Evaluating the Efficacy and Characterization of Plantago major Extract Microspheres for Novel drug Delivery

doi:10.21203/rs.3.rs-5260894/v1

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Evaluating the Efficacy and Characterization of Plantago major Extract Microspheres for Novel drug Delivery

https://doi.org/10.21203/rs.3.rs-5260894/v1

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Background

In the recent years, advancement in novel drug delivery system with new technologies have emerged to deliver plant bioactives and extracts. The delivery system offers enhanced efficacy, better compatibility and enhanced shelf life and stability of the product. The current study aimed to formulate microspheres containing Plantago major extract and its evaluation and characterization.

Methods

Extract loaded microspheres were prepared using the solvent evaporation technique and were characterized using sophisticated tools like FTIR, DSC, SEM etc.

Results

Phytochemical screening of PME confirmed presence of compounds like alkaloids, flavonoids and phenols etc. Total phenolic and flavonoid contents were found to be 126 mg GAE/g and 312.54 mg QE/g respectively. LCMS analysis confirmed the presence of bioactive compounds including luteolin, baicalein etc. SEM revealed spherical and porous microspheres and DSC/FTIR confirmed PME-polymer compatibility. Entrapment efficiency ranged from 7.211–19.687% and drug release was negligible in an acidic environment, sustained alkaline media. F1 with low polymer, content release was 64% and F5 with highest polymer, content release was 41% over 12 hours. The release profile of the microspheres involves polymer relaxation and erosion along with drug diffusion

Conclusion

PME-loaded microspheres show potential for sustained release of contents for inflammation.

Anti-inflammatory

Ethyl-cellulose microspheres

Inflammation

PME

Plantago major

quercetin-like compounds

The growing interest in herbal preparations over synthetic drugs is due to their less toxicity and more biological compatibility (Teja et al.2022). The plant kingdom is a treasure house of potential drugs, with drugs from plants being easily available, less expensive, safe, and rarely having side effects. Extracts from medicinal plants are recognized as excellent traditional remedies for treating many diseases including inflammation (Kim et al. 2023). Plantago major, a member of the Plantaginaceae family, is an herbaceous perennial plant with a rosette of leaves around 15–30 cm in diameter. Plantago major, has been traditionally used for wound healing, burns, infections, hemorrhoids, and fevers etc (Najafian et al. 2018). Plantgo major has been reported to have antioxidant, anti-inflammatory and hepatoprotective activity (Zubair et al.2019, Eldesoky et al.2018).

Microspheres are spherical microscopic particles and can be utilized for delivery of herbal extracts due to its ability to encapsulate bioactive compounds, improving bioavailability by enhancing stability. Microspheres are capable of encapsulating and delivering herbal extracts in a controlled or sustained released fashion consistently ensuring release of active content efficiently (Lengyel et al. 2019). Ethyl cellulose microspheres are ideal for delivery of herbal extracts due to their biocompatibility and are non-toxic in nature and can be manufactured by using cost effective methods (Seddiqi et al.2021). Moreover, ethyl cellulose microspheres offer good stability and desired release profile can be achieved by simply adjusting the polymer concentration.

The plant has been reported to possess multiple pharmacological actions including antioxidants, anti-inflammatory, hepatoprotective etc. However, as per our knowledge, no effort has been made for delivery of the herbal extract into a dosage form. Therefore, we have made an effort to standardize the extraction methods based on the preliminary detection of phytochemicals using different qualitative and quantitative phytochemical screening followed by Liquid Chromatography- Mass Spectroscopy (LCMS) analysis which enables detection of bioactive molecules. The plant extraction has been evaluated for in vitro anti-inflammatory activities followed by formulation of microspheres. Then microspheres were then evaluated for physicochemical characterization by different sophisticated instruments FTIR, SEM, DSC etc. Content estimation and release of the quercetin like compounds were also estimated to study the release profile of the active compounds.

2.1. Chemicals used

Quercetin, ascorbic acid, Gallic acid, diclofenac sodium were purchased from sigma and all other chemicals used were of analytical grades.

2.2. Collection and authentication of the plant materials

Fresh aerial parts of the Plantago major plant were collected from Golf Links Forest, Mawlai Mawroh, Shillong, Meghalaya on 14^th August 2023 and the collected plant was thoroughly washed with water to remove dirt and other debris. The Herbarium was prepared and sent to the Botanical Survey of India, Eastern Regional Centre, Shillong -793003, Meghalaya for Authentication. The plant was identified and authenticated as Plantago major L. vide reference no. BSI/ERC/Tech/2023-24/1375.

2.3. Preparation of the plant materials

Fresh plants were collected in bulk and were thoroughly washed in running water and dried under shade. Dried plants were ground to a coarse powder using a Grinder (Bajaj, Majesty ion, 500, India), and the resultant powder was stored in airtight containers for extraction.

2.4. Extraction of plant materials

The powdered plant material was extracted using Cold Maceration and Soxhlet extraction method using hydroalcoholic solvent (ethanol: water) in the ratio of 70:30.

2.4.1 Maceration

50 grams of the plant coarse powder was extracted in Ethanol-Water mixture for 72 hours at room temperature. After 72 hours, the mixture was filtered, and the supernatant was collected. The solvent was removed using rotary vacuum evaporator below 50°C to get a semi-solid extract. The extracts were stored at 4 °C for further use.

2.4.2 Soxhlet extraction

Powdered plant materials were extracted using hydroalcoholic solvent. The heating mantle was kept at 40-60 ⁰C. The extraction process with the solvent ran for 72 hours until the filtrate in the siphon tube became clear of any color. The resultant mixture was filtered and concentrated to a semi-solid mass. It was then stored in an appropriate container in a refrigerator at 4 °C.

2.5. Preliminary phytochemical screening

The qualitative chemical composition of crude ethanolic extracts of Plantago major was accessed through preliminary phytochemical screening as per the methods described by Kokate (Kokate.2004).

