Phytomedicine Plus 4 (2024) 100533 Available online 13 February 2024 2667-0313/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). In-vitro and in-vivo anti-inflammatory properties of extracts and isolates of Pangdahai Mahmood B. Oppong a,b,*, Shijie Cao b, Shi-Ming Fang b, Seth K. Amponsah c, Paul O. Donkor d, Michael Lartey a, Lawrence A. Adutwum a, Kwabena F.M. Opuni a, Feng Zhao e, Qiu Feng b a Department of Pharmaceutical Chemistry, School of Pharmacy, College of Health Sciences, University of Ghana, Legon, Ghana b Tianjin State Key Laboratory of Modern Chinese Medicine and School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, 10 Poyanghu Road, Jinghai District, Tianjin, 301617, China c Department of Medical Pharmacology, University of Ghana Medical School, Accra, Ghana d Department of Pharmacognosy and Herbal Medicine, School of Pharmacy, College of Health Sciences, University of Ghana, Legon, Ghana e School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, 264005, People’s Republic of China A R T I C L E I N F O Keywords: Pangdahai Anti-inflammation Cytotoxicity Secondary metabolites A B S T R A C T Background: : Pangdahai (matured, ripened, and dried seeds of Scaphium affine (Mast.) Pierre) is widely used in managing several diseases in countries like China, Vietnam, Japan, and India. This study evaluated the anti- inflammatory effects of the crude extracts (ethanol and aqueous) and isolated compounds of Pangdahai. Methods: : Xylene-induced ear edema in mice, carrageenan-induced paw edema in rats, and nitric oxide (NO) assay were used to evaluate and screen the crude extracts and isolated compounds from the ethanolic extracts of Pangdahai. TNF-α and IL-1β levels in the tissues of rat foot and ear were determined by ELISA. The cytotoxicity of the isolated compounds was also determined by MTT assay. Molecular docking studies using targets involved in the inflammatory process were also used to further evaluate the compounds. Results: : Both aqueous and ethanol extracts demonstrated significant anti-inflammatory effect and markedly attenuated vascular permeability in mice induced by acetic acid in a dose-independent manner. The ethanol extract also significantly inhibited levels of IL-1β and TNF-α. Four (4) compounds exhibited significant inhibitory effects on NO release without cytotoxicity on RAW 264.7 macrophage. These compounds also showed good binding affinities for COX-2, PLA2, IRAK-4 and NIK. Conclusions: This study validates, provides scientific evidence and justification for the use of the aqueous de- coctions of Pangdahai in pharyngitis traditionally. (+) – Pinoresinol, tiliroside, Z-caffeic acid, and 3,4-dihydrox- ybenzoic acid (protocatechuic acid) isolated from Pangdahai showed anti-inflammatory activities, which might be responsible for the actions of Pangdahai. Tiliroside showed high binding affinity comparable to the native ligands of inflammatory mediators. List of abbreviations COX-2 cyclooxygenase-2 DMSO Dimethylsulfoxide DMEM Dulbecco’s modified eagle medium ELISA enzyme-linked immunosorbent assay FBS Fetal Bovine Serum IL Interleukin iNOS inducible nitric oxide synthase IRAK-4 Interleukin-1 Receptor-Associated Kinase-4 LPS Lipopolysaccharide NIK NF-κB–Inducing Kinase NO nitric oxide NSAIDs non-steroidal anti-inflammatory agents NTF Tumor necrosis factor OD Optical density PBS Phosphate buffered saline PDH Pangdahai * Corresponding author at: Department of Pharmaceutical Chemistry, School of Pharmacy, College of Health Sciences, University of Ghana, P.O. Box LG 43, Legon, Ghana. E-mail address: mboppong@ug.edu.gh (M.B. Oppong). Contents lists available at ScienceDirect Phytomedicine Plus journal homepage: www.sciencedirect.com/journal/phytomedicine-plus https://doi.org/10.1016/j.phyplu.2024.100533 mailto:mboppong@ug.edu.gh www.sciencedirect.com/science/journal/26670313 https://www.sciencedirect.com/journal/phytomedicine-plus https://doi.org/10.1016/j.phyplu.2024.100533 https://doi.org/10.1016/j.phyplu.2024.100533 https://doi.org/10.1016/j.phyplu.2024.100533 http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ Phytomedicine Plus 4 (2024) 100533 2 PGE Prostaglandin E2 PMSF phenylmethylsulfonyl fluoride PLA2 Phospholipase A2 TCH Traditional Chinese Medicine WHO World Health Organization. 