Phytochemical composition and anti-inflammatory properties of Aegle marmelos, Bryophyllum pinnatum, and Carica papaya

Abstract

The leaves of Aegle marmelos (AML) and Bryophyllum pinnatum (BPL), and unripe fruit peels of Carica papaya (CPFP) are traditionally used, either alone or in combination, to treat inflammatory conditions, without substantial scientific evidence. Therefore, this study aimed to investigate their phytochemical constituents and anti-inflammatory activities. Petroleum ether, chloroform, and methanol extracts were prepared by sequential extraction and subjected to qualitative and quantitative phytochemical analyses. The extracts were screened for in vitro free radical scavenging and anti-inflammatory activities. The extracts showing higher activities were tested at 125, 250, and 500 mg/ kg doses for anti-inflammatory activity using carrageenan-, CFA-, and potassium oxonate-induced inflammation models. The three plants’ methanol extracts showed comparatively higher flavonoids, polyphenols, and glycosaponins contents. The same extracts showed higher antioxidant and anti-inflammatory activities (p < 0.05). The anti-inflammatory activity was dose-dependent, along with restoration of morphology and histology of the inflamed tissues. The current study’s findings provide chemical composition and evidence for these folklore medicinal plants in treating inflammatory conditions like arthritis and gout. These studies suggest bioassay-guided isolation of these ingredients.

Keywords:
Inflammation; Rheumatoid arthritis; Gout; Antioxidant; Medicinal plants.

INTRODUCTION

Inflammatory disorders like rheumatoid arthritis (RA) and gout are affecting millions of people globally equally in terms of quality of life and economic burden (Ashiq et al., 2023). RA is an autoimmune disease in which the host’s immune system erratically attacks the joints, leading to persistent inflammation, stiffness, and tissue degradation. Five out of every thousand individuals are thought to have RA, which, if left untreated, can cause irreparable joint degeneration and disability (Almutairi et al., 2021). The current treatment strategies for RA include NSAIDs, DMARDs, B-cell therapy, TNF-α blockers, IL-1, IL-6 inhibitors, and antiangiogenic medicines. However, these treatments need caution due to gastrointestinal issues, cardiovascular events, hepatorenal toxicity, and increased risk of infections (Lin, Anzaghe, Schülke, 2020; Ashiq et al., 2023). Gout, contrarily, is a metabolic, inflammatory disorder manifested by the deposition of urate crystals in the skin, kidneys, joints, and several other tissues (Ashiq et al., 2021a). Elevation of blood uric acid leads to the formation of urate crystals, kidney stones, and tophi, which collectively culminate in gouty arthritis. Gout increases mortality rates in people with other comorbidities (Ashiq, Ashiq, Shehzadi, 2022). Currently available treatments for gout - xanthine oxidase inhibitors, uricosuric agents, and non-steroidal and steroidal anti-inflammatory agents - cause gastric ulcers, renal failure, liver toxicity, myalgia, and allergic reactions (Ashiq et al., 2021b). This necessitates the discovery of safer, more effective, and alternative therapies for the long-term management of such disorders.

Oxidative stress - an imbalance between reactive oxygen species (ROS) and antioxidants in the body - can damage cells and contribute to several diseases. There is considerable evidence to show the involvement of oxidative stress in the development and progression of inflammatory disorders. In gout, the deposition of monosodium urate crystals triggers the immune response that causes increased production of ROS. Similarly, RA triggers elevated ROS production by activating inflammatory cells, hyperactivity of fibroblasts, and mitochondrial dysfunction. In both RA and gout, increased ROS levels initiate a vicious cycle of stimulating the release of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α), the nuclear factor kappa B (NF-κB), and interleukin-6 (IL-6), which exacerbate inflammation and tissue damage in afflicted joints. Moreover, previous studies show that patients with RA have lower concentrations of antioxidants, including glutathione and superoxide dismutase, and high levels of pro-inflammatory cytokines, indicating that oxidative damage is involved in the development of the disease (Liu et al., 2021; López-Armada, Fernández-Rodríguez, Blanco, 2022). Therefore, lowering oxidative damage and inflammation is a significant treatment modality for gout and arthritis (Lakht-e-Zehra et al., 2015).

Along with several medicines, plant-based products are also used in folklore medicine. Among such products, the leaves of Aegle marmelos (AML) and Bryophyllum pinnatum (BPL), and the unripe fruit peels of Carica papaya (CPFP) are common in many Asian countries. The literature review indicated several studies on the phytochemical composition of these plants (Sadia et al., 2018; Ashiq et al., 2021b). However, the studies of AML and BPL were limited to partial phytochemical analysis. Moreover, no phytochemical report was found on CPFP. Likewise, pharmacological information on their role in lowering oxidative stress and the inflammatory response in diseases like arthritis and gout is poorly understood. This indicates the need to explore these ingredients for phytochemical composition, as well as antioxidant and anti-inflammatory activities.

Plant materials need to be extracted using solvents of different polarities to explore their chemical composition. Various in vitro models are available for the rapid screening of extracts using appropriate standards. The extracts that show promising in vitro activity undergo further evaluation using animal models. Therefore, the study’s theoretical framework includes phytochemical analysis and in vitro screening. In the in vivo anti-inflammatory studies, three models were used to gain insight into the mechanism of action. This study may provide the chemical composition and anti-inflammatory evidence of the selected ingredients.

MATERIAL AND METHODS

Chemicals

The chemicals used in this study included carrageenan, triton-x, petroleum ether and Folin-Ciocalteu (FC) reagent (BDH Chemicals), chloroform, methanol, ethanol, 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide, quercetin, diclofenac sodium, ammonium molybdate, xanthine oxidase (XO), complete Freund’s adjuvant (CFA), potassium oxonate (PO) and xanthine (Sigma-Aldrich), potassium ferricyanide, glucose, bovine serum albumin (BSA), tri-chloroacetic acid, ascorbic acid, sodium carboxymethyl cellulose (CMC-Na), dimethyl sulphoxide (DMSO) and sodium hydroxide (Merck), allopurinol (TCI-Tokyo Chemical Industry) and gallic acid (Sinochem).

Plant material collection and authentication

The AML and BPL were collected in December 2022, while unripe CPFP was obtained in January 2023 from Kasur, Pakistan. The material was authenticated vide vouchers No. 3891, 3892, and 3893, respectively, by Prof. Dr. Zaheer-ud-Khan, Department of Botany, Government College University (GCU), Lahore, Pakistan.

Drying and extraction

The material was washed under tap water to remove extraneous matter. The materials were dried under shade for three weeks and pulverized. Each material (1 kg) was sequentially extracted with petroleum ether, chloroform, and methanol using the Soxhlet apparatus. The extracts were reduced in vacuo using a rotary evaporator at 40°C and dried in a hot-air oven until constant weights. The dried extracts were stored in screw-capped glass vials at 4°C till further analyses.

Phytochemical studies

The extracts were tested for proteins, carbohydrates, lipids, polyphenols, flavonoids, and saponins (Usman, Abdulrahman, Usman, 2009). Then, the extracts were subjected to the estimation of primary metabolites such as total proteins (Lowry, Rosebrough, Farr, 1951), total lipids (Besbes et al., 2004), total carbohydrates (Al-Hooti, Sidhu, Qabazard, 1998), and total polysaccharides (Hussain et al., 2008), and secondary metabolites, including total polyphenols (Slinkard, Singleton, 1977), total flavonoids (Chang et al., 2002), and total glycosaponins (Hussain et al., 2008).