2.6. Determination of total phenolic content

The total phenolic content was determined using the technique described previously by Ranatunge, et al. with slight modifications ( Ranatunge et al. 2017). Briefly, 1 mL of 1 mg/mL of PME and gallic acid (GA) with concentrations of 10, 20, 40, 60, 80, and 100µg/mL were mixed with 0.5 mL of 1N Folin-Ciocalteu reagent (FCR) and incubated at room temperature for 5 minutes before mixing it with 1 mL of 20% sodium carbonate. After a 10-minute incubation, absorbance was taken at 730 nm in UV-Visible spectrophotometer (Thermo Scientific, Evaluation, 201). GA was used as a standard, and phenolic content was expressed as gallic acid equivalents (GAE). Using a standard calibration curve of GA and its linear equation, the total phenolic contents were calculated. The amount of total phenolic components in dry extract is given as mg per gram gallic acid equivalent (GAE).

2.7. Determination of total flavonoid content

The total flavonoid content of PME was evaluated using a colorimetric technique with Aluminum chloride (AlCl3), following the approach of Bahorun et al., with slight modifications (Bahorun et al.1996). Briefly, 1 ml of extract (2 mg/ml) and Quercetin standards (with concentrations 10, 20, 40, 60, 80, 100 µg/mL) were mixed with 1 ml of 2% AlCl3 solution (methanol). After 10 minutes in the dark at ambient temperature, the absorbance was taken at 510 nm using a UV visible spectrophotometer (Thermo Scientific, Evaluation, 201)). The test was carried out three times. Total flavonoid contents are expressed in milligrams of quercetin equivalent per gram of dry extract (µg QAE/mg extract).

2.8. Liquid Chromatography - Mass Spectroscopy (LCMS/MS)

The PME were underwent Liquid Chromatography - Mass Spectroscopy analysis at CytoGene Research and Development, Jankipuram, Lucknow (U.P.). The plant sample was prepared and introduced into the system with the use of Sampler model G7129A with a draw speed of 200 µl/min and eject speed of 400 µl/min. The Mobile phase was pumped using a Quaternary pump (Model G7111B) with a flow capacity of 1.500 ml/min. The Column for the separation of compounds used was Column comp. (Model G7116A). The detector used was DAD (Diode Array Detector) (Model G7115A) and the Mass analyzer used was MS Q-TOF (Time of Flight). Different compounds were detected using this analysis method.

2.9 In-vitro anti-inflammatory activity:

2.9.1 Bovine Serum Albumin (BSA) Denaturation Inhibition Assay:

Briefly, 0.2 % w/v aqueous solution of bovine serum albumin (BSA) was prepared in tris buffer saline and pH was adjusted to 6.8 with glacial acetic acid. A stock solution of 1000 µg/ml was prepared using methanol. From these stock solutions, four different concentrations of 200,400,600 and 800 µg/ml were prepared. 50µl of these different concentrations was transferred to Eppendorf’s tubes and 5 ml of 0.25 w/v BSA was added to it. Diclofenac sodium at a concentration of 10 µg/ml in methanol with 5 ml 0.2% w/v BSA were used as standard. It was incubated at 72°C for 5 minutes and then cooled for 10 minutes. The absorbance was determined at 660 nm using a UV-Spectrophotometer (Williams et al.2008).

The percentage inhibition of protein denaturation was calculated using the provided formula:

% Inhibition = Abs. of control – Abs. of extract/Abs. of control x 100

2.9.2. Egg Albumin Denaturation Inhibition Assay:

With a few minor adjustments, the procedure by Chandra et al., was used for this experiment (Chandra et al. 2012). 2 ml of the extracts with concentrations 20, 40, 60, 80, 100, and 120 µg/mL were taken and 2.8 ml of pH 6.4 phosphate buffer, and 0.2 ml of fresh hen's egg albumin were added to each of them. Similarly, 2ml of diclofenac with different concentrations 20, 40, 60, 80, 100, and 120 µg/mL were taken, and 2 ml of phosphate buffer (pH 6.4), and 2 ml of egg albumin were added to it and were used as standard. NaOH was used to bring the extract's and the standard solutions' pH levels down to 6.4. The solutions were incubated for 15 minutes at 37°C, followed by 5 minutes at 70°C. The absorbance of the samples as well as for standards were measured with a UV Spectrophotometer at 660 nm. The percent inhibition of protein denaturation was estimated using the following formula:

% Inhibition = Abs. of control – Abs. of extract/Abs. of control x 100

2.10. Formulation of the PME-loaded microspheres

Solvent evaporation technique was used for preparation of microspheres with slight modification from the original methods of preparation (Akash et al.2013). Drugs and polymers in various ratios 1:2, 1:3, 1:4, 1:5, and 1:6 (Table 1) were weighed and dissolved in Methanol at room temperature with vigorous agitation. This was progressively put into a dispersion medium of light liquid paraffin (50 ml). To achieve thorough solvent evaporation, the mixture was agitated at 500 rpm with a 3-blade homogenizer (REMI, RQ-126D, India) at room temperature for 2-3 hours. After decanting the liquid paraffin, the microspheres were separated using a Whatman filter paper. They were then washed three times with 180 ml of n-hexane and air-dried for 24 hours and stored for further characterization.

Table 1

Formulation composition of microspheres
Formulation Code	Drug & Polymer ratio	Amount of extract taken (mg)	Amount of Polymer taken (mg)
F1	1:2	100	200
F2	1:3	100	300
F3	1:4	100	400
F4	1:5	100	500
F5	1:6	100	600

2.11. Evaluation of the microspheres:

2.11.1. Percentage yield of microspheres:

Calculating the percentage of practical yield is crucial for determining the effectiveness of a manufacturing technique and selecting the most suitable one. The percentage yield was computed using the following formula (Umakanthareddy et al.2012).

% Yield = Weight of microspheres obtained/Amount of drug and polymer used × 100

2.11.2. Bulk and tapped density:

A measured quantity of microspheres were introduced into a 10 ml measuring cylinder, and the volume was noted. After that, it was tapped using a tapping machine for 100 times and the volume was noted again. The loose and tapped density was determined by using the following formulas:

Bulk density = Weight of microspheres/ Bulk volume

Tapped density = Weight of microspheres/ Tapped volume

2.11.3. Hausner’s ratio:

Another index for measuring the flowability of materials. The Hausner’s ratio can be calculated from the Bulk density and tapped density.

