Development and Optimization of Indian Propolis Formulation for Enhanced Immunomodulatory Potential

Abstract

Propolis, a complex natural product that honey bees produce by mastication to protect and maintain their hive structures, comprises various bioactive constituents, including phenolic acids, flavonoids, diterpenes, sesquiterpenes, lignans, vitamins, minerals, etc. The objective of the current research was to extract crude propolis to enrich the total polyphenolic and flavonoid content, conduct preliminary phytochemical screening, and develop and evaluate dosage form to improve formulation characteristics and immunomodulatory potential. Total balsam, polyphenols, and flavonoids were found to be 46% w/w, 34.82 ± 0.078 mg equivalent of gallic acid/g, and 23.61 ± 0.045 mg equivalent of quercetin/g, respectively. DSC and FTIR studies demonstrated molecular dispersion of the propolis extract. Formulation was optimized with a 3² factorial design, and an optimized batch showed 92.20 ± 1.72% drug release in 1 h, an elevated hypersensitivity (DTH) response (p < 0.0001), increased phagocytic activity (p < 0.01), and a significantly (p < 0.001) higher total leukocyte count ((5.015 ± 0.19) × 10³/mm³). The developed formulation showed significantly modulated immune modulatory potential compared with the propolis extract and conventional levamisole. This study can be further extended for clinical evaluations.

1. Introduction

Natural products are well known for their potential therapeutic applications. Propolis is one of the natural products that honey bees produce by mastication to protect and maintain their hives. Bees create propolis by mixing beeswax and saliva, which serves as a defense mechanism to protect the hive [1]. Raw propolis typically comprises pollens, plant resins, essential and aromatic oils, waxes, and other organic substances [2]. Propolis has immune-protective and antioxidant qualities due to its bioactive phytochemical constituents. The composition of propolis varies depending on the region of origin and contains a diverse range of compounds, such as lignans, alcohols, diterpenes, sesquiterpenes, aromatic aldehydes, esters, flavonoids, amino acids, vitamins, fatty acids, and minerals [3]. It has been reported that propolis contains over 300 different compounds such as galangin, quercetin, caffeic acid phenethyl ester [4], caffeic acid, apigenin, pinocembrin [5], luteolin, curcumin [6], etc. Over the past few decades, studies have shown that propolis has numerous pharmacological and biological effects. These effects include antioxidant, antibacterial, anti-tumor, anti-inflammatory, and immunomodulatory properties [7]. Based on chemical composition as per geographical origin, propolis shows various mechanisms of action for antioxidant, anti-inflammatory, and immunomodulatory potential [8].

Conventional chemotherapeutic agents have mainly acted through immunosuppressive effects, showing various adverse effects and cytotoxicity. Cytokines, which include interleukins and interferon, are utilized as immune stimulants. However, due to their side effects and high cost, they are not very effective in the long run, due to which there is high demand and a growing trend of using natural products as an alternative source of immune modulatory agents [9]. Propolis and its chemical constituents are well proven to have synergistic immune modulatory effects on various types of immune cells. These effects are mediated by eukaryotic transcription factors, such as nuclear factor kappa B, activated T cells, the mitogen-activated protein kinase, and the extracellular signal-regulated kinase 2 signaling pathway. Various invivo and invitro studies also reported antimicrobial potential for a wide range of microorganisms. This was achieved by activating monocytes, neutrophils, and macrophages. Furthermore, research studies revealed that propolis exhibits anti-allergic properties by preventing the release of histamine from mast cells or basophils. These compounds also boost antibody production, potentially serving as a vaccine adjuvant. The anti-inflammatory effects of propolis may be linked to its ability to inhibit the proliferation of lymphocytes [10]. Despite these beneficial effects, propolis has several limitations in terms of solubility, palatability, stickiness, staining, etc., which hampers its bioavailability and overall efficacy. Therefore, it is worthwhile to extract propolis for the removal of beeswax and other debris material for the enrichment of the total polyphenol and flavonoid content and develop a suitable formulation for improved solubility, release profile, bioavailability, and immunomodulatory effects [11].

Hence, the present investigation focused on the extraction of Indian propolisusinga suitable method, characterization of the extract, formulation of propolis solid dispersion tablets, and evaluation of the in vivo immunomodulatory activity of the propolis formulation using animal models. Furthermore, this study can be extended to clinical trials for therapeutic applications.

2. Materials and Methods

2.1. Materials

Indian propolis was purchased and authenticated from the Central Bee Research and Training Institute (CBRTI) Pune. Mannitol, Talc Sodium starch glycolate, Avicel PH 102, PVP K30, and Magnesium stearate were purchased from HiMedia Lab Private Ltd. Thane, India. Cyclophosphamide and levamisole were purchased from Dr. D. Y. Patil Medical College, Hospital & Research Centre, Pharmacy store, Pune, India. Colloidal carbon was purchased from Indian ink, Camel India Private Limited, Hyderabad, India. Analytical-grade chemicals were used for all other purposes.