Pharmacological studies

In vitro antioxidant activity

The sample and standard (ascorbic acid) solutions of 1.0 mg/mL concentration were prepared in methanol. Suitable controls for each assay were prepared by replacing the sample/standard solutions with methanol and treating them similarly.

DPPH radical scavenging assay

The DPPH assay was performed using the reported method (Sánchez-Moreno, Larrauri, Saura-Calixto, 1998). The sample/standard solution (2.0 mL), methanol (4.0 mL), and 0.1 mM DPPH (2.0 mL) solution were mixed. The solutions were kept at room temperature for 30 min before measuring their absorbance at 517 nm against methanol. The radical scavenging activity was calculated using Equation 1.

(Equation 1) $$\text{Radical scavenging activity (\%)}=\text{[Ac - As /Ac] × 100}$$

Where A_c = Control absorbance and A_s = Sample/ standard absorbance

Ferric-reducing power assay (FRAP)

The ferric-reducing power assay (FRAP) was performed according to Baba and Malik (2015). The sample/standard solution (1.0 mL) mixed with phosphate buffer (2.5 mL, pH 6.6) and 1% potassium ferricyanide (2.5 mL) was incubated for 20 min at 50°C followed by the addition of 10% trichloroacetic acid (2.5 mL). The mixture was centrifuged for 10 min at 3000 rpm, and the supernatant (2.5 mL) was treated with 0.1% ferric chloride (0.5 mL) and distilled water (2.5 mL) for 10 min. Finally, the absorbance of the solutions was measured at 700 nm against phosphate buffer as a blank. The reducing power was determined using Equation 2.

(Equation 2) $$\text{Reducing power (\%)}=\text{[As - Ac /As] × 100}$$

Hydrogen peroxide (H₂O₂) radical scavenging assay

The hydrogen peroxide (H₂O₂) radical scavenging assay was performed using the previously reported method (Nabi et al., 2023). The sample/standard solution (1.0 mL) was mixed with phosphate buffer (2.4 mL) and H₂O₂ solution (40 mM, 0.6 mL) and incubated at room temperature for 10 min. Then, the absorbance was measured at 230 nm against phosphate buffer used as a blank. The activity was calculated using Equation 1.

Phosphomolybdenum assay

The assay was performed using the reported method (Prieto, Pineda, Aguilar, 1999). The sample/standard solution (0.5 mL) was mixed with molybdate reagent (4.5 mL) that consisted of sodium phosphate solution (28 mM, 1.5 mL), sulfuric acid (0.6 M, 1.5 mL), and ammonium molybdate (4 mM, 1.5 mL). The mixture was incubated for 90 min at 95°C, followed by immediate cooling under tap water. The absorbance of the resulting blue-colored mixture was recorded at 695 nm against methanol. The activity was then calculated using Equation 2.

Determination of median inhibitory concentration (IC₅₀) and antiradical power

Five working solutions (100.0-1000.0 µg/mL) of the extracts exhibiting the highest antioxidant activity were analyzed using the abovementioned assays, and the IC₅₀ values were determined from the linear regression equation of the plot between % antioxidant activity and the concentration (µg/mL). The antiradical power was then calculated using Equation 3.

(Equation 3) $$\text{Antiradical power}=\frac{1}{{\mathrm{IC}}_{50}}$$

In vitro anti-inflammatory activity

The extracts and standard (diclofenac sodium) solutions of 1.0 mg/mL concentration were made in phosphate-buffered saline (PBS, pH 6.4), which was also used as a blank.

Heat-induced protein denaturation assay: The sample/standard solution (2.0 mL) mixed with PBS (2.8 mL, pH 6.4) and 4% egg albumin (0.2 mL) was incubated for 15 min at 37±2°C. The denaturation of albumin was initiated by heating the mixture at 70°C for 5 min. The contents were allowed to cool before recording their absorbance at 660 nm against PBS (Shunmugaperumal, Kaur, 2016). The anti-denaturation activity was calculated using Equation 1. Later, the five working solutions of the extracts exhibiting the highest activity were further evaluated to determine the IC₅₀.

Xanthine oxidase (XO) inhibition assay

The extract or standard (allopurinol) solution of 1.0 mg/mL concentration (prepared in 5% DMSO) was mixed with phosphate buffer (2.9 mL, pH 7.5) and XO solution (0.1 mL). After pre-incubating for 15 min at 25°C, xanthine solution (2.0 mL) was added, and incubation was continued for an additional 30 min. Lastly, 1N HCl (1.0 mL) was added to stop the reaction, and the mixture’s absorbance was recorded at 290 nm against the PBS (Latif et al., 2020). The control, which had 5% DMSO in place of extract/standard, was processed similarly, but HCl was added before the 30 min incubation. The enzyme inhibition activity was calculated using Equation 4.

(Equation 4) $$\text{\% Inhibition of xanthine oxidase}=\text{[1 - (Abs extract / Abs without extract] × 100}$$

The extracts exhibiting the highest activity were further evaluated to determine the IC_50, as mentioned already.

In vivo anti-inflammatory activity

Ethical approval

The study was carried out according to the protocol approved by the Institutional Ethics Review Board of the University of the Punjab, Lahore, Pakistan, vide Ref. No. D/181/FIMS.

Animal housing and grouping

The animals were kept in polypropylene cages in the Animal House, Punjab University College of Pharmacy, University of the Punjab, Lahore, Pakistan, in a 12 h light/ dark cycle room at 22±2°C. They were given free access to food and water during the acclimatization period. However, one day before the experiment, all the animals were fasted and had access to water. Male, Wistar rats (180±30 g) aged 5 weeks were randomly segregated into 12 groups, each having 6 animals to perform following assays.

Carrageenan-induced paw edema assay

The vehicle (normal saline), extracts, and diclofenac sodium (0.1 mL) were given orally to the rats (Table I). After 1 h, the sub-plantar injection of carrageenan (1% w/v) was administered in the posterior right foot of all the rats - except the ones in the untreated control group - to initiate acute inflammation. The thickness of the paw (mm) was measured using a Vernier caliper immediately after the injection and then at regular intervals till 6 hours (Daram et al., 2021).

TABLE I
Grouping and treatment of rats for anti-inflammatory studies of methanol extracts of Aegle marmelos leaf (AML), Bryophyllum pinnatum leaf (BPL) and Carica papaya fruit peel (CPFP) (continues)

Group	Treatment
Untreated control	Normal saline (0.1 mL p.o) in carrageenan-induced paw edema and CFA-induced arthritis; 0.5% Carbo-xymethyl cellulose sodium (0.1 mL p.o) in potassium oxonate-induced hyperuricemia
Disease	Carrageenan (1%, 0.1 mL sub-plantar); CFA (1%, 0.2 mL sub-plantar); Potassium oxonate (250 mg/kg, i.p)
Positive control	Diclofenac sodium (10 mg/kg, p.o) in carrageenan-induced paw edema and CFA-induced arthritis; Allo-purinol (10 mg/kg, p.o) in potassium oxonate-induced hyperuricemia
AML-I	AML extract (125 mg/kg, p.o)
AML-II	AML extract (250 mg/kg, p.o)
AML-III	AML extract (500 mg/kg, p.o)
CPFP-I	CPFP extract (125 mg/kg, p.o)
CPFP-II	CPFP extract (250 mg/kg, p.o)
CPFP-III	CPFP extract (500 mg/kg, p.o)
BPL-I	BPL extract (125 mg/kg, p.o)
BPL-II	BPL extract (250 mg/kg, p.o)
BPL-III	BPL extract (500 mg/kg, p.o)

CFA-induced anti-arthritis assay

All the animals (Table I), except those in the untreated control group, received an injection of CFA (0.2 mL) - heat-killed Mycobacterium tuberculosis (1 mg/mL) - into their left hind paws (Swathi, Jayaram, Sugumar, 2021). The animals in disease, positive control, and extract-treated groups were given vehicle (0.1 mL), diclofenac sodium (10 mg/kg), and extracts (125-500 mg/kg), respectively, 1 h before the injection of CFA. The treatment was continued for 28 days. A digital Plethysmometer was used to measure the paw volume weekly from day 0 to 28. Moreover, the body weight of rats in all groups was tabulated weekly. These observations were used to grade the paw erythema and edema - on a scale of 0 to 4 - using the arthritic scoring system (Zhang et al., 2018). On the 29^th day, the animals were given deep anesthesia and sacrificed to collect blood and tissues for biochemical, radiographical, and histopathological studies.