Hausner's ratio = Tapped density/Bulk density

2.11.4. Carr’s index (compressibility index):

Indirect measurement of rheological properties like shape, size, surface area, moisture content, and cohesiveness of materials is done by using Carr’s index. It is also known as Carr’s compressibility index (Ci). It is calculated by the following equation:

Carr’s index = Tapped density - bulk density/Tapped density x 100

2.11.5. Angle of repose:

The measurement of the angle of repose were done to determine the micromeritic properties of the prepared microspheres. The maximum possible angle between the surface of the pile and the horizontal plane is known as the angle of repose. It was determined by the fixed funnel method using the following formula:

Tan Ɵ = h/r

2.12. Characterization of Microspheres

2.12.1. Particle shape and surface morphology:

Scanning electron microscopy (SEM) was used to determine particle distribution, surface topography, and texture, and to examine the morphology of fractured or sectioned surfaces. The SEM analysis was conducted at the Sophisticated Analytical Instrument Facility (SAIF) in North-Eastern Hill University (NEHU) Shillong, Meghalaya-793022. Scanning electron microscopy (SEM) JSM-6360 (JEOL) at an accelerating voltage of 20 KV was employed. The samples for the SEM analysis were prepared by sprinkling the microspheres on one side of a stub. The stub was coated with a layer of Carbon and was then visualized under the SEM.

2.12.2. Fourier-transform infrared spectroscopy (FTIR):

The FT-IR spectra were taken to investigate the possible drug-excipient interaction between the extract and the ethyl cellulose polymer in the microsphere formulation. The spectra were obtained with the use of 40 co-added scans at wavelength resolution of 8 cm-1 over a wavelength range of 4000-400 cm-1. The spectra of the extract, polymer, and extract-loaded microspheres were then compared.

2.12.3. Differential scanning calorimetry (DSC):

Thermal analysis of the pure extract, polymer, and extract-loaded formulation were performed with a differential scanning calorimeter to determine the thermal properties of the extract, polymer, and that of formulated microspheres. The samples were heated between 30º C and 250º C at a steady increased rate of 10º C per minute.

2.13. Entrapment efficiency:

Entrapment efficiency is a measure of the amount of a drug contained in the microspheres, to the initial quantity of drug used in the formulation. Drug entrapment efficiency study was performed according to the method described by Ayorinde et al. with some modifications (Ayorinde et al.2023). Briefly, 100 mg of microspheres of each batch were crushed in a mortar with the help of a pestle and suspended in 100 mL of methanol. After mixing for 30 minutes, the dispersion was filtered. Five drops of freshly prepared aluminium chloride solution were mixed to 5 mL of the filtrate, followed by 0.1 mL of 1M Potassium acetate to achieve a yellow coloration. The flavonoid content was then measured with a UV-Vis Spectrophotometer at 510 nm. All readings were recorded in triplicate.

The flavonoid content was estimated using the standard calibration plot of pure quercetin, and the entrapment efficiency (E) was computed using the formula below:

Entrapment Efficiency = Practical Drug content /Theoretical Drug content ×100

2.14. Content estimation

A quercetin calibration curve was made using a modified aluminum chloride colorimetric technique. Quercetin was used to create the calibration curve. The quantity of quercetin-like flavonoids present in the extract was determined using the colorimetric technique with aluminium chloride with slight modifications (Hadjiioannou et al., 1993). 5 ml of each of the microsphere formulations (prepared in 1mg/ml concentration) was mixed with 5 drops of freshly made aluminium chloride (10%) and 0.1 mL of 1M potassium acetate. The reaction was allowed to stand for 30 minutes. The combination was analyzed spectrophotometrically at 510 nm to evaluate its quercetin-like flavonoid content. Absorbances were measured three times using a spectrophotometer. The Content estimation was performed for all the five microsphere formulations (F1 to F5).

2.15. In-vitro release studies and release kinetics

The flavonoid release character of formulated microspheres was carried out by using a USP dissolution test apparatus type II rotating paddle (Electrolab, India). The test was performed in two different dissolution media, 0.1N HCl pH of 1.2 for 2 hours and then in PBS of pH 6.8 for 10 hours at 100 rpm with a maintained temperature of 37°C ± 0.5°C. 5ml of each sample was taken out at scheduled periods and replaced with an equal volume to maintain sink condition and then filtered. After adding five drops of aluminium chloride solution and 0.5 ml of potassium acetate, the samples were analyzed spectrophotometrically at 510 nm. Finally, the Cumulative Percentage of drug release was determined from the standard curve of quercetin (Ayorinde et al. 2023).

Data obtained from the release study was fitted to various empirical and non-empirical kinetic equations to find the pharmacokinetic model followed by the drug while released from the prepared microsphere in-vitro.

Zero-order model: The equation is employed to represent the drug dissolution of medications which will not disaggregate, and which will slowly release the drug.

Qt = Q0 + K0t

Where,

Qt is the quantity of drug dissolved in time t,

Q0 is the initial amount of drug in the solution (usually = 0), and

K0 is the zero-order release constant

Data observations recorded from the drug release experiments were plotted as Cumulative % released versus time to analyze the release kinetics [16]

First Oder model: This model has been employed to describe drug absorption and excretion. The equation for first-order kinetic drug release is

Dc/dT = - Kc

Where,

K represents the rate constant represented in units of time-1

The equation may be represented by the following:

log C = log C0 - Kt / 2.303

Where,

C0 represents the starting drug concentration, and ‘t’ is the time (Bourne .2002).

The data is displayed as a log cumulative % of drug remaining vs. time.

Higuchi Model: This model assumes that drug concentration in the matrix is higher than solubility, diffusion occurs only in one dimension, drug particles are smaller than system thickness, matrix swelling, and dissolution are negligible, drug diffusivity is constant, and perfect sink conditions are always achieved in the release environment. The model is represented by the equation:

ft = Q = A √D(2C-Cs) Cs t.

Where,

` Q represents the amount of medication released per unit area during time t.

A represents the drug's starting concentration,

C represents its solubility in the matrix media, and

D stands for the drug's diffusion coefficient.

The above equation may be reduced as follows:

f t = Q = KH ×t1/2

where KH represents the Higuchi dissolution constant.