2.2. Total Balsam Content

To determine the percentage of balsam, 1 g of crude propolis was accurately weighed and dissolved in 10 mL of ethanol. The solution was then filtered and the ethanol-soluble portion was obtained. The percentage of balsam in the crude propolis was calculated by drying the filtrate until a constant weight was obtained [8].

2.3. Extraction and Standardization of Propolis Extract

A total of 10 g of crude propolis was pretreated by sonication with 30 mL of hexane to remove the debris and wax and then filtered. The mark was further extracted with ethanol by the Soxhlet method at 60 °C to obtain an ethanolic extract of Indian propolis (EEIP). This was further filtrated, and the collected filtrate was heated to between 40 and 60 degrees Celsius using a rotary evaporator to obtain an extract that was rich in flavonoids, polyphenols, and other chemical constituents. The extract was stored at 2–8 °C and used for further evaluation. Qualitative analysis and standardization was carried out by developing the RP-HPLC method w.r.t chrysin, caffeic acid, and quercetin. The percent flavonoid and polyphenolic contents of extract were also determined [10,12,13,14].

2.4. RP-HPLC Method Development and Validation

2.4.1. Chromatographic Conditions

A 250 mm × 4.6 mm, 5 µm reverse phase Kromacil C18 column was used for method development. The experiment utilized a UV–visible detector to detect the wavelength of the mobile phase, which was set at 370 nm. The column was kept at a constant temperature of 35 °C, while the mobile phase, consisting of a mixture of methanol and 0.1% orthophosphoric acid in distilled water at a ratio of 65:35, was run for 20 min at a flow rate of 1.0 mL/min.

2.4.2. Preparation of Standard Solution

The individual stock solutions of chrysin, caffeic acid, and quercetin of 100, 40 and 100 µg/mL, respectively, were prepared and stored in a refrigerator at 4 °C until further use. To optimize HPLC separation, aliquots of chrysin and caffeic acid ranging from 10 to 60 µg/mL and aliquots of quercetin ranging from 4 to 24 µg/mL were used.

2.4.3. Sample Preparation

A total of 10 mg of extract was dissolved in 1 mL of mobile phase, and the mixture was then diluted to make the 1000 µg/mL solution.

2.4.4. Method Validation

An analytical method’s performance characteristics are validated in compliance with ICH guidelines in terms of accuracy, linearity, method precision, system precision, robustness, limit of quantitation (LOQ), and limit of detection (LOD) parameters.

2.5. UV Absorbance Spectrum

To find the wavelength of maximum absorption (λ max) of propolis extract, the UV spectrum was recorded in the 200–800 nm range using a JASCO double beam UV–visible spectrophotometer (Japan)against a blank.

2.6. Solid Dispersion

A solid dispersion of propolis extract was formulated with the solvent evaporation method by combining PVP K30 and propolis in a 1:1 ratio and dissolving them in ethanol. The solution was stirred until homogeneous and evaporated to obtain a dry solid. The solid was ground into a fine powder and sieved for uniform particle size distribution. The final product was stored in an airtight container [15].

2.7. Evaluation of Solid Dispersion

2.7.1. DSC

Thermograms of propolis extract and formulation were recorded using a differential scanning calorimetry (DSC 821e) instrument, Mettler-Toledo, Greifensee, Switzerland. The 5 mg samples were heated in a hermetically sealed aluminum pan under nitrogen atmosphere at a heating rate of 10 °C/min. [16].

2.7.2. FTIR Study

FTIR investigations were conducted using the KBR dispersion technique for identification of functional groups present in the propolis extract and formulation. The spectral analysis was performed after recording the spectrum using Shimadzu Model-8400S [17].

2.7.3. Percent Yield

The percent yield of the finished product was determined using weight of solid dispersion/theoretical weight (carrier +EEIP) × 100.

2.7.4. Percent Propolis Extract Content

Percent propolis extract content was determined by measurement of UV absorbance at 370 nm using the following formula: (actual amount of EEIP in solid dispersion/theoretical amount of EEIP in solid dispersion) × 100.

2.7.5. Saturation Solubility

The saturated solubility of propolis extract and solid dispersion was evaluated in both distilled water and 0.1N HCl. The 5 mL glass vials were filled with 3 mL of each solvent and excess propolis extract or solid dispersion. The vials were immersed in a water bath and vigorously shaken at 50 rpm for 48 h at 37 ± 0.5 °C. The final samples were diluted in the same solvent after being filtered through syringe filters with a pore size of 0.22 µm. Using a UV–visible spectrophotometer, the EEIP absorbance was measured at the pre-scanned λ max. The absorbance was correlated into concentration for each solvent using the standard calibration curve of the EEIP [17].