1. Introduction Inflammation can be said to be one of the body’s reaction to tissue damage or infection and is typically characterized by redness, swelling, heat, and pain (Megha et al., 2021). In inflammation, there can be activation of inflammatory mediators like chemokines and cytokines (Zhu et al., 2018). There are three main phases of inflammation. Phase 1is marked by an increase in vascular permeability leading to the exudation of fluids from the blood into the interstitial space; phase 2 involves the leukocytes infiltration from the blood into the tissues, and phase 3 is distinctively shown by granuloma formation and tissue repair (Mukhopadhyay et al., 2019). Edema is also a characteristic feature of the acute inflammation (Li et al., 2021; Tian et al., 2021). Inflammation is a common symptom for most disease conditions, some of which include pharyngitis, bowel diseases, arthritis, allergic rhinitis, and atopic dermatitis (Zhu et al., 2018). Therefore, regulating mediators of inflammation could decrease disease severity or progression. Conventionally, inflammatory conditions are managed with anti- inflammatory agents. These agents could be steroidal or non-steroidal in nature. The steroidal agents include glucocorticoids, such as predni- sone, prednisolone, triamcinolone, methylprednisolone, and dexa- methasone. Glucocorticoids can cause side effects like high blood sugar, difficulty responding to insulin, high blood pressure, muscle weakness, vulnerability to infections, Cushing’s syndrome, stomach ulcers, and mental health issues (Yang and Yu, 2021). The non-steroidal anti-in- flammatory agents (NSAIDs), such as piroxicam, aspirin, aceclofenac, ibuprofen, diclofenac, naproxen, indomethacin, and celecoxib, mainly inhibit the synthesis of prostaglandin or cyclooxygenase. Despite their clinical utility, NSAIDs are also known to cause gastric ulcers, liver and kidney damage (Olry de Labry et al., 2021). NSAIDs elevate blood pressure and increase the risk of myocardial infarction (Patrono, 2016). The contribution of natural products to maintaining health and wellbeing is underestimated. Natural products are the engine behind the successes of traditional medicine and/or herbal medicine practices. Medicinal plants are good sources of secondary metabolites which form the basis for most commercially produced pharmaceuticals and herbal remedies (Li et al., 2020). The use of medicinal plants in preventing and treating/curing human diseases dates back to antiquity. The analgesic and antipyretic properties of the bark of the willow tree have long been documented by the Greeks and Romans (Montinari et al., 2019). Empirical knowledge of these medicinal plants and their potential toxic effects were passed on by oral tradition and sometimes recorded in texts (Jansen et al., 2021). Monographs on specific herbs are accessible from several sources, for example, the European Scientific Cooperative on Phytotherapy and the World Health Organization (WHO, 2019). Furthermore, Traditional Chinese Medicine (TCM) has attracted inter- est, acceptability, and significance in many countries. TCM continues to play a major role in the management of diseases and is also an excellent source in the discovery of natural bioactive compounds or lead com- pounds (Wang et al., 2018). Pangdahai (PDH) is the dried seeds of Scaphium affine (Mast.) Pierre, of the family Malvaceae (Medicinal Plant Name Services, 2021). In the Chinese Pharmacopoeia, it is recorded as Sterculia lychnophora Hance Pierre (scientific synonym) (Chinese Pharmacopoeia Commission, 2015). PDH is famously used in traditional/folk medicine in Asia (China, Japan, Vietnam, Thailand, and India). Decoctions of PDH are used for treating pharyngitis, laryngitis, constipation, cough, menorrhagia, and pain. The crude extracts and isolates of PDH have shown diverse phar- macologic effects, including anti-inflammatory, neuroprotective, anti-microbial, anti-hypertensive, analgesic, antipyretic, anti-ulcer, and anti-oxidative effects (Oppong et al., 2018). Clinically, PDH is notable for treating chronic pharyngitis in China (Oppong et al., 2018). Data also suggest that it contains many secondary metabolites such as lignans, phenylpropanoids, flavonoids, nitrogenous bases, phenolic acids, het- erocyclic aromatic acids, phytosteroids, glycosides, sesquiterpenoids, and nucleosides (Oppong et al., 2020). Indeed, continuous in- vestigations must be done to ascertain and validate the traditional or folkloric uses of plants and their extract. This work reports, the anti-inflammatory properties of the aqueous and ethanol extracts and some isolated secondary metabolites of PDH for the first time. 2. Methods 2.1. Chemicals and reagents Dexamethasone acetate was purchased from Zhejiang Xianjun Pharma Ltd., China. The water used was purified with Millipore Milli Q plus purification system (Thermo Fisher Scientific, USA) Carrageenan, xylene, physiological normal saline solution, 0.6 %v/v acetic acid solu- tion, 0.5 %w/v Evans blue solution, Griess reagent were purchased from Sigma, USA. Bacterial lipopolysaccharide (LPS) was purchased from Sigma, USA. RAW 264.7 murine macrophage cell line was bought from the American Type Culture Collection (USA). Fetal Bovine Serum (FBS) was obtained from Hyclone (USA), Dimetylsulfoxide (DMSO) from Solarbio (China), and Dulbecco’s modified eagle medium (DMEM) from Thermo Fisher Scientific (USA). All other reagents used were of analytical grade and commercially available. 