The data was shown as cumulative % drug release versus square root of time.

Korsmeyer-Peppas model: Korsmeyer developed a simple equation to represent drug release character from a polymeric system. The model is given by the following equation:

Mt / M∞ = Ktn.

Where

Mt / M∞ is a proportion of drug released at time t,

k is the release rate constant; n is the release factor.

The choice of ‘n’ in the Higuchi model determines the drug release mechanism. The data from drug release trials were plotted as a log cumulative % drug release versus log time. The relationship between the ‘n’ value and the drug release mechanism is given in Table 2.

Table 2

Relationship between release exponent ‘n’ and drug transport mechanism in the Korsmeyer-Peppas model (Bourne et al.2002)
Release exponent (n)	Drug transport mechanism	Rate as a function of time
≤ 0.5	Fickian diffusion	t^− 0.5
0.5 < n < 1.0	Non-Fickian diffusion (Anomalous transport)	Tⁿ⁻¹
1.0	Case II transport	Zero-order release
Higher than 1.0	Super case II transport	Tⁿ⁻¹

Data interpretation and construction of graphs were done by using Microsoft Excel.

4.1. Extractive value and Preliminary phytochemical analysis

The dried material plant of Plantago major was successfully extracted by cold maceration and Soxhlet extraction. The extractive % yield of Cold maceration and Soxhlet extraction are 20.49 ± 3.596 and 30.926 ± 4.93% respectively (Table 3).

Table 3

Phytochemical Screening of two types of PME and their Percentage yield.
Parameter	Maceration	Soxhlet extraction
Extractive Yield	20.49 ± 3.59	30.926 ± 4.93%
Test for Steroids and Triterpenoids	+	+
Test for Proteins	+	-
Test for Carbohydrates	+	+
Test for Alkaloids	+	+
Test for Phenols	+	+
Test for Flavonoids	+	-
Test for Tannins	+	+
Test for Saponins	+	-
Test for Glycosides	+	+

Preliminary phytochemical analysis of PME by maceration shows presence of steroids and triterpenoids, proteins, carbohydrates, alkaloids, phenols, flavonoids, tannins, Saponins and glycosides. In Soxhlet extraction, similar profile of phytochemicals were present except saponins, flavonoids and proteins (Table 3)

4.2. Total Phenolic and flavonoid Content

The total phenolic content and flavonoid content of PME were calculated from the standard calibration curve of GA (Fig. 1A) and quercetin (Fig. 1B) respectively and the values were found to be 126.9635 ± 0.022 mg GAE/g and 312.54 ± 0.004 mg QE/g respectively.

4.3. Liquid chromatography-mass spectroscopy (LC-MS)

LC-MS report states a wide range of phytoconstituents present in the extract. Compounds reported in the LC-MS analysis shows presence of baicalein, luteolin, velloquercetin (a derivative of quercetin) etc. The chromatogram of the PME, baicalein and luteolin are given in Fig. 2(A), 2(B) and 2(C) respectively.

4.4 In-vitro anti-inflammatory activity of the extract:

4.4.1. Bovine serum albumin (BSA) and egg albumin denaturation inhibition Assay:

The % inhibition of BSA denaturation by PME is comparable with the standard anti-inflammatory drug diclofenac and the % inhibition increases with increasing concentrations (Fig. 3A). The IC₅₀ value for standard diclofenac was found to be 162.413 µg/ml while the IC₅₀ value of the PME was found to be 267.11 µg/ml. PME shows similar dose dependent inhibition in egg albumin denaturation assay (Fig. 3B). The IC₅₀ value of the standard diclofenac was found to be 32.641 µg/ml while the IC ₅₀ value of the PME was found to be 50.169 µg/ml.

4.5. Evaluation of microspheres:

4.5.1. Percentage yield and micromeritics evaluation of microspheres

The percentage yield of different formulations (F1 to F5) was determined by weighing the microspheres after drying. The percentage yield of the different formulations was in the range of 77.125 ± 0.085% to 87.6 ± 0.074% (Table 4). The formulation F3 had the highest percentage yield of 87.6 ± 0.074% while formulation F2 had the lowest percentage yield of 77.125 ± 0.085%.

Table 4

Micromeritic properties of microspheres and their percentage yield
Formulation Code	Hausner’s ratio	Carr’s index	Angle of Repose (Ɵ)	Percentage Yield
F1	1.139 ± 0.002	12.254 ± 0.02	16..858 ± 0.76	79.33 ± 0.10%
F2	1.2311 ± 0.008	18.773 ± 0.09	27.474 ± 0.77	77.125 ± 0.08%
F3	1.0684 ± 0.006	6.410 ± 0.03	17.744 ± 0.78	87.6 ± 0.07%
F4	1.2080 ± 0.007	17.223 ± 0.05	18.434 ± 0.17	77.83 ± 0.07%
F5	1.153 ± 0.005	13.337 ± 0.07	23.962 ± 0.18	83.142 ± 0.06%
Values are represented as Mean ± SD in three independent variables

Carr's index, Hausner's ratio, and Angle of repose were evaluated for the formulated microspheres. Carr's index ranges from 6.410 ± 0.036% to 18.773 ± 0.094% (Table 4), showing strong and acceptable compressibility. Hausner's ratio of all the formulations was less than 1.25 (Table 4), indicating good and passable flowability (Biswas and Sen.2018).

4.6. Scanning Electron Microscopy (SEM):

SEM was used to determine particle distribution, surface topography, and texture, and to examine the morphology of fractured or sectioned surfaces. The SEM images of microspheres depicted in Fig. 4 show that the ethyl cellulose microspheres were spherical and porous, with a smooth and dense exterior surface (Biswas and Sen.2018).

4.7. FT-IR study

The obtained spectra of the PME, ethyl cellulose, and the formulated microspheres loaded with the PME were studied individually and their characteristic peaks and functional groups were then compared, and their interaction if present were discussed. The FTIR spectra for PME, ethyl cellulose and the microspheres containing PME extract has been depicted as Fig. 5A, 5B and 5C respectively.