2.8. Formulation and Optimization by DoE

The propolis solid dispersion was precisely weighed; excipients were added, blended, and passed through sieve no. 80. Tablets were compressed with a tablet punching machine (make: Mini press Karnawati, model: Rimek) using a 10 mm punch (

Table 1. Formula for the preparation of immunomodulatory propolis immediate-release tablets as per experimental design (trial batches).

Ingredients (% w/w)	Formulation
	T-1	T-2	T-3	T-4
Propolis	40	40	40	40
Amla Powder	20	20	20	20
Mannitol	q.s	q.s	q.s	q.s
Cross povidone	-	8	-	-
Cross carmellose sodium	-	-	8	-
Sodium starch glycolate	-	-	-	8
Avicel PH 102	8	8	8	8
Magnesium stearate	1	1	1	1
Talc	1	1	1	1

Total weight of tablet is 500 mg.

).

2.9. Full Factorial Design

Experimental runs were conducted using 3² factorial designs to better understand the effect of variables on responses. The independent variables chosen were hardness and SSG concentration, while the dependent variables were drug release in 1 hrand disintegration time. The resulting data were statistically analyzed using analysis of variance (ANOVA) and Design Expert software (DoE version 13). A 3D response surface methodology was used to determine how material and process attributes affected the responses. The designs of the trial runs are presented in

Table 2. Different independent and dependent variables.

Independent Variable	Dependent Variable
X1 = Disintegrant Concentration (%)	Y1 = Drug Released in 1 h
X2 = Hardness (kg/cm2)	Y2 = Disintegration Time

,

Table 3. Coded and actual values of formulations as per 3²factorial design.

Codes Values	Actual Value
	X1	X2
+1	4	3
0	6	5
−1	8	7

and

Table 4. Design matrix as per 3²factorial designs.

Run	Coded Levels		Actual Value
	X1	X2	Disintegrant Conc. (%)	Hardness (kg/cm2)
F1	−1	−1	4	3
F2	−1	0	4	5
F3	−1	+1	4	7
F4	0	−1	6	3
F5	0	0	6	5
F6	0	+1	6	7
F7	+1	−1	8	3
F8	+1	0	8	5
F9	+1	+1	8	7

.

2.10. Characterization

Formulated powder blends were assessed for various compression and flow parameters prior to tablet compression [18,19,20]. Various tablet parameters, including organoleptic properties, friability, hardness, weight variation, thickness, disintegration, and in vitro drug release, were assessed as per methods described by Khar et al. 2013 [21]. Organoleptic properties play a vital role in patient compliance. Important organoleptic characteristics evaluated to make the dosage form more pleasant are color, odor, shape, etc. For the in vitro drug release study, a USP Type II paddle dissolution test apparatus was used with 900 mL of 0.1NHCl of dissolution media at 37 ± 0.5 °C at 50 rpm. The dissolution profile of the developed formulation was compared with the marketed formulation.

2.11. Immunomodulatory Activity

2.11.1. Animals

For the investigation, male and female Swiss albino mice weighing 20–25 g were used. They were adjusted to the 12 h light/dark cycle, 25 ± 2 °C temperature, and 50–60% relative humidity in the lab. Normal pellet meal from Nutrivet Life Sciences, Pune, and water were given to the mice. The experimental procedure was carried out in accordance with guidelines of the committee for control and supervision of experimentation on animals (CPCSEA) when providing care for the animals.

2.11.2. Preparation of Drug and Cyclophosphamide

A homogeneous suspension of propolis tablets was prepared using 1% (w/v) carboxymethyl cellulose (CMC). Levamisole and cyclophosphamide were dissolved in water to form solutions.

2.11.3. Preparation of Sheep RBC (SRBC) Suspension

Blood from sheep was obtained in a 1:1 sterile solution of Alsevere’s. The obtained blood was centrifuged for ten minutes at 2500 rpm in order to separate the plasma.Further, the blood was centrifuged three more times and RBCs were suspended in 0.9% w/v pyrogen-free normal saline and stored.

2.11.4. Acute Oral Toxicity Studies

The Organization for Economic Cooperation and Development (OECD) recommendation and draft guidelines 425 from CPCSEA, the Ministry of Social Justice and Empowerment, Government of India, were followed for acute toxicity studies. Over the course of 14 days, propolis tablets were given orally at a dose of 2000 mg/kg equivalent of propolis, and the effects on mortality and physical and behavioral changes were monitored [22].