Biochemical studies

The hematological parameters such as hemoglobin, total blood cell count, and percentage packed cell volume were determined using the Beckman Coulter Blood Analyzer (USA) immediately after collection of the blood samples, while the Wintrobe method was used to determine the erythrocyte sedimentation rate (ESR). Serum was used for determining liver function markers (total bilirubin, direct and indirect bilirubin, total protein, ALT, AST, and ALP), CRP, and RA factor using the Beckman Coulter kits (BCKs, USA).

Radiological studies

The hind limbs of the animals excised on the 29^th day of the study were subjected to radiography to determine the degree of bone erosion, joint space destruction, and joint space narrowing.

Histopathological studies

The paws’ sections (5 μm) were stained using hematoxylin and eosin and observed under a light microscope - integrated with a camera at 10x magnification - to document necrosis, joint spaces, and/ or inflammatory cell infiltration.

Potassium-oxonate-induced gout assay

The activity was performed according to the reported method (Abdulhafiz et al., 2022). Briefly, all the animals except the untreated control group (Table I) received 250 mg/kg potassium oxonate intraperitoneally, 1 h following allopurinol and/or extract dose. This treatment was continued daily for 2 weeks. On the 14^th day, 2 h following the dosing, animals were given anesthesia to collect blood and tissues for biochemical and histopathological studies.

Biochemical studies

The blood was centrifuged at 3000 × g for 15 min at 4°C to separate serum. Then, BCKs (USA) were used to assess uric acid, blood urea nitrogen (BUN), and creatinine.

Histopathological studies

The kidneys were washed with 0.9% cold saline and fixed in formalin. Their 5 μm thick sections were stained and observed under a light microscope.

Statistical analysis

The data were analyzed using GraphPad Prism (6.0) and Microsoft Excel (2010). A linear regression analysis with a correlation coefficient (R²) was used to quantify phytoconstituents and find IC₅₀. The results were calculated as mean ± SD for in vitro studies and mean ± SEM for in vivo studies. Post hoc Tukey’s test was carried out after a one-way ANOVA to compute the biochemical parameters, while a two-way ANOVA with Bonferroni’s post hoc (multiple comparison) test was used to compare in vivo anti-inflammatory activity. A p-value less than 0.05 was considered statistically significant.

RESULTS

Yield of extract

The results of the extraction yield of AML, CPFP, and BPL are given in Table II. Three solvents were used to prepare extracts of varying polarity so that pharmacological activity could be assigned to a particular group of phytochemicals. The yield of methanol extracts was higher than the other two extracts, which indicated that the plant parts contained polar metabolites in significantly higher quantities (p < 0.05). It is also interesting to note that the pattern of extractive yield of the leaves and fruit peels was similar (increased by raising polarity).

TABLE II
Yield of extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL)

Plant material	Extract yield (%±SD)
	Petroleum ether	Chloroform	Methanol
AML	8.03±0.77	5.14±0.41	36.5±0.49
CPFP	13.00±0.38	2.25±0.52	40.20±0.71
BPL	5.20±0.18	3.22±0.64	31.4±0.53

Phytochemical studies

Qualitative phytochemical studies

The results of the qualitative phytochemical analysis of extracts of AML, CPFP, and BPL are given in Table III. The methanol extracts contained proteins, carbohydrates, lipids, polyphenols, flavonoids, and saponins, while the latter three were not found in the petroleum ether extract of the plants. The chloroform extract of AML and the other two plants showed positive tests for flavonoids and saponins, respectively. This varied phytochemical composition of the extracts was due to the varied polarity solvents used for extraction.

TABLE III
Phytochemical constituents in extracts of Aegle marmelos leaf (AML), Carica papaya unripe fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL)

Phytochemical tests	Petroleum ether extract			Chloroform extract			Methanol extract
	AML	CPFP	BPL	AML	CPFP	BPL	AML	CPFP	BPL
Proteins	+	+	+	+	+	+	+	+	+
Carbohydrates	+	+	+	+	+	+	+	+	+
Lipids	+	+	+	+	+	+	+	+	+
Polyphenols	˗	˗	˗	˗	˗	˗	+	+	+
Flavonoids	˗	˗	˗	+	˗	˗	+	+	+
Saponins	˗	˗	˗	˗	+	+	+	+	+

Quantitative phytochemical studies

Primary metabolites

The amounts of the primary metabolites in the sequential extracts are given in Table IV. The BSA standard curves with linear regression equation y = 0.0066x + 0.0212; R² = 0.9941, and glucose calibration curve with the linear regression equation y = 0.0006x + 0.0003; R² = 0.9891 were used to estimate total protein and total polysaccharides, respectively. Moreover, carbohydrates were calculated by subtracting total proteins and lipids from the total dry weight. As indicated earlier in the qualitative tests, the methanol extract of the three plants contained appreciable amounts of protein, carbohydrate, and polysaccharides compared to the petroleum ether and chloroform extracts. On the other hand, petroleum ether extracts had higher lipid content because of the defatting nature of the solvent.

TABLE IV
Primary and secondary metabolites in extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL) (n=3; mg/g±SD)

Phytochemical class	Petroleum ether extract			Chloroform extract			Methanol extract
	AML	CPFP	BPL	AML	CPFP	BPL	AML	CPFP	BPL
Total proteins	106.13±0.70	114.50±0.12	47.91±0.35	155.42±0.21	167.63±0.26	170.84±0.78	293.32±0.4	269.71±0.58	339.01±0.57
Total lipids	71.28±0.88	138.72±0.55	97.60±0.64	43.33±0.67	77.62±0.82	56.70±0.75	67.84±0.43	114.33±0.65	86.92±0.69
Total carbohydrates	41.33±0.53	77.39±0.49	54.52±0.58	118.93±0.41	94.81±0.68	132.92±0.73	492.73±0.75	576.00±0.59	524.11±0.67
Total polysaccharides	10.10±0.15	10.30±0.11	56.67±0.21	220.00±0.23	87.78±0.82	390.00±0.03	490.20±0.24	600.20±0. 61	546.67±0.23
Total polyphenol	5.33±0.18	24.02±2.94	15.25±6.3	36.38±1.40	45.33±3.25	61.43±3.68	101.51±3.58	98.04±2.64	112.20±3.05
Total glycosaponins	2.33±2.86	2.53±3.03	2.14±2.40	8.57±3.30	4.34±3.29	5.80±3.45	42.97±5.08	33.55±7.96	39.12±5.51
Total flavonoids	9.33±2.28	9.92±3.51	7.79±3.18	29.57±2.02	27.72±1.61	32.18±4.2	106.20±2.55	96.67±2.15	99.32±2.55

Secondary metabolites

The results of the quantitative tests for secondary metabolites are summarized in Table IV. To calculate the total polyphenol content, the gallic acid calibration curve with regression equation y = 0.0060x + 0.0355; R² = 0.9930 was used, while the flavonoid content was determined from the quercetin calibration curve with the regression equation y = 0.0051x + 0.0081; R² = 0.9988. The polyphenols, flavonoids, and glycosaponins were present in higher amounts in the methanol extracts than in petroleum ether and chloroform extracts of the leaves and peels. These results were not consistent with our observations in qualitative screening. This could be due to the high sensitivity of the instrumental method for determining the metabolites.