4.8 Differential Scanning Chromatography (DSC)

DSC is a thermal analysis technique that measures the heat flow associated with phase transitions of materials as a function of temperature. DSC was performed for pure extract, polymer, and extract-loaded microspheres (METTLER SW 16.00, USA). DSC curves show an early endothermic peak of about 60–80°C, suggesting moisture in the PME, ethyl cellulose, and loaded microsphere formulation (Fig. 6A, 6B and 6C). The large endothermic peaks observed at 100–200°C indicate that both the PME and ethyl cellulose are thermally degraded at this temperature range. The exothermic peak at 280°C in the loaded microsphere formulation indicates a chemical contact, maybe owing to the encapsulation process or a reaction between the extract and the polymer.

4.9. Entrapment Efficiency and Content Estimation

All the formulations were evaluated for entrapment efficiency (EE). The entrapment efficiency of the extract-loaded microspheres of different polymer-to-drug ratios is depicted in Table 5.

Table 5

% Drug Entrapment and Content of Quercetin-like compounds in the formulations
Formulation Code	Mg Quercetin Equivalent/ gram	% Drug Entrapment
F1	133.86 ± 0.0012	7.211 ± 0.022%
F2	138.54 ± 0.00124	11.009 ± 0.040%
F3	148.54 ± 0.0017	14.264 ± 0.028%
F4	153.86 ± 0.00125	18.058 ± 0.052%
F5	157.86 ± 0.0017	19.687 ± 0.34%
Values are represented as Mean ± SD in three independent variables

The colorimetric method of aluminium chloride was used to determine the amount of quercetin-like flavonoids present in the extract. The content determination of flavonoid-like compounds was done for all formulations F1 to F5 using the standard calibration curve of Quercetin (Fig. 1B) and the results are given in Table 5.

4.10. In-Vitro Drug Release and kinetics study

PME-loaded ethyl cellulose microspheres were tested for content release in vitro at simulated stomach fluid (pH 1.2) and intestinal fluid (pH 6.8) pH levels. The drug release pattern and profile are shown in Fig. 7. At pH 1.2, all formulations discharged less than 8% of their drug (quercetin-like flavonoids) content which is negligible. However, at pH 6.8, drug releases were observed 64% (F1), 63%( F2), 57.5% (F3), 45.5% (F4), and 41% (F5) demonstrating a sustained release pattern from microspheres. Formulations F1 to F3 with drug-polymer ratios of 1:2, 1:3, and 1:4 resulted in 64%, 63%, and 57.5% cumulative drug release after 12 hours. Decreased polymer concentration for F1 to F3 may be the reason for the increased cumulative drug release percentage. The formulations with higher polymer ratios F4 (1:5) and F5 (1:6) result in delayed/sustained drug release due to the longer diffusion paths that the drug must traverse to reach the dissolving liquid (Ayorinde et al.2023).

In our study, we have used two different extraction methods namely Soxhlet using heat and another cold maceration at room temperature. Although the extractive yield of Soxhlet extraction is higher compared to maceration, it is found that compounds including saponins, flavonoids and proteins were absent in the Soxhlet extract. However, all these compounds were present in macerated extract. This may be due to the loss of heat-sensitive compounds which might have degraded during the entire extraction process. Since many pharmacological activities including antioxidant, anti-inflammatory etc are exhibited by saponins and flavonoids, phenols, the macerated extracts were taken into consideration for further evaluation and formulation development (Rodríguez De Luna et al. 2020).

Phenols and flavonoids are the most important compounds in the plant. They have multiple pharmacological actions. They decrease the oxidative stress by scavenging the Reactive oxygen species (ROS), a free radical species mainly responsible for cellular damage in the body. Phenols and flavonoids have direct relation with antioxidant and anti-inflammatory activities (Rudrapal et al. 2022). These compounds are also responsible for other activities either by working synergistically or individually.

LCMS data revealed the presence of flavonoids and related polyphenols in the extract and may be responsible for the biological activities. Luteolin, a flavone, has been found to suppress inflammatory response by blocking IKKb/NF-kB activation and efficiently enabling insulin signaling transduction along the IRS-1/Akt/eNOS pathway in the endothelium (Deqiu et al.2011). Another study reported anti-inflammatory effects on dsRNA-induced macrophages by inhibiting the production of NO, cytokines, chemokines, and growth factors via the endoplasmic reticulum stress–CHOP/STAT pathway (Kim et al.2018). Similarly, baicalein has been reported to show anti-inflammatory and immunomodulatory actions by inhibiting NF-κB signaling pathway and nucleotide-binding oligomerization domain-like receptor pyrin domain protein 3 (NLRP3) inflammasome and suppress the proinflammatory markers such as interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor α (TNF-α) etc (Wen et al.2023, Liao et al.2021, Dinda et al.2017).

Protein denaturation is the loss of biological characteristics of protein molecules. This denaturation process is linked to inflammatory disorders such as rheumatoid arthritis and diabetes etc. Preventing protein denaturation may help avoid inflammatory diseases (Mendez-Encinas et al.2023). Protein denaturation occurs when external forces such as heat, strong acids or bases, organic solvents, or concentrated inorganic salts disturb the tertiary and secondary structures of proteins. Preventing protein denaturation may reduce the risk of inflammation. Protein will be called denatured, when its natural shape is lost along with its biological functions. Protein denaturation at the site of inflammation may trigger a series of inflammatory responses followed by complex and immunological reactions to the damage sites with a series of molecular events (Gonfa et al.2023). Plant extracts contain various mixtures of different compounds including phenols and flavonoids. The presence of active compounds in plant extracts are mainly responsible for the inhibition and hence shows anti-inflammatory action (Osman et al. 2016, Habila et al. 2018). Our results also support the notion of protein denaturation and its anti-inflammatory activities.

The micromeritic evaluation of microspheres were found to be free-flowing, with an angle of repose below 30° in all the formulations possibly due to their spherical shape and low moisture content. From the observations, all the formulated microspheres had good micromeritic properties and it was seen that microspheres of formulation F3 had the best micromeritic properties out of all the formulations. The SEM images of the microspheres show microspheres were similar in size with a uniform size distribution in the range of 10–100 µm. The surface of the microspheres revealed the presence of numerous pores suggesting that the drug might be released through these pores and the mechanism of entrapped drug release may be diffusion controlled (Rama et al.2005).