2.11.5. Delayed Type Hypersensitivity Test (DTH)

Saline solution was given to group I (n = 6) as a normal control; cyclophosphamide (30 mg/kg, p.o.) was given to group II (n = 6) as a disease control; levamisole (2.5 mg/kg, p.o.) and cyclophosphamide (30 mg/kg, p.o.) were given to group III (n = 6); propolis extract (50 mg/kg, p.o.) and cyclophosphamide (30 mg/kg, p.o.) were given to group IV (n = 6); propolis tablets were given to groups V (n = 6)and VI (n = 6) at a dose of 50 mg/kg, p.o. and 100 mg/kg, p.o., and cyclophosphamide (30 mg/kg, p.o.), respectively. Propolis extract, propolis tablets, and levamisole were administered orally every day for seven days; cyclophosphamide was given orally only once on the fourth day. After 24 h since the final dose of propolis extract, propolis tablet, and levamisole, six mice from each group were given subcutaneous injections of 50 µL of 0.15 M phosphate buffer saline (PBS) (pH 7.2) into the left hind footpad and 50 µL 1 × 10⁸ SRBC into the right hind footpad. Using a digital vernier caliper (Mitutoyo Manufacturing Company), the DTH response, i.e., footpad edema brought on by the hypersensitive reaction, was quantified 24 h later as an increase in footpad thickness. The change in thickness (mm) between the left and right footpads injected with PBS and SRBC, respectively, was considered as a measure of footpad edema reaction. For each group, mean thickness difference values were computed and compared to the vehicle-treated control group [22,23].

2.11.6. Carbon Clearance Test

Group I (n = 6) normal group, Group II (n = 6) disease group, and Group III (n = 6) standard treatment group were allocated. Group IV(n = 6) was given propolis extract (50 mg/kg, p.o.), while Groups V(n = 6)and VI (n = 6) were given propolis tablets at a dose of 50 mg/kg, p.o.,and 100 mg/kg, p.o., respectively. Propolis extract, propolis tablets, and levamisole were given orally for seven days. On the fourth day, cyclophosphamide was given only once (p.o.). Test samples were given for seven days. A 0.1 mL carbon ink suspension was administered to each group via the tail vein on day eight. In a carbon suspension, blood was extracted from each animal by retro-orbital plexuses at 0 and 15 min. Two milliliters of 0.1% sodium carbonate were used to lyse 25 microliters of blood, and the absorbance at 675 nm was measured by spectroscopy to calculate the optical densities [23,24].

2.11.7. Effect on Total Leukocyte Count

Animals were grouped in a manner similar to the DTH response test, and propolis extract, propolis tablets, and levamisole were given orally every day for a duration of 14 days. On the fourth and ninth days, cyclophosphamide was given only once orally (p.o.) using a hemocytometer, and the total leukocyte count was determined [25].

2.12. Statistical Analysis

The mean ± SEM was determined for all observations. GraphPad Prism software Version 8.0.2 was used to analyze the data using Tukey’s multiple comparisons test and one-way ANOVA. The threshold of p < 0.0001 indicated that the difference between the groups was significant.

3. Results

3.1. Total Balsam Content

Total balsam content was determined to be 46% w/w, which denotes the ethanol-soluble fraction of crude propolis.

3.2. Characterization of Propolis Extract

A total extraction yield of 49.47% w/w was obtained. The propolis extract was found to have a total polyphenolic content of 34.82 ± 0.078 mg equivalent of gallic acid/g and flavonoid content of 23.61 ± 0.045 mg equivalent of quercetin/g.

Table 5. Qualitative phytochemical analysis of propolis extract.

Parameters	Test	Present (+)/ Absent (−)
Carbohydrates	Molisch test	+
Proteins	Ninhydrin test Biuret test	+
Flavonoids	H2SO4 test	+
Sterols	Liebermann–Burchard test	+
Alkaloids	Mayer’s test Wagner’s test Hager’s test	+
Tannins	Dil. Potassium permanganate test Lead acetate test	+
Phenols	Bromine water test	+
Saponins	Foam test	+
Anthraquinone	Bontrager’s test	+

presents the findings from qualitative investigations of the EEIP.

3.3. RP-HPLC Method Development and Validation

At wavelength 370 nm, an acceptable response and detection of the markers were obtained. A linear range of 10–60 mg/mL for caffeic acid and chrysin and 4–24 mg/mL for quercetin, with a desired correlation coefficient (n = 3), was observed.

Table 6. Summary of validation parameters.

Parameters	Caffeic Acid	Quercetin	Chrysin
Linearity range (µg/mL)	10–60 µg/mL	4–16 µg/mL	10–60 µg/mL
Regression equation	y = 7778.9x − 3325.1	y = 127,114x − 24268	y = 161,463x – 161192
R2	0.9938	0.9933	0.9917
Slope	7778.9	127,114	161,463
Intercept	3325.1	24,268	161,192
Retention time (min)	2.8 + 0.002	4.7 + 0.01	15.2 + 0.04
Theoretical plates	7403 + 73	7545 + 121	12,681 + 515
LOD (µg/mL)	1.055	0.527	0.843
LOQ (µg/mL)	3.197	1.599	2.555
Precision	0.819888	0.987063	0.661134
Intra-day	1.772298	1.023244	1.193007
Inter-day	1.533429	1.981705	1.678114
Accuracy%	97.12 + 0.20 to 102.20 + 1.5	98.30 + 1.72 to 101.89 + 0.39	99.27 + 0.86 to 102.45 + 0.99
Robustness	≤2	≤2	≤2

represents the linear regression results, average retention time, LOD, and LOQ for each marker. Figure 1 displays a typical representative RP-HPLC chromatogram. For all markers, the percent relative standard deviation (% RSD) for intra-day and inter-day precision was determined to be less than 2. Table 6 illustrates that all three markers had satisfactory recoveries. The suggested method is robust, as evidenced by the robustness result, which indicated that the peak regions remained unchanged (% RSD ≤ 2).

