Formulation and Characterization of Herbal Tablets for the Management of Anti-microbial Activity

Herbal medicines have gained significant attention in the modern pharmaceutical industry due to their wide therapeutic benefits, biocompatibility, and lower risk of side effects compared to synthetic drugs. With the growing concern over antibiotic resistance and adverse reactions to conventional antimicrobial agents, there is a renewed interest in exploring plant-based alternatives. Among the various dosage forms, tablets are one of the most widely used and accepted forms of drug delivery due to their convenience, stability, ease of administration, and patient compliance._[1] Euphorbia hirta, a medicinal herb belonging to the Euphorbiaceous family, is traditionally used in various systems of medicine, especially in tropical and subtropical regions. It is known for its diverse pharmacological activities such as antimicrobial, anti-inflammatory, antiasthmatic, antidiarrheal, and wound healing properties. The plant contains active phytochemicals such as flavonoids, tannins, phenolic compounds, and essential oils, which are responsible for its biological activities. Its antimicrobial effect has been particularly noted against both grampositive and gram-negative bacteria, making it a promising candidate for developing alternative therapies for microbial infections. _[7] In the present study, an attempt has been made to formulate and characterize herbal tablets containing Euphorbia hirta extract using the wet granulation technique. The formulation involves the use of various pharmaceutical excipients like diluents, binders, disintegrants, and lubricants to ensure uniformity and acceptable mechanical properties of the tablets. Postformulation, the tablets were evaluated for standard quality control parameters such as hardness, friability, disintegration time, weight variation, thickness, and organoleptic properties. _[7] Additionally, the antimicrobial activity of the Euphorbia hirta extract was assessed using methods like agar well diffusion or disc diffusion, targeting common microbial strains to validate its therapeutic efficacy. The primary objective of this work is to develop a standardized, effective, and patient-friendly herbal dosage form that can be used in the management and prevention of microbial infections, especially in the context of rising antimicrobial resistance. _[8] This study aims to bridge the gap between traditional herbal knowledge and modern pharmaceutical sciences by developing a reliable, natural antimicrobial agent in tablet form, which can contribute to growing field of phytopharmaceuticals and integrative medicine. _[8]

MATERIALS AND METHODS:

Materials:

Raw Material:- Euphorbia hirta Extract, Fractional Flavonoidal content of Euphorbia hirta Extract.

Chemicals/ Reagents:- Methanol Petroleum Ether, Aluminum Chloride, Sodium Hydroxide, Ethyl Acetate, Dil. Ammonia  HCL  Peptone Beef Extract, Sodium Chloride, Dichloromethane, Starch, Aerosil, Magnesium Stearate , Talc, Mannitol, Sodium Bicarbonate, NH4OH, Distilled Water, Citric acid, Chloroform, PVP, etc. 

Instruments/ Accessories:- Electronic Balance, Soxhlet Apparatus, Water Bath, Sonicator, Hot Air Oven, Colorimeter, Laminar Air Flow, Incubator, Tablet Hardness Tester, Disintegration Test Apparatus, Friability Tester, Dissolution Tester, Tablet Thickness and Diameter Gauge, Analytical Balance (for Weight Variation Test), Powder Flow Tester, Vernier Caliper (for dimension measurement), Bulk Density Apparatus, Tapped Density Tester, Angle of Repose Apparatus, Tablet Compression Machine,etc. 

 Methods:

PREPARATION OF EUPHORBIA PLANT EXTRACTS:

1. 1. 1. Determination of Alcohol Soluble Extractive Value: Weighed accurately 4grams of sample was macerated with 100ml of alcohol in conical flask for 24hour, with frequent shaking at an interval of 6hours. It was then allowed to stand for 18hours and filtered rapidly to prevent any loss during evaporation. 25ml of filtrate was evaporated until it dries in a porcelain dish and dried at 105°C to a constant weight. The percentage of alcohol soluble extract was calculated with reference to air-dried material. _[1]
      2. Extraction Method: Coarsely powdered plant material was subjected to cold maceration with methanol with occasional shaking. Extract was filtered through Whatman’s filter paper and the filtrate was collected in clean glass container. Mare was dried and subjected to Soxhlet extraction using methanol (till the colored extract become colorless) at 40°c. Extract was mixed with filtrate of cold maceration. A total extract was concentrated by using water bath. Obtained concentrated extract was dried and stored in air tight container. _[2]
      3. Fractionation of Flavonoid: Extract the sample (crude plant material) in suitable solvent. Evaporate it at 40°c to obtain residue. The obtaining residue is dissolved in 2 diff. parts of citric acid & dichloromethane. X gm. of sample dissolved in X ml of 5% citric acid. Add 25ml of dichloromethane keep it for 10 min for separation. Separate the dichloromethane & aqueous layer in separating funnel. Evaporate both liquid to dryness. Dichloromethane residue is dissolve in petroleum Ether & 90% methanol in the ratio of (1:1). In next step we get two layers of petroleum ether and methanol. In petroleum ether consist of lipids & waxes whereas methanol layer contains phenolic, terpenes & sterols. The obtained fractions are used for further phytochemical testing. _[3]
      4. Phytochemical Screening:
         1. Alkaline reagent test- Add 5 drops of dilute sodium hydroxide (NaOH) to 2 ml of plant extract, then add diluted hydrochloric acid (HCl). If the yellow solution turns colorless, flavonoids are present.
         2. Ammonium test- Heat 10 ml of ethyl acetate with the extraction in boiling water for 3 minutes, then filter. Mix the filtrate with 1 ml of dilute ammonia solution, then shake. If the ammonia layer is not yellow, flavonoids are present.
         3. Shinoda test- Add 2-3 ml of filtrate extraction with magnesium metal, then add HCL concentrate. If the color is magenta, flavonoids are present. _[4]
      5. Determination of Total flavonoid content: The total flavonoid content (TFC) in plants can be determined using a colorimetric assay with aluminum chloride. Add 100 microliters of extract to 4 milliliters of distilled water. Add 0.3 milliliters of 5% sodium nitrite. After 5 minutes, add 0.3 milliliters of 10% aluminum chloride. After 6 minutes, add 2 milliliters of 1 M sodium hydroxide. Immediately, add 3.3 milliliters of distilled water and mix thoroughly. Then the sample solutions were prepared in concentration range of (0, 0.2, 0.4, 0.6, 0.8, 1) mg/ml. Determine the absorbance at 510 nanometers versus a blank. Similarly, the standard solution of quercetin was prepared in range of (0, 0.2, 0.4, 0.6, 0.8, 1) mg/ml. Calculate the TFC using a calibration curve. _[4]

FORMULATION OF HERBAL TABLETS:

Method of Preparation of Herbal Tablet: Herbal tablets were prepared using the wet granulation method. Wet dough mass was passed through sieve no. 16 for uniform granules. Granules were air dried for 3–4 hours. Further drying was done in a hot air oven at 45°C–50°C for 20–30 minutes. Dried granules were sieved again for uniformity. Magnesium stearate and talc were added as lubricants. Granules were compressed using a tablet compression machine. Tablet hardness was maintained at a maximum of 4 kg/cm². _[9]  

Table No. 1: The formula for the Herbal Anti-microbial tablet (per 500mg) in mg

EVALUATION PARAMETERS OF TABLETS: _[9]

1) Pre-compression Evaluation Parameters:

• • • • Angle of Repose: Powder is poured from a funnel onto a horizontal surface; it will form a cone due to gravitational forces. The angle between the sides of the cone and the horizontal is referred to as the angle of repose. The angle of repose is a relatively simple technique for estimation of the flow property of powder. Powders with low angle of repose are free flowing and those with a high angle of repose are poorly flowing powders. 10gm of granules were passed through funnel and the pile was formed.

Angle of Repose (θ) = tan?¹ (h / r)

• • • • Bulk Density: This is obtained to know the exact volume of the granules that is being placed in the cylinder. Initials are used in the formula. Bulk density is also known as the fluff and poured density.

Bulk Density = Mass (M) / Volume (V)

• • • • Tapped Density: It is obtained with the help of tap density apparatus, in which the powder is filled in the cylinders and the tapping is done. After few times of intervals the volume of the cylinder is noted done and the tapped density of the granules.

Tapped Density = Weight (W) / Volume after 50 taps (V??)

• • • • Carr’s Index: After obtaining the tapped and fluff density, the Carr’s Index is being calculated by using 100ml measuring cylinder.

% Compressibility = [(Tapped Density − Bulk Density) / Tapped Density] × 100

• • • • Hausner’s Ratio: Calculate the ratio of tapped density to poured density.

Hausner’s Ratio = Tapped Density / Bulk Density

• • • • Void Volume: This volume of the granules is obtained by using the values of bulk volume and tapped density. This will indicates the air volumes that is being created in the granules during tapping.

Void Volume = Bulk Volume − Tapped Volume

2) Post-compression Evaluation Parameters:

• Weight Variation: 10 tablets were selected randomly and weight individually. The average of tablets is calculated using formula and the Standard deviation is calculated by using formula.