FTIR spectra of PME shows a broad peak that may be representing hydroxyl groups (O-H stretching around 3400 − 3200 cm-1), which are common in many natural compounds such as alcohols and phenols. C-H Stretching (around 2900 cm⁻¹) was seen and Peaks in this region are due to aliphatic C-H bonds. C = O Stretching (around 1730 cm⁻¹) was also seen indicating the presence of carbonyl groups, such as ketones, aldehydes, or carboxylic acids. C = C Stretching (around 1600 cm⁻¹) has Peaks that are indicative of aromatic or alkene groups.

Similarly, for ethyl cellulose a broad peak was observed at around 3500 cm-1 indicating the presence of hydroxyl groups, which could be due to residual moisture or hydrogen bonding. C-H Stretching (around 2900 cm⁻¹) observed was characteristic of C-H bonds in methylene and methyl groups. C-O-C Stretching (around 1100 cm⁻¹) seen in this region is indicative of the ether linkages present in ethyl cellulose. Various other peaks in the fingerprint region (below 1500 cm⁻¹) correspond to the bending and wagging modes of the functional groups.

In the formulated microspheres containing PME, O-H Stretching (around 3400 − 3200 cm⁻¹) was observed, and a broad peak that could be a combination of hydroxyl groups from both the ethyl cellulose and the plant extract. C-H Stretching (around 2900 cm⁻¹) similar to the ethyl cellulose spectrum was also seen, indicating the presence of aliphatic C-H bonds. C = O Stretching (around 1730 cm⁻¹) was noted, indicating the presence of carbonyl groups (Yasin et al.2021). By comparing the spectra’s with PME, ethyl cellulose, and the microspheres loaded with PME, no interaction between the extracts and excipients was observed.

Based on the DSC spectra, the plant extract and ethyl cellulose exhibit similar thermal degradation patterns. The loaded microsphere formulation shows significant interactions between the plant extract and ethyl cellulose, indicating good compatibility. The exothermic peak in the microsphere formulation may suggest a more stable compound formation or encapsulation efficiency. These interpretations indicate that the ethyl cellulose is compatible with the PME, making it a suitable polymer for encapsulating and stabilizing the extract in microsphere formulations (Li et al.2008).

For a formulation to be successful, it requires entrap sufficient amounts of active ingredients or extract in the formulation core. The microspheres formulated using solvent evaporation techniques are evaluated for % EE. The EE% was determined based on the entrapment of flavonoids content in the microspheres by quantifying the quercetin-like flavonoid content in the formulation (Ayorinde et al.2023). The %EE of the flavonoids was dependent on the amount of polymer used i.e. the polymer concentration is directly proportional to the EE. The EE of the flavonoid was poor and was in the range of 7.211 ± 0.022% to 19.687 ± 0.34%. Formulations with high concentrations of ethyl cellulose polymer (F3, F4, and F5) had better entrapment than F1 and F2. For content estimation, aluminium chloride methods were used to estimate the quercetin-like flavonoid content. The quercetin-like flavonoid content from the prepared concentration of microspheres (1mg/ml) shows the actual content of flavonoids present in the formulation.

Dissolution data of all formulations were fitted into different mathematical models (Zero-order, First-order, Higuchi, and Peppas) to predict the kinetics of the drug release rate. The kinetic model with the highest coefficient of determination (R2) value was considered a more suitable model for the dissolution profile. The data for analysis of the drug release mechanism from PME-loaded microspheres is given in Table 6.

Table 6

Data for analysis of content release mechanism from PME-loaded microspheres
Formulation Code	Zero Order	First Order	Higuchi	Korsmeyer-Peppas		Best fit Model
Formulation Code	R²	R²	R²	R²	n	Best fit Model
F1	0.9901	0.9926	0.9589	0.8169	1.2089	First Order
F2	0.9917	0.9918	0.9000	0.9645	1.4853	First Order
F3	0.9863	0.9515	0.8893	0.8809	1.2158	Zero Order
F4	0.9957	0.9895	0.9146	0.9465	1.3045	Zero Order
F5	0.9844	0.9904	0.9121	0.9628	1.5718	First Order

The coefficient of determination (R²) is used as an indicator of the best-fit models considered. The kinetic data analysis of most of the formulations reached a higher coefficient of determination with the First-Order model (R² = 0.9904 to 0.9926) except for the formulations F3 and F4 which followed the Zero-order model (Table 6). Formulations F1, F2, and F5 followed the First Order release kinetics, suggesting concentration-dependent flavonoid content release. In first-order kinetics, the amount of content released depends on the concentration of the drug. Formulations F3 and F4 follow Zero-Order release kinetics, suggesting a constant flavonoid release rate over time. All formulations show “n” values higher than 1.0 indicating super case-II transport, suggesting complex release mechanisms involving polymer relaxation and erosion along with drug diffusion (Ayorinde et al. 2023, Lengyel et al.2019).

Plantago major being one of the most abundant plants and generally being considered as a weed has been successfully employed to elucidate its plethora of medicinal benefits in particular anti-inflammatory agents. From the preliminary phytochemical screening to total flavonoid & phenolic content determination, it was evident that Plantago major contained significant amounts of flavonoids and phenolic compounds which is the base for their potential anti-inflammatory activities either working synergistically or individually.

LCMS analysis of PME shows presence of flavonoid compounds such as Luteolin and Baicalein which are proven to have anti-inflammatory activity. In-vitro protein denaturation inhibition results of the extract also support the notion of anti-inflammatory activity. The concept of formulating microspheres loaded with Plantago major extract was feasible and it offers a suitable, practical, and novel approach to achieve a prolonged therapeutic effect by continuously releasing the medicament over an extended or sustained period. The microspheres showed First-order release kinetics as well as zero-order release kinetics over an extended period (12 hours). The “n” value from the Korsmeyer-Peppas plot of all the formulations showed values higher than 1.0 which indicated a Super Case-II transport and suggested a complex release mechanism involving polymer relaxation as well as polymer erosion along with drug diffusion. The formulated microsphere has achieved zero order release kinetics and therefore there is huge scope for the development of a novel anti-inflammatory drug delivery system for prolonged periods with minimized side effects, unlike NSAIDs could be achieved. However, further research is warranted for the safety profile of the plants, their chemical characterization, and also studies in non-human primates for safety and efficacy evaluation at molecular levels.