3.4. Standardization of Extracts

It was found that the propolis extract contained 0.910 ± 0.026%, 1.344 ± 0.069%, and 3.749 ± 0.058% of quercetin, caffeic acid, and chrysin, respectively, as shown in Figure 2.

3.5. Formulation and Evaluation of Solid Dispersion

The percent yield of solid dispersion was obtained as 98.35% w/w. The saturation solubility of propolis and propolis solid dispersion was investigated in 0.1 N HCl and distilled water. The saturation solubility of propolis was observed to be 8.74% in 0.1 N HCl and 7.17% in distilled water. The solubility of propolis solid dispersion was observed to be 90.21% in distilled water and 93.22% in 0.1 N HCl. These findings demonstrate the enhanced solubility of propolis extract when formulated as a solid dispersion, suggesting its potential for an improved dissolution pattern.

3.6. DSC

DSC analysis was carried out for the propolis extract, PVP K 30, and solid dispersion in order to determine the molecular state of the extract (Figure 3A–C). In the case of propolis extract, due to various polyphenols and flavonoids, different melting transitions were observed at 48.2 °C, 67.4 °C, and 127.3 °C. The PVP K 30 melting transition was observed at 84.5 °C. Solid dispersion showed melting transition at 84.5 °C. The disappearance of the melting transition of propolis extract indicates the molecular dispersion of propolis extract in the solid dispersion formulation.

3.7. FTIR

Figure 4A–C represent the FTIR spectra of propolis extract (A), PVP K30 (B), solid dispersion. Several peaks at 3333 cm⁻¹, 3485.7 cm⁻¹, 3541.63 cm⁻¹, 3571.52 cm⁻¹ in the propolis extract FT-IR spectrum were attributed to –OH stretching. The C–H stretch is represented by a band that measures 3083.62 cm⁻¹. C=C, C=O, and C–O stretching was identified as the causes of the strong and narrow peaks at 1594.8 cm⁻¹, 1639.2 cm⁻¹, and 1164.7 cm⁻¹, respectively. At 1658 cm⁻¹ in the PVP K30 band, the FTIR spectrum shows the presence of a carbonyl group. The presence of moisture is indicated by the very broad band at 3440 cm⁻¹, which highlights PVP K30′s hygroscopic character. However, FTIR spectra of solid dispersion showed all major peaks of propolis extract with reduced intensity, confirming the dispersion of propolis extract in PVP30.

3.8. Evaluation of Preliminary Batches of Propolis Tablets

Preliminary trials were conducted, and results were obtained as shown in

Table 7. Results of preliminary batches.

Evaluation Parameters	Preliminary Batches
	T-1	T-2	T-3	T-4
Disintegration time (s)	186 + 62	685 + 3	550 + 2	240 + 3
Drug release (1 h) (% w/w)	11.84 + 1.8	51.46 + 1.4	62.84 + 0.8	87.21 + 1.6

. The disintegration times of T-1, T-2, and T-3 were more than 500 s, and the disintegration time of T-4 was 240 s. Based on these findings, SSG was taken into consideration for additional investigation. The hardness of the tablets was optimized, as it affects both the amount of drug released from the tablets and the disintegration time. A 3² factorial design was used in this investigation to examine the effects of the SSG concentration and the hardness of the tablet systematically.

3.9. 3² Full Factorial Design

Two factors were selected for a 3² full factorial design:the amount of super disintegrant (SSG, X1) and the hardness (X2). The responses were assessed using a statistical model that included polynomial and interaction components. The results showed that all nine batches (F1 to F9) show significant variance, indicating that all dependent variables are strongly dependent on the selected independent factors. The results are summarized in

Table 8. Effect of dependent variables.

Batch Code	Variable Levels in Coded Form		Dissolution at 1 h (% w/w)	Disintegration Time (c)
	X1 (%)	X2 (kg/cm2)
F-1	−1	−1	43.16 + 1.2	404 + 3.0
F-2	−1	0	38.50 + 0.7	457 + 1.0
F-3	−1	+1	33.71 + 1.4	482 + 2.0
F-4	0	−1	64.27 + 0.8	286 + 4.0
F-5	0	0	61.42 + 1.7	320 + 4.0
F-6	0	+1	53.47 + 1.3	361 + 3.0
F-7	+1	−1	89.47 + 0.6	224 + 4.0
F-8	+1	0	92.20 + 0.8	246 + 2.0
F-9	+1	+1	78.64 + 1.1	304 + 3.0

n = 3, all values represent mean ± SD.