Pharmacological studies

In vitro antioxidant activity

The antioxidant activity of the petroleum ether, chloroform, and methanol extracts of AML, CPFP, and BPL in four in vitro assays is given in Table V. The highest activity was exhibited by methanol extracts, followed by chloroform and petroleum ether extracts. This was due to the higher polyphenol and flavonoid content in the AML, CPFP, and BPL methanol extracts. The methanol extracts also showed concentration-dependent antioxidant effects in all the assays. The IC_50s of the extracts are given in Table VI.

TABLE V
In vitro antioxidant and anti-inflammatory activities of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP, and Bryophyllum pinnatum leaf (BPL) (n=3; % activity±SD) (continues)

Extract	Plant	DPPH assay	FRAP assay	Phosphomolybdenum assay	H2O2 assay	Anti-denaturation assay	Xanthine oxidase inhibitory assay
Petroleum ether	AML	8.24 ± 2.40	27.32 ± 0.93	29.98 ± 0.72	31.38 ± 0.48	15.46 ± 0.33	10.10 ± 0.44
	CPFP	9.04 ± 1.60	28.64 ± 3.16	10.87 ± 2.23	49.64 ± 1.04	7.08± 0.17	10.40 ± 0.67
	BPL	7.79 ± 1.77	31.49 ± 6.82	16.80 ± 0.69	50.12 ± 0.53	8.45 ± 0.19	20.80 ± 0.36
Chloroform	AML	6.24 ± 2.35	43.66 ± 3.29	35.41 ± 1.34	45.82 ± 0.36	23.09 ± 0.34	3.86 ± 0.16
	CPFP	9.15 ± 1.38	37.56 ± 1.19	30.78 ± 0.89	37.83 ± 0.83	24.81 ± 0.30	26.48± 0.58
	BPL	10.04 ± 2.13	41.91 ± 6.09	38.83 ± 1.08	45.94 ± 0.81	25.64 ± 0.31	35.51 ± 0.58
Methanol	AML	74.26 ± 2.76	71.69 ± 4.58	78.07 ± 0.87	73.99 ± 0.77	78.73 ± 0.11	70.12 ± 0.02
	CPFP	67.38 ± 3.49	74.89 ± 2.70	75.96 ± 0.48	70.88 ± 0.74	72.75 ± 0.26	73.52 ± 0.77
	BPL	75.44 ± 1.75	77.84 ± 3.06	76.16 ± 0.99	72.43 ± 1.23	75.05 ± 0.10	76.92 ± 0.26
Standards	Vitamin C	78.43 ± 1.81	83.29 ± 3.27	81.69 ± 0.53	77.21 ± 0.85
	Diclofenac sodium					84.64 ± 0.14
	Allopurinol						80.11 ± 0.23

TABLE VI
Median inhibitory concentration (IC₅₀) and antiradical and protein/enzyme inhibitory power of methanol extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL) (n=3; activity±SD)

Extract	DPPH assay		FRAP assay		Phosphomolybdenum assay		H2O2 assay		Protein denaturation assay		Xanthine oxidase inhibitory assay
	IC50 (µg/ mL)	Antiradi-cal power (mL/µg)	IC50 (µg/mL)	Antiradi-cal power (mL/µg)	IC50 (µg/ mL)	Antiradical power (mL/ µg)	IC50 (µg/ mL)	Antiradi-cal power (mL/µg)	IC50 (µg/ mL)	Inhibitory power (mL/µg)	IC50 (µg/ mL)	Inhibitory power (mL/µg)
AML	659.73± 0.16	0.0015 ± 0.57	702.26± 0.22	0.0014 ± 0.43	633.83 ± 0.18	0.0016± 0.71	669.19 ± 0.37	0.0015± 0.41	399.00 ± 0.53	0.0025 ± 0.61	633.10 ± 0.61	0.0016 ± 0.56
CPFP	696.70 ± 0.43	0.0014 ± 0.34	646.40 ± 0.56	0.0015 ± 0.58	697.88 ± 0.13	0.0014 ± 0.49	664.53 ± 0.21	0.0015 ± 0.61	459.94 ± 0.39	0.0022± 0.73	653.95 ± 0.34	0.0015 ± 0.43
BPL	627.42 ± 0.81	0.0016 ± 0.27	600.17 ± 0.31	0.0017 ± 0.65	608.40 ± 0.26	0.0016 ± 0.18	588.49 ± 0.42	0.0017 ± 0.55	447.70 ± 0.48	0.0022 ± 0.19	625.71 ± 0.53	0.0016 ± 0.22

In vitro anti-inflammatory activity

Heat-induced protein denaturation assay: The protection of heat-induced protein denaturation offered by the sequential extracts of AML, CPFP, and BPL is given in Table V. All the extracts inhibited the heat-induced denaturation of albumin in a concentration-dependent manner. The least active extracts were petroleum ether (7.08±0.17% to 15.46±0.33%), while chloroform extracts showed moderate inhibition (23.09±0.34% to 25.64±0.31%). On the other hand, the methanol extracts of AML, CPFP, and BPL significantly suppressed the protein denaturation, and their effects were comparable to diclofenac sodium. Moreover, the anti-denaturation effects of the methanol extracts were concentration-dependent, and IC_50s are given in Table VI.

Xanthine oxidase (XO) inhibition assay

The inhibitory effects of the extracts of AML, CPFP, and BPL for xanthine oxidase are summarized in Table V. Consistent with the previous observations, the petroleum ether extracts were the least active (10.10±0.44% to 20.80±0.36%). The chloroform extract of AML offered negligible inhibition (3.86±0.16%) compared to the CPFP and BPL, which showed 26.48±0.58% and 35.51±0.58% activity, respectively. Moreover, the methanol extracts of the plants were the most active. These extracts inhibited XO in a concentration-dependent manner, and IC_50s are given in Table VI.

Effect on carrageenan-induced paw edema

The effects of the methanol extracts of AML, CPFP, and BPL on carrageenan-induced paw edema are shown in Figure 1. A significant increase in paw thickness was observed after 1 h of carrageenan injection, which continued to rise till 6 h. The extracts effectively reduced the inflamed paw’s diameter in a dose-dependent manner. After treatment with 500 mg/kg doses of the methanol extracts, a significant decrease in paw thickness was observed from 4 h (p < 0.01), and it continued to decrease till 6 h (p < 0.001). These results were comparable to diclofenac sodium, which also showed a considerable reduction of inflammation at the 4 h (p < 0.01) and continued till the 6 h (p < 0.001). Contrary to this, low doses (125 mg/kg and 250 mg/kg) of the extracts did not bring any significant reduction in the inflamed paw size till 4h after which the paw diameter was reduced significantly at 6 h (p < 0.01).