Ethics approval and consent to participate: Not applicable

Consent for publication:All the authors involved in this article have read and given their consent to publish

Availability of data and material: Not applicable

Competing interests: The authors declare no competing interest associated with this manuscript

Funding: Not applicable

Authors' contributions: Mr. BN actively conducted the research work and wrote the manuscript. The concept, design and writing and arrangement of the manuscript was done by Mr PK Roy Ms. Lalruatsangi has also actively worked with Ms. Lalruatsangi conducted the research and also helped her to write. Mr Laldinchhana guided Mr. BN and Ms. Lalruatsangi and provided necessary support and helping hand in conducting research. Dr. H Lalhlenmawia provided the correct guidance with helping hand in editing and analysing the data of the manuscript. Dr. Sonjit helped in writing the discussion and conducting some parts of the analysis of the manuscript. Prof. Sanjay D. Sawant has provided necessary guidance, concepts for the study and facilities necessary to conduct the tests and analysis.

Acknowledgements: The authors would like to thank all the staff of the Department of Pharmacy, RIPANS for their unconditional support for conducting the research work.

Teja, P. K., Mithiya, J., Kate, A. S., Bairwa, K., & Chauthe, S. K. (2022). Herbal nanomedicines: Recent advancements, challenges, opportunities and regulatory overview. Phytomedicine : international journal of phytotherapy and phytopharmacology, 96, 153890. https://doi.org/10.1016/j.phymed.2021.153890
Kim, Y. J., & Kang, K. S. (2023). The Phytochemical Constituents of Medicinal Plants for the Treatment of Chronic Inflammation. Biomolecules, 13(8), 1162. https://doi.org/10.3390/biom13081162
Najafian, Y., Hamedi, S. S., Farshchi, M. K., & Feyzabadi, Z. (2018). Plantago major in Traditional Persian Medicine and modern phytotherapy: a narrative review. Electronic physician, 10(2), 6390–6399. https://doi.org/10.19082/6390
Zubair, M., Widén, C., Renvert, S., & Rumpunen, K. (2018). Water and ethanol extracts of Plantago major leaves show anti-inflammatory activity on oral epithelial cells. Journal of traditional and complementary medicine, 9(3), 169–171. https://doi.org/10.1016/j.jtcme.2017.09.002
Eldesoky, A.H., Abdel-Rahman, R.F., Ahmed, O.K., Soliman, G.A., Saeedan, A.S., Elzorba, H.Y., Elansary, A.A. and Hattori, M. (2018) Antioxidant and Hepatoprotective Potential of Plantago major Growing in Egypt and Its Major Phenylethanoid Glycoside, Acteoside. Journal of Food Biochemistry, 42, e12567. https://doi.org/10.1111/jfbc.12567
Lengyel, M., Kállai-Szabó, N., Antal, V., Laki, A. J., & Antal, I. (2019). Microparticles, microspheres, and microcapsules for advanced drug delivery. Scientia Pharmaceutica, 87(3), 20. https://doi.org/10.3390/scipharm87030020
Seddiqi, H., Oliaei, E., Honarkar, H., Jin, J., Geonzon, L. C., Bacabac, R. G., & Klein-Nulend, J. (2021). Cellulose and its derivatives: towards biomedical applications. Cellulose, 28(4), 1893-1931. https://doi.org/10.1007/s10570-020-03674-w
Kokate CK. Practical Pharmacognosy. Published by Nirali Prakashan New Delhi;2004: 29-107.
Ranatunge, I., Adikary, S., Dasanayake, P., Fernando, C. D., & Soysa, P. (2017). Development of a Rapid and Simple Method to Remove Polyphenols from Plant Extracts. International journal of analytical chemistry, 2017, 7230145. https://doi.org/10.1155/2017/7230145
Bahorun, T., Gressier, B., Trotin, F., Brunet, C., Dine, T., Luyckx, M., Vasseur, J., Cazin, M., Cazin, J. C., & Pinkas, M. (1996). Oxygen species scavenging activity of phenolic extracts from hawthorn fresh plant organs and pharmaceutical preparations. Arzneimittel-Forschung, 46(11), 1086–1089.
Williams, L. A., O'Connar, A., Latore, L., Dennis, O., Ringer, S., Whittaker, J. A., Conrad, J., Vogler, B., Rosner, H., & Kraus, W. (2008). The in vitro anti-denaturation effects induced by natural products and non-steroidal compounds in heat treated (immunogenic) bovine serum albumin is proposed as a screening assay for the detection of anti-inflammatory compounds, without the use of animals, in the early stages of the drug discovery process. The West Indian medical journal, 57(4), 327–331.
Chandra, S., Chatterjee, P., Dey, P., & Bhattacharya, S. (2012). Evaluation of in vitro anti-inflammatory activity of coffee against the denaturation of protein. Asian Pacific Journal of Tropical Biomedicine, 2(1), S178-S180. https://doi.org/10.1016/S2221-1691(12)60154-3
Akash, M. S. H., Iqbal, F., Raza, M., Rehman, K., Ahmed, S., Shahzad, Y., & Shah, S. (2013). Characterization of ethylcellulose and hydroxypropyl methylcellulose microspheres for controlled release of Flurbiprofen. J Pharm Drug Deliv Res, 2(1), 1-10. doi:10.4172/2325-9604.1000113
Umakanthareddy, M. A., Sreeramulu, J., & Satyanarayana Punna, S. P. (2012). Formulation development of Losartan potassium microspheres using natural polysaccharides and their in-vitro evaluation. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 2012, Vol. 3, No. 2, 725-734.
Ayorinde, J. O., & Okonye, E. O. (2023). Antioxidant properties and effects of formulation variables on Ceiba pentandra microspheres. Nigerian Journal of Pharmaceutical Research, 19(1), 1-16. Doi: 10.4314/njpr.v19i1.1s
Hadjiioannou, T. P., Christian, G. D., Koupparis, M. A., & Macheras, P. E. (1993). Quantitative calculations in pharmaceutical practice and research (pp. 345-348). New York: VCH.