,

Table 9. Results of pre-compression parameters of propolis immediate-release tablet formulation (F1–F9) powders.

Batches	Angle of Repose	Bulk Density	Tapped Density	Hausner Ratio	Carr’s Index (%)
F1	28.92 + 0.32	0.653 + 0.002	0.740 + 0.02	1.133 + 0.004	13.32 + 0.29
F2	27.52 + 0.41	0.641 + 0.003	0.735 + 0.02	1.146 + 0.003	14.66 + 0.28
F3	23.51 + 0.23	0.654 + 0.004	0.728 + 0.01	1.094 + 0.001	11.31 + 0.24
F4	31.14 + 0.27	0.587 + 0.003	0.685 + 0.03	1.166 + 0.002	16.69 + 0.31
F5	22.87 + 0.32	0.638 + 0.002	0.725 + 0.02	1.136 + 0.002	13.63 + 0.27
F6	24.41 + 0.34	0.661 + 0.002	0.742 + 0.03	1.122 + 0.003	12.25 + 0.23
F7	30.84 + 0.41	0.647 + 0.004	0.749 + 0.03	1.157 + 0.004	15.76 + 0.28
F8	25.61 + 0.23	0.648 + 0.002	0.724 + 0.02	1.117 + 0.002	11.72 + 0.22
F9	27.84 + 0.28	0.589 + 0.003	0.664 + 0.04	1.127 + 0.003	12.73 + 0.31

n = 3, all values represent mean ± SD.

and

Table 10. Post-compression parameters of propolis tablets.

Batches	Thickness (mm)	Hardness (kg/cm2)	Friability (%)	Weight Variation (mg)	Drug Content (%)
F1	5.79+ 0.227	3.2 + 0.1	1.3 + 0.2	504 + 3.4	93.17 + 0.35
F2	5.54 + 0.031	5.3 + 0.2	0.9 + 0.1	510 + 2.4	97.14 + 0.24
F3	5.26 + 0.025	7.4 + 0.1	0.7 + 0.1	497 + 5.7	96.38 + 0.27
F4	5.81 + 0.018	3.1 + 0.1	1.2 + 0.2	507 + 4.1	92.91 + 0.31
F5	5.64 + 0.027	4.9 + 0.3	0.8 + 0.3	511 + 3.8	95.14 + 0.25
F6	5.32 + 0.018	7.2 + 0.2	0.9 + 0.1	495 + 2.7	97.18 + 0.37
F7	5.73 + 0.023	3.2 + 0.1	0.9 + 0.2	512 + 2.6	94.28 + 0.38
F8	5.38 + 0.017	5.4 + 0.1	0.6 + 0.1	506 + 2.2	97.45 + 0.27
F9	5.17 + 0.019	6.8 + 0.3	0.5 + 0.1	513 + 4.6	96.47 + 0.22

n = 3, all values represent mean ± SD.

. After taking into account the coefficient’s magnitude and the mathematical sign (positive or negative), conclusions were drawn using the polynomial equations.

The data below represent the polynomial equation for response variables:

Y1 (DT) = +298.67 − 90.00X1 + 22.17X2 + 0.25X1X2 + 37.00X12 + 3.50X22

Y2 (DR 1 h) = +60.69 + 22.73X1 − 4.68X2

3.10. Optimization

The F8 batch, having a hardness of 5 kg/cm² and 8% SSG as the disintegrating agent, demonstrated desirable disintegration time and drug release properties, selected as optimized batch. The drug release in 30 min was more than 60%, indicating rapid drug release. At the 1 h mark, the drug release percentage was measured to be 92.20 + 0.8%, suggesting a significant drug release within the desired timeframe. This indicates that the tablet was designed as an immediate-release formulation, where the drug is intended to be released quickly after administration.

3.11. Comparison of the Optimized Batch with Marketed Tablets

The optimized propolis immediate-release tablet formulation (F8) was compared with marketed immediate-release superbee propolis tablet formulations for in vitro drug release profile. Results are shown in

Table 11. Comparison of F7 formulation with marketed tablets.

Product	% Drug Release in 1 h (DR 1 h)
Superbee Propolis Tablet Marketed Formulation	96.83 + 1.98
Formulated Immunomodulatory Propolis Tablets	92.20 + 1.72

, and a comparative release profile is given in Figure 5.

3.12. Quantification of Markers Present in Formulation

RP HPLC analysis of the developed formulation revealed that the propolis tablet formulation contains 0.903 ± 0.002 percent caffeic acid, 1.323 ± 0.043 percent quercetin, and 3.592 ± 0.047 percent chrysin.