2.2. Preparation of PDH extracts The PDH was obtained in March 2016 from the Guangxi province (China). A voucher specimen (No.: 20161205SL) was kept at the Tianjin State Key Laboratory of Modern Chinese Medicine at Tianjin University of Tradition Chinese Medicine, China. Briefly, 1 kg each of PDH was extracted separately with water and 95 %v/v ethanol, concentrated and dried in vacuo to yield 20.80 and 6.50 %w/w of aqueous and ethanol extracts, respectively as previously described in our work Oppong et al., 2020. 2.3. Acquisition of animals Male Sprague–Dawley rats, SPF grade (200–220 g) were obtained from Shandong Yantai Raphael Biotechnology Co. (China). Male Kunming mice (20 ± 2 g) were obtained from Shandong Yantai Raphael Biotechnology Co. (China). The experimental animals were kept in a temperature- and humidity-controlled room (23 ◦C, 60 % air humidity). They had unrestricted access to standard diet and water. They were kept in separate metabolic cages with no food but unrestricted access to water for 12 h before the experiment. All procedures conformed to the Guidelines associated with Care and Use of Laboratory Animals (Na- tional Institutes of Health). Xylene–induced ear edema in mice In brief, 35 male Kunming mice, weighing averagely 20 ± 2 g, were randomly grouped into 5: the model (negative control), dexamethasone (positive control), low dose of PDH, medium dose of PDH and high dose of PDH groups. With the aqueous extract of PDH, the low, medium, and high dose groups were treated with 200, 400, and 800 mg/kg.d (bw) of the extract. With the ethanol extract, the low, medium, and high dose groups were treated with 20, 40, and 80 mg/kg.d (bw) of the extract. For both extracts, 6 mg/kg.d (bw) of dexamethasone was used as a positive control. The test agents (extracts and dexamethasone) were adminis- tered directly into the stomach by oral gavage at a volume of 0.2 mL/10 g using normal saline as the vehicle. The negative control group was given 2 mL of normal saline once M.B. Oppong et al. Phytomedicine Plus 4 (2024) 100533 3 daily. After the fifth day of treatment, 0.1 mL of xylene was evenly smeared on both the inner and outer sides of the right ear of each of the mice (to induce edema), and the left ear was left as the control. The mice were sacrificed after 4 h. The left and right ears were cut along the ear line. Ear discs were cut from both ears from the same part of each ear of the same mouse with a stainless-steel perforator (diameter: 6 mm). The ear discs were then weighed with an analytical balance (Zhao et al., 2018). The degree of edema was evaluated by the difference in weight between the right and left ear discs of the same mice. The degree of edema inhibition was used as an index of the anti-inflammatory activity of the extracts. Ear edema(mg) = weight of left ear disc − − weight of right ear disc The ear tissues were stored in a refrigerator at -80 ℃. 2.5. Carrageenan-induced paw edema in rats Male Sprague Dawley rats (SPF grade), weighing an average of 200 ± 20 g, were randomly put into 5 groups of 7 animals. One set of the animals were assessed using the aqueous extract of PDH, and another set of animals were assessed using the ethanol extract of PDH. For the first set (aqueous extract), the grouping included 7 rats put in 5 group: negative control (2 mL normal saline), dexamethasone (positive control - 6 mg/kg⋅day bw), low dose of aqueous extract of PDH (200 mg/kg⋅day bw), medium dose PDH (400 mg/kg⋅day bw), and high dose PDH (800 mg/kg⋅day bw). The extracts were administered directly into the stomach by oral gavage at a volume of 0.2 mL/10 g using normal saline as the vehicle. The ethanol extract of PDH followed the same procedure (7 rats in 5 groups) as done for the aqueous extract: low, medium, and high dose groups received 20, 40, and 80 mg/kg⋅day (bw). Dexamethasone was used as a positive control at 6 mg/kg⋅day (bw). The negative control group received 2 mL of normal saline once daily. Two (2) hours after the last treatment, the volumes of both hind- paws up to the ankle joint of the rats were measured with a plethys- mometer. Afterwards, the rats were injected with 1 %w/v carrageenan solution (0.1 mL each) into the