Bourne D.W.: Pharmacokinetics. in: Modern pharmaceutics. 4th ed. Banker GS, Rhodes CT, Eds., Marcel Dekker Inc, New York, 2002.
Biswas, R. A. N. U., & Sen, K. K. (2018). Development and characterization of novel herbal formulation (Polymeric microspheres) of syzygium cumini seed extract. Int J Appl Pharm, 10(5), 226-34.
Rodríguez De Luna, S. L., Ramírez-Garza, R. E., & Serna Saldívar, S. O. (2020). Environmentally friendly methods for flavonoid extraction from plant material: Impact of their operating conditions on yield and antioxidant properties. The Scientific World Journal, 2020(1), 6792069. https://doi.org/10.1155/2020/6792069
Rudrapal, M., Khairnar, S. J., Khan, J., Dukhyil, A. B., Ansari, M. A., Alomary, M. N., ... & Devi, R. (2022). Dietary polyphenols and their role in oxidative stress-induced human diseases: Insights into protective effects, antioxidant potentials and mechanism (s) of action. Frontiers in pharmacology, 13, 806470. https://doi.org/10.3389/fphar.2022.806470
Deqiu, Z., Kang, L., Jiali, Y., Baolin, L., & Gaolin, L. (2011). Luteolin inhibits inflammatory response and improves insulin sensitivity in the endothelium. Biochimie, 93(3), 506-512. https://doi.org/10.1016/j.biochi.2010.11.002
Kim, Y. J., Kim, H. J., Lee, J. Y., Kim, D. H., Kang, M. S., & Park, W. (2018). Anti-inflammatory effect of baicalein on polyinosinic–polycytidylic acid-induced RAW 264.7 mouse macrophages. Viruses, 10(5), 224. https://doi.org/10.3390/v10050224
Wen, Y., Wang, Y., Zhao, C., Zhao, B., & Wang, J. (2023). The Pharmacological Efficacy of Baicalin in Inflammatory Diseases. International journal of molecular sciences, 24(11), 9317. https://doi.org/10.3390/ijms24119317
Liao, H., Ye, J., Gao, L., & Liu, Y. (2021). The main bioactive compounds of Scutellaria baicalensis Georgi. for alleviation of inflammatory cytokines: A comprehensive review. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 133, 110917. https://doi.org/10.1016/j.biopha.2020.110917
Dinda, B., Dinda, S., DasSharma, S., Banik, R., Chakraborty, A., & Dinda, M. (2017). Therapeutic potentials of baicalin and its aglycone, baicalein against inflammatory disorders. European journal of medicinal chemistry, 131, 68–80. https://doi.org/10.1016/j.ejmech.2017.03.004
Mendez-Encinas, M. A., Valencia, D., Ortega-García, J., Carvajal-Millan, E., Díaz-Ríos, J. C., Mendez-Pfeiffer, P., Soto-Bracamontes, C. M., Garibay-Escobar, A., Alday, E., & Velazquez, C. (2023). Anti-Inflammatory Potential of Seasonal Sonoran Propolis Extracts and Some of Their Main Constituents. Molecules (Basel, Switzerland), 28(11), 4496. https://doi.org/10.3390/molecules28114496
Gonfa, Y. H., Tessema, F. B., Bachheti, A., Rai, N., Tadesse, M. G., Singab, A. N., ... & KumarBachheti, R. (2023). Anti-inflammatory activity of phytochemicals from medicinal plants and their nanoparticles: A review. Current Research in Biotechnology, 100152. https://doi.org/10.1016/j.crbiot.2023.100152
Osman, N. I., Sidik, N. J., Awal, A., Adam, N. A., & Rezali, N. I. (2016). In vitro xanthine oxidase and albumin denaturation inhibition assay of Barringtonia racemosa L. and total phenolic content analysis for potential anti-inflammatory use in gouty arthritis. Journal of intercultural ethnopharmacology, 5(4), 343–349. https://doi.org/10.5455/jice.20160731025522
Habila, J. D., Isah, Y., Adeyemi, M. M., Mahmoud, A. Y., Habila, A. J., & Hamza, M. H. (2018). In Vitro Anti-Inflammation Studies of Jakaranda mimosifoliaExtract Using Protein Denaturation Technique. Journal of Chemical Society of Nigeria, 43(3).
Rao, K. R., Senapati, P., & Das, M. K. (2005). Formulation and in vitro evaluation of ethyl cellulose microspheres containing zidovudine. Journal of microencapsulation, 22(8), 863–876. https://doi.org/10.1080/02652040500273498
Yasin, H., Al-Taani, B., & Salem, M. S. (2020). Preparation and characterization of ethylcellulose microspheres for sustained-release of pregabalin. Research in pharmaceutical sciences, 16(1), 1–15. https://doi.org/10.4103/1735-5362.305184
Li, X. W., & Yang, T. F. (2008). Fabrication of ethyl cellulose microspheres: Chitosan solution as a stabilizer. Korean Journal of Chemical Engineering, 25, 1201-1204. https://doi.org/10.1007/s11814-008-0198-8

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Evaluating the Efficacy and Characterization of Plantago major Extract Microspheres for Novel drug Delivery

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Abstract

Background

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and Methods

3. Data interpretation and graph

4. Results

4.1. Extractive value and Preliminary phytochemical analysis

4.2. Total Phenolic and flavonoid Content

4.3. Liquid chromatography-mass spectroscopy (LC-MS)

4.4 In-vitro anti-inflammatory activity of the extract:

4.4.1. Bovine serum albumin (BSA) and egg albumin denaturation inhibition Assay:

4.5. Evaluation of microspheres:

4.5.1. Percentage yield and micromeritics evaluation of microspheres

4.6. Scanning Electron Microscopy (SEM):

4.7. FT-IR study

4.8 Differential Scanning Chromatography (DSC)

4.9. Entrapment Efficiency and Content Estimation

4.10. In-Vitro Drug Release and kinetics study

5. Discussion

6. Conclusion

Declarations

References

Additional Declarations

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