3.13. In Vivo Immunomodulatory Study

3.13.1. Acute Oral Toxicity Study

The study employed the up-down regulation method to assess acute oral toxicity. It was found that the propolis tablets were safe for the subjects, as no death was seen even at a maximum dose of 2000 mg/kg. Based on the literature reviewed earlier, two doses of the propolis tablets, i.e., 50 mg/kg and 100 mg/kg, were used in the subsequent investigation.

3.13.2. Delayed Type Hypersensitivity (DTH) Response

In the DTH test, groups treated with propolis extract, propolis tablet, and levamisole showed a dose-dependent increase in DTH response compared to the vehicle. This improvement in DTH response indicated the stimulation of the cell-mediated response to SRBC. This may be due to the induction of lymphocytes and accessory cell types that are essential for the DTH response. Another possible reason may be the generation of memory T cells during SRBC sensitization, which becomes activated in response to lymphoblasts. The propolis tablet at a dose of 100 mg/kg exhibited the highest (0.5402 ± 0.033) and statistically significant (p < 0.0001) DTH response compared to the vehicle. The elevated DTH response may be due to the presence of alkaloids, carbohydrates, flavonoids, tannins, and saponins. Results obtained are shown in

Table 12. Effect on delayed hypersensitivity response.

Group	DTH Response
NC	0.4301 + 0.026
DC	0.2833 + 0.028
Std + Cyp	0.6316 + 0.027
PE50	0.4716 + 0.040
Tab50 + Cyp	0.3951 + 0.039
Tab100 + Cyp	0.5402 + 0.033

All values are expressed as mean + SEM, n = 6. Abbreviations: DTH = delayedtype hypersensitivity, NC = normal control, DC = disease control, Std = standard (levamisole), Cyp = cyclophosphamide, PE = propolis extract, Tab50 = propolis tablet containing 50 mg equivalent of propolis, Tab100 = propolis tablet containing 100 mg equivalent of propolis.

and Figure 6.

3.13.3. Carbon Clearance Test

The test is employed to monitor the reticuloendothelial system’s (RES) activity in removing colloidal carbon suspension from the bloodstream. The increase in the carbon clearance index indicates the improvement of non-specific immunity and the phagocytic function of macrophages. Macrophages play a vital role in the body’s defense mechanisms at every stage of both innate and acquired immunity. The phagocytic index measures the rate at which ingested macrophages remove carbon from the blood. The faster removal of carbon particles is linked to increased phagocytic activity. Propolis tablets have been shown to increase phagocytic activity.Animals given 100 mg/kg propolis tablets had a substantial (p <0.01) increase in phagocytic activity (0.0391 ± 0.002) in comparison to the control group. Results obtained are shown in

Table 13. Carbon clearance assay.

Group	Phagocytic Index
NC	0.0318 + 0.003
DC	0.0183 + 0.002
Std	0.0501 + 0.002
PE50	0.0296 + 0.001
Tab50	0.0322 + 0.003
Tab100	0.0391 + 0.002

All values are expressed as mean + SEM, n = 6. Abbreviations: NC = normal control, DC = disease control, Std = standard (levamisole), DTH = delayedtype hypersensitivity, PE = propolis extract, NC = normal control, DC = disease control, Tab50 = propolis tablet containing 50 mg equivalent of propolis, Cyp = cyclophosphamide, Tab100 = propolis tablet containing 100 mg equivalent of propolis.

and Figure 7.

3.13.4. Effect on Total Leucocyte Count

Animals treated with 100 mg/kg propolis tablets had a significantly (p < 0.001) higher total leukocyte count (5.015 + 0.19) than the control group. Results obtained are shown in

Table 14. Effect on total leucocyte count.

Groups	Total Leucocyte Count (×103/mm3)
NC	4.265 + 0.10
DC	2.725 + 0.20
Std + Cyp	5.710 + 0.31
PE50	3.768 + 0.26
Tab50 + Cyp	3.080 + 0.32
Tab100 + Cyp	5.015 + 0.19

All values are expressed as mean + SEM, n = 6. Abbreviations: DTH = delayedtype hypersensitivity, NC = normal control, DC = disease control, Std = standard (levamisole), Cyp = cyclophosphamide, PE = propolis extract, Tab50 = propolis tablet containing 50 mg equivalent of propolis, Tab100 = propolis tablet containing 100 mg equivalent of propolis.

and Figure 8.

4. Discussion

As a potential natural product with a wide range of chemical constituents, propolis possesses various nutraceutical and pharmacological activities. It also has been shown to have immunomodulatory effects, including enhancing the production of specific immune cells and cytokines while inhibiting other immune cells’ activity that may cause inflammation. Despite these beneficial effects, some limitations in terms of solubility, palatability, stickiness, and staining hamper its bioavailability and overall efficacy. The approach in the present study was to overcome these issues and develop a suitable formulation of propolis extract to improve its immunomodulatory activity. Raw propolis, in its composition, is waxy and consists of a portion of debris material. The developed extraction technique removes all the waxes and other debris matter, producing a polyphenol- and flavonoid-enriched propolis extract. UV analysis, FTIR, and melting point results gave the identification background of the propolis and validated a novel analytical method for characterization. Formulated solid dispersion of propolis using PVP K30 as a hydrophilic polymer resulted in enhanced solubility, which in turn improved dissolution and may enhance subsequent absorption of the active ingredients into the bloodstream, thereby improving their therapeutic effectiveness.

