°C to obtain a thick paste or dried extract.

5)Store the extract in airtight amber bottles for further use. Label with date and type of extract.

Fig. Extraction method (Reflux)

3) Preparation of antimicrobial granules

Formulation of antimicrobial granules (10gm)

4) Phytochemical test for Brahmi

5) Method Of Preparation

Wet granulation methods

Step 1: In a mortar pestle, add a Brahmi and neem powder triturate and then add a starch, magnesium stearate and talc powder and triturate it and then add water and make a soft paste.

Fig Wet Granulation Method

Step 2:  Then sieve no.20 make a uniform size granules

STEP 3: Then keep in oven at 50-60 C for 20 mins

Fig. Granules Forming

Step 4: Repeat the same procedure for preparation of different batches

6) Microbial assay

The antimicrobial activity was carried out with 24 hrs bacterial cultures of Escherichia coli, Staphylococcus aureus with solvents of 80% methanol extracts of Bacopa monnieri was tested separately using Agar well diffusion method. The nutrient agar plates were swabbed with bacterial strains and were individually inoculated and maintained. A well of 6mm diameter was made using a sterile cork borer. The concentrations (100μl) of plant extracts were introduced into the well. The plates were incubated at 37±2°C for 24hrs the results were measured and tabulated.

Agar Diffusion Methods

Preparation of culture media Nutrient Agar (NA)

Ingredients (per 100 mL):

Peptone – 0.5 g Beef extract – 0.3 g

Sodium chloride – 0.5 g    Agar – 1.5 g

Distilled water – up to 100 mL

???? Preparation Steps:

1.Dissolve all the ingredients in 100mL of distilled water with gentle heating

2.Sterilize by autoclaving at 121°C for 15 minutes.

3.Cool to 45–50°C and pour into sterile Petri dishes (~20 mL/plate).

4.Let the agar solidify and dry before inoculating.                                         

Fig. Culture Media

Streaking for Isolation:

Flame the loop again until red-hot and let it cool. Rotate the plate about 90°. Drag the loop from the edge of the first streak area and streak into the second quadrant. Repeat this process for the third and fourth quadrants, flaming the loop and rotating the plate each time

Incubation: Close and seal the plate with parafilm or tape. Invert the plate and incubate at 37°C for 24 hours.

Microorganisms Used:

Staphylococcus aureus (Gram-positive) & Escherichia coli (Gram-negative) 

Fig. Growth of Bacteria

Preparation of standard and test sample

Standard solution is made up of tablet norfloxacin with NaOH and test sample can be made up of saline or distilled water.  After the bacterial growth makes a 9mm diameter of four quadrants out of two is for standard solution two for test solution. Add 100 µL of granule extract solution into each well. Incubate at 37°C for 24 hours. Measure the zone of inhibition in mm.

Fig. Effect of antimicrobial activity on Brahmi

Table No. 8 Zone of Inhibition Table (In Mm)

7.) Evaluation Parameter

7.1). Organoleptic Character

?Color: Color examination is done by observing the powder directly with our naked eye

?Odour: Powder was smelled individually then the Odour can be detected

?Taste: A pinch of powder was taken on the taste bud of the tongue to detect the taste.

7.2). Flow property test

Angle of Repose (θ):

θ=tan−1(h/r)

where h is the height and r the radius of the powder cone. Lower θ indicates better flow due to reduced interparticle friction and cohesion.

Bulk Density (ρ_bulk) and Tapped Density (ρtap):

1. Bulk density = mass/ volume, 
2. Tapped density = mass/ tapped volume

Differences reflect the packability of granules under vibration.

Carr’s Index (CI) and Hausner’s Ratio (HR):

 A) CI = tap density - bulk density/ tap density, 

 B) HR= tap density/bulk density

CI < 15% and HR < 1.25 denote good flow

RESULT AND DISCUSSION

According to various studies and reports and by comparing through standard the batch first (Batch 01) found to be more acceptable results.

Table No. 9 Evaluation Parameter