FIGURE 1
Reduction in paw thickness (mm) over time (hours) treated with methanol extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL) following carrageenan-induced edema (n=6).

CFA-induced arthritis activity

Effect on paw volume

The effects of methanol extracts of AML, CPFP, and BPL on paw volume (mL) following CFA injection are presented in Figure 2. A significant increase in paw edema was observed on the 7^th day after CFA injection, which continued to persist till the 28^th day. The methanol extracts of the plants reduced the paw volume in a dose-dependent manner. After treatment with 500 mg/kg of the methanol extracts of AML and CPFP, the paw volume significantly decreased from the 14^th day (p < 0.05) and continued declining till the 28^th day (p < 0.001). The anti-inflammatory effects of methanol extract of BPL (500 mg/kg) were observed from the 7^th day (p < 0.05) to the end of the study (p < 0.001). In comparison to the above, at doses of 250 mg/kg, methanol extracts of BPL and CPFP brought a significant reduction in paw volume from the 14^th day (p < 0.05) to the 28^th day (p < 0.01) but AML started showing the anti-inflammatory response from the 21^st day to the 28^th day (p < 0.01). The results of the extracts were comparable to the diclofenac sodium. At low doses (125 mg/kg), methanol extracts showed a delayed response and a substantial reduction in paw volume was observed from the 21^st day till the 28^th day (p < 0.05).

FIGURE 2
Rat paw volume (mL) over the time (days) treated with methanol extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL) following CFA-induced arthritis (n=6).

Effect on body weight

The effects of methanol extracts of AML, CPFP, and BPL on the body weight of rats suffering from CFA-induced arthritis are presented in Figure 3. The body weight of the CFA-treated group started reducing from the 7^th day (p < 0.05) and continuously dropped till the 28^th day (p < 0.001). The treatment with methanol extracts effectively improved the body weight in a dose-dependent manner. The improvement in body weight of rats was observed right from the 7^th day till the end of the study (p < 0.05). The results of methanol extracts were comparable to the diclofenac sodium-treated group.

FIGURE 3
Rat body weight (grams) over the time (days) treated with methanol extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL) following CFA-induced arthritis (n=6).

Effect on arthritic score

The effects of AML, CPFP, and BPL methanol extracts on arthritic scores following CFA injection are presented in Figure 4. The arthritic score was significantly high after injecting CFA, which demonstrates a progressive arthritic state. The methanol extracts improved the arthritic score in a dose-dependent manner. After treatment with 500 mg/kg doses of the methanol extracts, a significant reduction in arthritic score was observed from the 14^th day (p < 0.01). The results were comparable to the diclofenac sodium. At doses of 250 mg/kg, methanol extracts of AML, CPFP, and BPL didn’t show any considerable results till the 21^st day. Moreover, doses of 125 mg/kg of methanol extracts did not bring a significant reduction in the arthritic score.

FIGURE 4
Rat arthritic score over the time (days) treated with methanol extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL) following CFA-induced arthritis (n=6).

Effects on hematological and biochemical parameters

The effects of methanol extracts of AML, CPFP, and BPL on hematological and biochemical parameters are summarized in Tables VII-IX. In the CFA-treated group, WBC count, ESR, and platelets were significantly increased (p < 0.001), while RBC count and Hb level were significantly decreased (p < 0.001) compared to the untreated group. The methanol extracts of AML, CPFP, and BPL significantly improved the hematological and biochemical parameters in dose-dependent manners (Table VII). However, the most prominent effect was observed at doses of 500 mg/ kg of the methanol extracts. The findings of LFTs are presented in Table VIII, and the results of RA factor and CRP are given in Table IX. In the CFA-treated group, the levels of bilirubin, ALT, AST, ALP, CRP, and RA factors were remarkably increased (p < 0.001), while a significant decline in protein levels was also noticed (p < 0.001). The methanol extracts at all administered doses restored normal LFTs, RA factors, and CRP levels. However, more pronounced effects were seen at 500 mg/kg doses of the extracts. These results were comparable to the diclofenac sodium-treated group.

TABLE VII
Hematological parameters of rats treated with methanol extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL) following CFA-induced arthritis (n=6)

Group	WBC/TLC (x10^3/uL)	RBC’s (x10^6/uL)	Hb (g/dL)	ESR (mm/ 1st. hour)	HCT (%)	MCV (fl)	MCH (pg)	MCHC (g/ dL)	MPV (fl)	Platelets (x10^3/uL
Untreated control	11.41± 0.66	9.70 ± 0.44	15.72 ± 0.67	1.17 ± 0.17	32.10 ± 0.95	47.36 ± 0.63	20.65± 0.70	30.49± 0.69	9.88± 0.33	1183 ± 0.43
Disease	60.79 ± 0.26 a***	3.49 ± 0.17a***	8.19 ± 0.51a***	4.69 ± 0.15a***	16.10 ± 0.38a***	28.07 ± 0.62a***	14.61 ± 0.58a***	24.98 ± 0.72a***	3.12 ± 0.16a***	2891 ± 0.65a***
Positive control	14.65 ± 0.62b***	7.80 ± 0.15b***	15.08 ± 0.79b***	1.17 ± 0.17b***	29.72 ± 0.21b***	47.16 ± 0.70b***	28.43 ± 0.13b***	32.24 ± 0.73b***	9.56 ± 0.29b***	1254 ± 0.31b***
AML 125 mg/kg	58.27 ± 0.21b*	3.49 ± 0.22ns	11.39 ± 0.16b**	3.83± 0.13ns	24.53 ± 0.45b***	31.74 ± 0.35b**	16.80 ± 0.53ns	32.06 ± 0.55 b***	10.43 ± 0.13b***	2220 ± 0.72b**
AML 250 mg/kg	35.75 ± 0.31b***	7.87 ± 0.31b***	14.88 ± 0.29b***	2.57 ± 0.21ns	43.91 ± 0.38b***	42.67 ± 0.62b***	25.31 ± 0.39b***	32.51 ± 0.10b***	10.98 ± 0.29b***	875 ± 0.34b***
AML 500 mg/kg	13.41 ± 0.33b***	7.91 ± 0.34 b***	14.25 ± 0.33b***	1.54 ± 0.12b***	40.73 ± 0.33b***	54.04 ± 0.29b***	17.17 ± 0.31b*	32.93 ± 0.36 b***	8.78 ± 0.31 b***	860 ± 0.20b***
CPFP 125 mg/kg	55.24 ± 0.18b*	6.70 ± 0.24b**	13.31 ± 0.16b***	6.63 ± 0.13ns	35.53 ± 0.22b***	45.29 ± 0.27b***	15.89 ± 0.11ns	28.21 ± 0.17b***	7.61 ±s 0.08 b***	2498 ± 0.51b**
CPFP (250 mg/kg)	32.39 ± 0.16b**	6.59 ± 0.30 b**	11.86 ± 0.32b***	2.75 ± 0.14ns	28.33 ± 0.42b***	45.46 ± 0.36b***	18.72 ± 0.29b**	31.75 ± 0.28b***	7.88 ± 0.32b***	2145 ± 0.43b**
CPFP 500 mg/kg	15.52 ± 0.52b***	7.02 ± 0.77b***	13.63 ± 0.69 b***	1.39 ± 0.25b***	61.65 ± 1.88b***	45.44 ± 0.45b***	25.70 ± 0.32b***	36.10 ± 0.85b***	10.45 ± 0.58b***	1180 ± 0.21b***
BPL 125 mg/kg	45.19 ± 1.05b**	5.54 ± 0.65ns	9.49 ± 0.67ns	4.43 ± 0.29ns	17.46 ± 0.57ns	44.95 ± 0.71b***	17.35 ± 0.46b**	34.04 ± 0.67b***	7.50 ± 0.67b***	2107 ± 0.31b**
BPL 250 mg/kg	19.11 ± 0.66b***	5.73 ± 0.51b*	12.36 ± 0.56b***	3.50 ± 0.22ns	17.78 ± 0.50ns	43.06 ± 1.19b***	17.53 ± 0.39b**	33.92 1.30b***	7.54 ± 0.61b***	1450 ± 0.61b***
BPL 500 mg/kg	17.44 ± 0.84b***	6.78 ± 0.35 b***	13.74 ± 0.73b***	1.80 ± 0.17 b**	16.65 ± 0.90ns	42.52 ± 1.27b***	17.43 0.71b**	28.85 1.64b***	7.64 0.34b***	1280 ± 0.53b***