S.D. = √ (Σ D² / N)

• • • • Hardness Test: This test is done using the Monsanto and Pfizer apparatus. In this the tablet is kept in its place in the apparatus and the pressure is applied to it. The pressure is noted down which have been recorded by the pressure gauge and average hardness is calculated.
• • • • Friability Test: This test is carried out by using Friability apparatus. The weighted tablets are placed in the apparatus and it is rotated at 25 rpm for 5 minutes. After sometimes tablets are removed out from apparatus and again they are weight.

Friability (%) = [(W? − W_f) / W?] × 100

• • • • Disintegration Test: Take 3 tablets are taken for the evaluation of the disintegration time. The tablets are placed in the disintegration apparatus and the time is observed till the tablet gets totally disintegrated. The temperature of the apparatus is maintained at 37º C.
      • Tablet Thickness Test: Tablets are placed one by one in a Vernier caliper or digital thickness gauge. The thickness is measured in millimetres (mm).
      • Dissolution Study: At each time point, an equal volume of the sample was withdrawn. It was replaced with the same volume of pre-warmed fresh dissolution medium to maintain sink condition. The amount of drug released was determined spectrophotometrically at the selected wavelength of 203 nm. [10]

 MICROBIAL ASSAY:

1) Assay of anti-bacterial activity: Assay of anti-microbial activity of herbal tablet and standard solution of azithromycin tablet was done by Disc Diffusion method. In this method 100ml of sterilized Nutrient Agar was equally poured into 2 sterile petri plates, after solidification, 120µl of bacterial culture poured on the plates and the culture was spread on plates using spreader. Then, the Whatman’s filter paper discs (6mm in diameter) were kept over the 2 agar plates using sterile forceps at various concentrations. First prepare plate containing the standard azithromycin solution, second plate containing the herbal tablet solution. The anti-bacterial assay plates were kept incubator, where all the plates were incubated at 37ºc for 24hours. The diameter of inhibition zone was noted down. _[3]

Table No.2: Formula for Nutrient Agar

 RESULTS AND DISCUSSION:

Determination of Alcohol Soluble Extractive Value:

The Extractive Value (Methanol as a solvent) of the Euphorbia Hirta was found to be 7.2 % w/w. The Standard Extractive Value of the Euphorbia Hirta is 9.71`% w/w.

Extraction and Fractionation of Plant Material:

The methanol extract of Euphorbia Hirta Plant was prepared and used for further studies. Extract was dark green in colour. Obtained extract was subjected to fractionation for separation of Flavonoids. Methanol fraction was used for study the anti-microbial activity.

Phytochemical Screening:

Obtained result of phytochemical screening of Isolated Flavonoids from Euphorbia are as follow;

Table No. 3: Phytochemical Tests

Figure 1: Phytochemicals Tests

Determination of Total Flavonoid Content:

The total flavonoid content of euphorbia hirta was found to be 11.57 mg QE/g of sample by colorimetric assay method. The TFC of Quercetin can range from 87.53 ± 0.30 mg QE/g. Thus, the total flavonoid content (TFC) of Euphorbia hirta is lies between the TFC of Quercetin. 

Table No.4: Observation Table

Figure 2: Calibration curve for quercetin

Pre-compression Evaluation Parameters:

The Evaluation Parameters Angle of Repose, Tapped Density, Bulk Density, Carr’s Index, Hausner’s Ratio and Void Volume Were Carried Out for the Granules of F1, F2, F3, F4, & F5 are showed as follow;

Table No. 5: Showing Different Evaluation Parameters of Granules of F1, F2, F3, F4 & F5.

Post-compression Evaluation Parameters:

The Evaluation Parameters like Physical Appearance, Weight Variation, Friability, Hardness, Thickness and Disintegration Test Were Carried for the F1, F2, F3, F4 & F5 are shown as follow;

Table No. 6: Showing Different Evaluation Parameters of Tablets of F1, F2, F3, F4 & F5.

Dissolution Study:

The percentage drug release record mentioned in the table as per particular seconds.The graph shows that the formulation number F1 (80%) has the highest drug release and formulation number F5 (32.60%) is the very less drug release. So, we choose the formulation number F1 for study the anti-microbial activity.

Table No. 7: The % drug release record mentioned in the table as per particular seconds.

Figure 3: The Percentage drug release record mentioned in the graph

Microbial Assay:

Antimicrobial activity refers to any action that destroys bacteria or inhibits their growth and reproduction. This can be achieved through heat, chemicals such as chlorine, and antibiotic drugs, all of which possess antimicrobial properties. Antimicrobial are commonly used to treat bacterial infections and are generally considered to have low toxicity in humans and other animals. However, they may have negative health effects in some cases. The Herbal Tablets (Batch No. F1) prepared from Isolated Flavonoid of Euphorbia hirta shows the anti-microbial activity against the Escherichia coli.