Tablet formulation of the solid dispersion was developed with the direct compression method. Formulation technology was optimized based on the material and process attributes. Powdered amla was added to the tablet as an antioxidant. The batch containing SSG showed the fastest disintegration and desired release profile, according to the initial screening investigations. Based on the results, attributes were selected for further studies. In order to understand the effects of the amount of super disintegrant and hardness, a 3² factorial design was employed. It was observed that tablets with a moderate hardness and a high SSG content had more effective drug release. It is expected that reducing the hardness and increasing the SSG concentration will result in a shorter disintegration time. The F8 batch was found to be optimized with an 8% concentration of SSG and hardness of 5 kg/cm². Formulated tablets were evaluated for various tablet evaluation parameters. Results obtained in a desirability range in terms of hardness, thickness, friability, disintegration and in vitro release compared with marketed dosage forms confirm the efficiency of formulation technology.

A few in vitro and in vivo study attempts have been made for the assessment of immune modulatory effects of propolis extract samples from Brazil, Turkey, Bulgaria, etc. [26,27,28,29,30]. Touzani, S et al., 2019 assessed in vitro immune modulatory activity of ethanolic extract propolis at doses of 125 and 250 µg/mL. Study results revealed suppression of the TNF-α and IL-6 production in LPS-stimulated PBMNCs; this increases IL-10 in a dose-dependent manner [31]. Conti, B.J et al., 2016 evaluated in vitro immune modulation of ethanolic extract propolis at doses of 5, 10, 20 and 40 µg/mL. Study results revealed activation of human DCs; induction of the NF-kB signaling pathway and TNF-α, IL-6, and IL-10 production; inhibition of the expression of hsamiR-148a and hsa-miR-148b; and increased miR-155 expression [32]. Girgin, G et al., 2009 reported dose-dependent downregulation by induction of neopterin production and tryptophan degradation and inhibition of TNF-α and IFN-γ levels by ethanolic extract of propolis [33].Overall, study attempts supported the immunomodulatory effects of propolis; however, Indian propolis has not been explored as a drug delivery system for immune modulation. The desirable formulation reflects its efficacy in in vivo evaluation for immune modulatory effects in animal models to obtain scientific evidence.

In the present study, the immune modulatory effects were explored for the developed formulation. The thickness of a footpad was measured to access the reaction of delayedtype hypersensitivity (DTH). The animal showed a marked increase in paw edema after receiving SRBC immunization and an antigenic challenge (24 h) due to the development of antibodies in reaction to the antigen. In animals treated with cyclophosphamide, the immune regulatory system’s suppressor T-cells were found to be damaged, leading to an enhanced DTH response. Lymphocytes and other essential cell types required for the expression of the reaction have been found to be stimulated by the developed formulation. A dose-dependent and significant increase in the DTH response was observed. The phagocytic index is a measure of how quickly ink (or carbon particles) is removed from the bloodstream after being injected intravenously. The ink contains colloidal carbon, which is consumed by macrophages. A propolis tablet showed a significant increase in the phagocytic index by stimulating the reticuloendothelial system.

The first cells that respond to foreign substances invading the body are blood cells. Any substance that affects the immune system would initially cause a shift in the number of white blood cells. Study results for group VI, which received a propolis tablet with a higher concentration of 100 mg/kg, had the highest white blood cell count of 5015 WBC/mm³. These results revealed that the blood cells were stimulated first to mount a strong immune response. The study also showed that administering drugs at a higher concentration (100 mg/kg) produces better immune responses than at lower concentrations (50 mg/kg).

The overall study results also conclude that the immediate-release tablets containing propolis solid dispersion demonstrated significantly improved immunostimulatory effects on both non-specific and specific immune mechanisms. The high content of flavonoids, polyphenols, saponin, steroids, proteins, carbohydrates, and phenols in in the developed propolis tablets might be responsible for their notable increase in immunostimulatory activity.

5. Conclusions

Immunomodulation plays a crucial role in the improvement of the immune response, which further leads to a more robust defense against pathogens. The current attempt was made to overcome the limitations of propolis in its therapeutic applications. The developed extraction approach enriched the polyphenols and flavonoid contents of the propolis, which were essential to improving immune modulatory potential. The developed optimized drug delivery system showed desirable finished product specifications as well as immune modulatory activity in terms of delayed hypersensitivity, phagocytic response, and total leukocyte count. Overall, the present study can be further extended for clinical trials for therapeutic applications.