• mean ± SEM;
• ^a
  (disease and untreated control groups comparison);
• ^b
  (comparison of extract-treated groups (AML, CPFP, and BPL) and positive control group (diclofenac sodium) with the disease group);
• ^***
  (p < 0.001);
• ^**
  (p < 0.01);
• ^*
  (p < 0.05);
• ^ns
  (non-significant)

TABLE VIII
Liver function tests of rats treated with methanol extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (PBL) following CFA-induced arthritis (n=6)

Group	Bilirubin total (mg/ dL)	Bilirubin direct (mg/ dL)	Bilirubin indirect (mg/dL)	ALT (U/L)	AST (U/L)	ALP (U/L)	Total protein (g/ dL)	Albumin (g/dL)	Globulin (g/dL)
Untreated control	0.30 ± 0.03	0.17 ± 0.01	0.12 ± 0.01	52.00 ± 0.55	66.00 ± 0.58	125.00 ± 0.54	6.85 ± 0.18	3.61 ± 0.27	3.23 ± 0.29
Disease	2.74 ± 0.12 a***	1.32 ± 0.04a***	1.42 ± 0.09a***	77.00 ± 1.73a***	141.00 ± 1.14 a***	206.33 ± 0.33 a***	2.34 ± 0.13a***	1.14 ± 0.03a***	1.25 ± 0.10a***
Positive control	0.57 ± 0.04b***	0.29 ± 0.43b***	0.28 ± 0.13b***	54.00 ± 0.58b***	85.00 ± 0.58 b***	131.00 ± 0.58b***	5.76 ± 0.01b***	2.90 ± 0.01 b***	2.31 ± 0.03 b***
AML 125 mg/kg	2.63 ± 0.10ns	1.30 ± 0.10ns	1.33 ± 0.04ns	77.00 ± 0.58ns	104.67 ± 0.33b***	145.00 ± 0.58b***	2.24 ± 0.34ns	1.90 ± 0.31b*	1.35 ± 0.10ns
AML 250 mg/kg	1.65 ± 0.19b***	0.64 ± 0.33b**	1.01 ± 0.31b*	55.67 ± 1.20b***	84.33 ± 1.45b***	129.33 ± 1.20b***	4.50 ± 0.10b***	3.40 ± 0.32b***	3.03 ± 0.48 b***
AML 500 mg/kg	0.70 ± 0.02b***	0.32 ± 0.03b***	0.37 ± 0.01b***	47.33 ± 0.88b***	81.33± 1.45b***	117.67 ± 1.45b***	6.30 ± 0.16b***	2.47± 0.03 b***	3.83± 0.12 b***
CPFP 125 mg/kg	1.63 ± 0.01b***	0.64 ± 0.34b**	0.99 ± 0.33b**	55.00± 1.73b***	86.33 ± 1.20b***	143.33 ± 2.02b***	5.35± 0.02b***	3.13 ± 0.13b***	2.23 ± 0.11b***
CPFP 250 mg/kg	0.65 ± 0.02b***	0.32 ± 0.08b***	0.33 ± 0.07b***	49.00 ± 1.16b***	82.00 ± 1.53b***	135.33 ± 0.67b***	5.24± 0.04b***	3.07 ± 0.03b***	2.17 ± 0.07 b***
CPFP 500 mg/kg	0.45 ± 0.05b***	0.23 ± 0.01b***	0.22 ± 0.01b***	47.00 ± 1.16b**	78.67± 1.45b***	116.33 ± 1.20b***	5.33 ± 0.07b***	2.90 ± 0.10b***	2.43± 0.17b***
BPL 125 mg/kg	2.75 ± 0.02ns	1.41 ± 0.02ns	1.34 ± 0.10ns	65.00 ± 1.00b**	114.00 ± 2.08b***	184.33 ± 1.76b***	2.23 ± 0.09ns	1.20 ± 0.16ns	1.03 ± 0.20ns
BPL 250 mg/kg	1.53 ± 0.04b***	0.90 ± 0.34b*	0.63 ± 0.34b***	62.67 ± 0.68 b***	87.33 ± 1.88b***	113.00 ± 1.73b***	3.28 ± 0.02b**	1.94 ± 0.21b**	1.34 ±0.23b*
BPL 500 mg/kg	0.65 ± 0.02b***	0.29 ± 0.01b***	0.36 ± 0.02b***	46.67 ± 1.20b***	84.00 ± 2.31b***	122.67 ± 1.76b***	5.61 ± 0.39b***	3.11 ± 0.17b***	2.51 ± 0.26b***

• mean ± SEM;
• ^a
  (disease and untreated control groups comparison);
• ^b
  (comparison of extract-treated groups (AML, CPFP, and BPL) and positive control group (diclofenac sodium) with the disease group);
• ^***
  (p < 0.001);
• ^**
  (p < 0.01);
• ^*
  (p < 0.05);
• ^ns
  (non-significant)

TABLE IX
C-reactive protein (CRP) and rheumatoid arthritis (RA) factor of rats treated with methanol extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL) following CFA-induced arthritis (n=6)

Group	CRP (mg/L)	RA factor (u/mL)
Untreated control	7.51 ± 0.31	5.35 ± 0.25
Disease	28.31 ± 0.40a***	26.63 ± 0.43a***
Positive control	8.24 ± 0.21b***	8.73 ± 0.90b***
AML 125 mg/kg	17.31 ± 0.34b**	19.65 ± 0.36b**
AML 250 mg/kg	15.75 ± 0.48 b***	12.37 ± 0.53b***
AML 500 mg/kg	5.17 ± 0.27b***	11.88 ± 0.35b***
CPFP 125 mg/kg	18.31 ± 0.21b**	14.23 ± 0.19b***
CPFP 250 mg/kg	14.97 ± 0.27b***	12.85 ± 0.20b***
CPFP 500 mg/kg	8.65 ± 0.29b***	12.24 ± 1.16b***
BPL 125 mg/kg	15.54 ± 0.34b***	18.23 ± 0.19b**
BPL 250 mg/kg	12.60 ± 0.26b***	10.71 ± 0.26b***
BPL 500 mg/kg	11.01 ± 0.28b***	8.49 ± 0.29b***

• mean ± SEM;
• ^a
  (disease and untreated control groups comparison);
• ^b
  (comparison of extract-treated groups (AML, CPFP, and BPL) and positive control group (diclofenac sodium) with the disease group);
• ^***
  (p < 0.001);
• ^**
  (p < 0.01);
• ^*
  (p < 0.05);
• ^ns
  (non-significant)

Radiological examination

Rats injected with CFA presented a reduction in joint spaces, edema, and bone degradation, whereas control rats displayed normal morphology (Figure 5). These impairments were either insignificant or absent in the positive control and methanol extracts treated groups, particularly at 500 mg/kg doses.

FIGURE 5
Radiological findings of rat’s paw treated with methanol extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL) following CFA-induced arthritis.

Histopathological studies of rats’ paw

The normal morphology of rats in the untreated control group is shown in Figure 6. Rats injected with CFA showed cartilage destruction, vascular proliferation, benign bony trabeculae, pannus formation, and infiltration by severe chronic inflammatory cells. At lower doses (125 mg/kg and 250 mg/kg) of methanol extracts, histopathological features indicated mild to moderate arthritis. However, methanol extracts at doses of 500 mg/kg showed nearly normal morphology with fewer inflammatory cells, well-preserved joint spaces, reduced pannus formation, and minimum synovial hyperplasia, and these findings were comparable to the diclofenac sodium-treated group.

FIGURE 6
Histopathological findings of rat’s paw treated with methanol extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL) following CFA-induced arthritis.

PO-induced hyperuricemia activity

Effects on RFTs and serum uric acid levels

In the disease group, urea, BUN, creatinine, and uric acid levels were significantly increased (p < 0.001) compared to the untreated control group, as given in Table X. The methanol extracts at all administered doses restored urea, BUN, creatinine, and uric acid levels.

TABLE X
Renal function tests and serum uric acid of rats treated with methanol extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL) following potassium oxonate-induced hyperuricemia (n=6)

Group	Urea (mg/dL)	BUN (mg/dL)	Creatinine (µmol/L)	Uric acid mg/dl
Untreated control	22.89 ± 0.10	8.52 ± 0.29	31.00 ± 0.58	2.92 ± 0.10
Disease	27.16 ± 0.22a***	16.08 ± 0.20a***	73.67 ± 1.20a***	5.89 ± 0.12a***
Positive control	22.59 ± 0.26b***	12.08 ± 0.17b***	36.00 ± 0.58b***	2.96 ± 0.13b***
AML 125 mg/kg	24.85 ± 0.14b*	14.17 ± 0.20ns	49.33 ± 0.68b***	5.38 ± 0.12ns
AML 250 mg/kg	18.42 ± 0.22b***	12.34 ± 0.23b**	33.00 ± 1.16b***	4.66 ± 0.09b**
AML 500 mg/kg	18.69 ± 0.22b***	9.15 ± 0.09b***	17.83 ± 0.88b***	3.07± 0.08b***
CPFP 125 mg/kg	28.83 ± 0.55ns	14.72 ± 0.23ns	65.33 ± 0.60b***	5.20 ± 0.11b*
CPFP 250 mg/kg	22.88 ± 0.25b***	11.53 ± 0.23b***	60.00 ± 0.29b***	3.97± 0.06b***
CPFP 500 mg/kg	18.32 ± 0.18b***	9.83 ± 0.27b***	57.00 ± 1.16b***	2.34± 0.15b***
BPL 125 mg/kg	22.39 ± 0.33b***	12.64 ± 0.49b***	55.00 ± 1.732 b***	4.98 ± 0.12b**
BPL 250 mg/kg	22.68± 0.35b***	11.52 ± 0.33b***	45.00 ± 1.16b***	3.41± 0.20b***
BPL 500 mg/kg	18.97 ± 0.21b***	9.52 ± 0.27b***	37.00 ± 1.14b***	2.64 ± 0.16b***

• mean ± SEM;
• ^a
  (disease and untreated control groups comparison);
• ^b
  (comparison of extract-treated groups (AML, CPFP, and BPL) and positive control group (allopurinol) with the disease group);
• ^***
  (p < 0.001);
• ^**
  (p < 0.01);
• ^*
  (p < 0.05);
• ^ns
  (non-significant)

Histopathological studies of rats’ kidney

The normal morphology of the rat’s kidney in the untreated control group is shown in Figure 7. PO-treated rats presented several histopathological changes consistent with acute interstitial disease and glomeruli atrophy. The renal section of PO-treated rats revealed that the interstitium was infiltrated by chronic inflammatory cells, sclerosis, congested blood vessels, and bleeding with degeneration. Moreover, renal parenchyma showed glomeruli atrophy with few dilated renal tubules. The methanol extracts’ lower doses (125 mg/kg and 250 mg/kg) reduced severity and showed moderate improvement in the kidneys’ tissues. On the other hand, doses of 500 mg/kg of the methanol extracts restored normal morphology with reduced inflammatory cells, preserved renal parenchyma, and no glomeruli atrophy with intact renal tubules.

FIGURE 7
Histopathological findings of rat’s kidney treated with methanol extracts of Aegle marmelos leaf (AML), Carica papaya fruit peel (CPFP), and Bryophyllum pinnatum leaf (BPL) following potassium oxonate-induced hyperuricemia.

DISCUSSION

The present study provides a comprehensive phytochemical analysis, antioxidant, and anti-inflammatory properties of the leaves of A. marmelos and B. pinnatum and unripe fruit peels of C. papaya. Sequential extraction was employed to prepare extracts using solvents in ascending polarity. This type of extraction provides the separation of phytochemicals based on polarity. The plant materials and even different parts of plants vary in the nature of compounds. The solvents also possess different extraction capabilities. In the present study, methanol extract yield was higher. The higher extraction yield was due to methanol’s ability to dissolve various phytochemicals due to its miscibility with both polar and moderately non-polar compounds. This is why it contained higher amounts of proteins, carbohydrates, polysaccharides, flavonoids, polyphenols, and glycosaponins than petroleum ether and chloroform extracts. High contents of flavonoids, glycosaponins, and polyphenols impart medical benefits. Both polyphenols and flavonoids have been extensively reported for antioxidant, anti-inflammatory, antidiabetic, cardioprotective, and anti-cancer effects (Di Lorenzo, Colombo, Biella, 2021; Al-Khayri et al., 2022). Moreover, glycosaponins (due to their unique chemical characteristics: a variety of glycone and aglycone) contribute to the anti-inflammatory, anti-tumor, and immune-boosting properties (Jolly, Hour, Lee, 2024). The results of the phytochemical analyses suggested that these plants can scavenge free radicals, suppress inflammation, and inhibit xanthine oxidase activity.

Previous studies indicated that flavonoids, polyphenols, and glycosaponins regulate inflammatory processes by suppressing NF-κB activation and decreasing pro-inflammatory cytokine gene expression (TNF-α, IL-6, IL-1β). These compounds also have antioxidant properties by scavenging free radicals and increasing endogenous enzymes such as SOD and CAT. Moreover, polyphenols and flavonoids suppress xanthine oxidase, decreasing the levels of uric acid and alleviating inflammation in hyperuricemia (Zhao et al., 2020; Sobhani et al., 2021; Swathi, Jayaram, Sugumar, 2021).

Oxidative stress is considered to be the primary cause of many chronic inflammatory diseases. According to the results of the present study, methanol extracts of leaves and peels exhibited strong antioxidant activity compared to petroleum ether and chloroform extracts. Therefore, these extracts may prove helpful in treating inflammatory conditions in which oxidative stress plays a pivotal role. The ability of methanol extracts to scavenge free radicals is associated with the presence of polyphenols and flavonoids. The biochemical scavenger theory states that dietary polyphenols and flavonoids are potent scavengers of free radicals and reactive oxygen species (ROS) because of hydroxyl groups, aromatic rings, and extensively conjugated systems. Polyphenols have the potential to neutralize ROS and reduce cellular oxidative stress. They also shield DNA, lipids, and proteins from oxidative damage, which lessens tissue inflammation (Rudrapal et al., 2022). Furthermore, surfactant-like properties of glycosaponins mitigate the damage that inflammatory processes inflict on cells (Latif et al., 2020).

The heat-induced protein denaturation assay is commonly used to evaluate in vitro anti-inflammatory activity. Heat, toxins, and stress can cause protein denaturation and provoke inflammatory responses. Thus, preventing or inhibiting protein denaturation is a unique approach to treating inflammation (Shunmugaperumal, Kaur, 2016). In the current study, methanol extracts were more effective in inhibiting protein denaturation than petroleum ether and chloroform extracts. The ability of methanol extract to inhibit heat-induced denaturation of proteins undermines the plants’ efficacy in treating inflammation. The presence of polyphenols, flavonoids, and glycosaponins in the extracts could be the reason for this activity (Nabi et al., 2023).

The XO inhibition assay is an in vitro technique to determine anti-gout effects. XO is involved in the metabolism of purines to uric acid. The overactivity of this enzyme leads to elevated uric acid levels and gout. Therefore, inhibition of XO activity is one of the options for treating gout (Ashiq et al., 2021b). According to the results of the present study, the methanol extracts outperformed petroleum ether and chloroform extracts in inhibiting XO. It could be due to the tendency of the flavonoids and polyphenols to bind directly to the enzyme to inhibit uric acid synthesis and reduce the likelihood of developing gout (Liu et al., 2020; Zhao et al., 2020).

The carrageenan-induced paw edema assay is used extensively to assess and discover new anti-inflammatory drugs. There are two distinct phases in the inflammatory response caused by carrageenan: an early and a delayed phase. The early phase, which lasts for an hour after injection, is mainly driven by the release of serotonin, bradykinin, and histamine. Elevated capillary permeability and increased blood flow are the hallmarks of this stage, leading to edema development. Following the initial hour, a delayed phase ensues, marked by the migration of leukocytes and the production of prostaglandins, two critical steps towards acute inflammatory response (Borsani et al., 2021; Semis, Gur, Ileriturk, 2021). The results of the present study revealed that methanol extracts of the AML, BPL, and CPFP - at all studied doses - reduced paw thickness. Particularly, the anti-inflammatory effects of the extract, at a 500 mg/kg dose, were comparable to the standard. The anti-inflammatory effects of the serial extracts of AML, phosphate-buffered saline extract of CPF, and aqueous extracts of BPL extracts have been documented previously, and our results correlate very well with the reported data (Ojewole, 2005; Arul, Miyazaki, Dhananjayan, 2005; Nafiu, Rahman, 2015). However, the preventive effects of AML, CPFP, and BPL against CFA-induced arthritis are reported for the first time in the present study. The methanol extracts of the plants significantly reduced inflammation and restored normal morphology in rats with arthritic conditions produced by CFA. Additionally, all of the extracts normalized hematological parameters as well as ALT, AST, ALP, CRP, and RA factor. The extracts exhibited a dose-dependent response, with the maximum effect at 500 mg/kg.

A change in body weight is a crucial indicator of disease progress. CFA injection significantly reduces body weight in rats and indicates that arthritis affects weight, even when normal and arthritic rats consume the same amount of food. This weight reduction is due to rheumatoid cachexia, a condition where RA progression leads to muscle atrophy and weakness. Furthermore, inflammation impairs the small intestine’s ability to absorb nutrients, contributing to weight loss. Chronic inflammation in RA affects anabolic factors like insulin-like growth factor-1, increases catabolic hormones like glucocorticoids, and disrupts the neuroendocrine response. These effects and elevated inflammatory cytokines result in hypermetabolism and decreased body mass (Noh et al., 2021). The results of the present study indicate that methanol extracts, especially at a dose of 500 mg/kg, help in the recovery of average body weight.

The arthritic score is a measure to quantify the degree of joint inflammation. The methanol extracts-treated groups showed lower arthritic scores than the CFA-treated groups, suggesting that the former has effectively reduced disease progression. The histopathological data also supported these effects, as the rats treated with methanol extracts had significantly improved paw tissue morphology.

Further, compared to healthy rats, the CFA-treated rats had reduced hemoglobin and RBC counts, suggesting anemia from early-stage RBC destruction and decreased erythropoietin. The elevated platelets, ESR, and WBC levels in these rats also indicated the overexpression of inflammatory proteins in the bloodstream. The elevated levels of AST, ALT, and ALP in the disease control indicated liver tissue injury, leading to the release of enzymes into the bloodstream. Moreover, increased serum CRP and RA factor suggested the progression of the disease, as systemic inflammation is linked explicitly to these factors owing to their capability to release pro-inflammatory cytokines. The methanol extracts of the plants significantly improved the symptoms of CFA-induced arthritis by returning these markers to normal.

Potassium oxonate is a hyperuricemic agent that inhibits uricase, an enzyme that converts uric acid into a more soluble form called allantoin for removal through the kidneys. The uricase inhibition results in uric acid accumulation in the blood and leads to gout. Elevated serum uric acid levels are reported to be an independent risk factor for decreasing overall renal function (Alrashdi et al., 2022). Moreover, excessive creatinine in serum can reduce the glomerular filtration rate (GFR) and precipitate gouty attacks. Following PO injection, renal impairment in rats was indicated by higher serum levels of renal markers and disturbed renal histology. The AML, BPL, and CPFP methanol extracts decreased urea and creatinine levels and improved renal function. Additionally, the renal histology indicated reduced signs of injury, such as interstitial inflammation, tubular atrophy, and glomerular hypertrophy. These histological changes suggested that methanol extracts have the potential to restore the normal integrity and structure of renal tissue and mitigate kidney damage caused by hyperuricemia (Hao, Zhou, 2023; An et al., 2023).

The current study has certain limitations as it evaluates individual plants, necessitating future research to assess their anti-inflammatory potential in combination. This research is restricted to in vitro and in vivo studies, and additional clinical trials are required to verify the effectiveness and safety of these plant extracts in humans. In the future, extensive research is recommended to understand the mechanisms of anti-inflammatory action, safety, and bioassay-guided isolation of active compounds from these plants.

CONCLUSION

The study reveals that A. marmelos leaf, C. papaya fruit peel, and B. pinnatum leaf are rich in phytochemicals like flavonoids, polyphenols, and glycosaponins, and show promising radical scavenging, antiarthritic, and anti-gout properties. This study opens the door for their use as complementary or alternative medicines, warranting further pharmacological and clinical studies.

ACKNOWLEDGEMENTS

None

• Funding
  The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Ethics Approval

The study was carried out according to the protocol approved by the Institutional Ethics Review Board of the University of the Punjab, Lahore, Pakistan vide Ref. No. D/181/FIMS.

Data Availability

The data generated during and/or analyzed during the current study are available from the corresponding author and first author upon reasonable request.

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Edited by

• Associate Editor:
  Camila Manoel Crnkovic

Publication Dates

• Publication in this collection
  17 July 2026
• Date of issue
  2026

History

• Received
  17 Dec 2024
• Accepted
  07 Mar 2025