distal end of their left hind limbs. The paw volumes were measured again after 1, 2, and 4 h. Each measure- ment was done in triplicate (Rezq et al., 2021). The rats were then sacrificed by injecting 3 mL of 10 %v/v chloral hydrate solution into their abdominal cavity. The paw tissues were removed and stored in a refrigerator at -80 ℃. The degree of edema in the rats was calculated as the difference in weight between the paw volumes measured before carrageenan injec- tion (basal volumes (VB)) and after carrageenan injection (pathological volumes (VA)). Edema inhibition, relative to the percentage increase in paw volume, was used as an index of the anti-inflammatory activity of the extracts. Percentage increase in paw volume = {(VA − VB) /VB} × 100 Where VA: Rat paw volume after carrageenan injection VB: Rat paw volume before carrageenan injection 2.6. Acetic acid-induced vascular permeability in mice Thirty-five male Kunming mice, weighing averagely 20 ± 2 g, were randomly grouped into 5: the negative control, dexamethasone, low, medium, and high groups. For the aqueous extract, mice in the low, medium, and high dose groups were given 200, 400, and 800 mg/kg⋅day (bw) of the extract. The extracts were administered directly into the stomach by oral gavage at a volume of 0.2 mL/10 g using normal saline as the vehicle. For the ethanolic extract, mice in the low, medium, and high dose groups were given 20, 40, and 80 mg/kg⋅day (bw) of extracts. The positive control was Dexamethasone at 6 mg/kg⋅day (bw) in both cases. After the fifth day of treatment, the tails of the mice were injected with 0.5 %w/v Evans blue saline solution (0.2 mL), followed by an in- jection of 0.2 mL 0.6 %v/v acetic acid 0.2 mL intraperitoneally. After sacrificing the mice, 10 mL saline solution was to wash their peritoneal cavities (3x). The saline washings were pooled, filtered and centrifuged (3000 rpm, 10 min) to obtain the supernatant (5 mL). The optical den- sity (OD) values of the supernatant were measured at 590 nm with a UV–Vis spectrophotometer (Rezq et al., 2021). The intraperitoneal injection of dilute acetic acid causes an increase in capillary permeability, and this can cause Evans blue to extrude into the abdominal cavity. The amount of Evans blue represented the capil- lary permeability, which was estimated by measuring the optical density values of the supernatant. 2.7. Histo-pathological study of sections of mice edematous ear induced by xylene The ear tissues of the negative control group, dexamethasone group, and PDH ethanol extract (low – high dose) groups were kept in 10 %v/v formaldehyde solution for 24 h to prepare paraffin sections. The paraffin sections were dewaxed and then stained with hematoxylin and eosin. The pathological changes of the local tissues of the mice auricles were observed under a light microscope to ascertain the degree of inflam- mation (Huang et al., 2011). 2.8. Determination of the levels of TNF-α and IL-1β in the rat foot tissue The rat foot tissues stored at -80 ◦C were obtained and crushed into centrifuge tubes. Afterwards, 500 μL PBS and 5 μL PMSF (100 mM) were added and placed on an ice water bath for 30 s and then centrifuged at 4 ◦C, 13,000 r/min for 6 min. The supernatant was collected, and the amount of protein was estimated with Bradford method kit. The TNF-α and IL-1β levels contained in 1 mg protein of rat foot tissue were measured using enzyme-linked immunosorbent assay (ELISA) (Huang et al., 2011). This was repeated for samples obtained from rats treated with the ethanolic extract of PDH (showed the highest activity). 2.9. Determination of the levels of TNF-α and IL-1β in mouse ear tissue The ear tissues of the mice stored at -80 ◦C were removed, crushed and placed in centrifuge tubes. Afterwards, 500 μL PBS and 5 μL PMSF (100 mM) were added and placed on an ice water bath for 30 s and then centrifuged at 4 ◦C, 13,000 r/min for 6 min. The supernatant was collected, and the amount of protein was estimated with Bradford method kit. TNF-α and IL-1β levels in 1 mg protein of mice ear tissue were measured using enzyme-linked immunosorbent assay (ELISA), according to manufacturer’s protocol (Huang et al., 2011). This was repeated for samples obtained from mice treated with the ethanolic extract of PDH that showed the highest activity. 2.10. Isolation and characterization of compounds from Pangdahai Compounds from PDH extracts were isolated and characterized using various chromatographic and spectroscopic techniques described in our previous work (Oppong et al., 2020). 2.11. In vitro anti-inflammatory screening of isolated compounds 2.11.1. Cell culture and MTT assay Complete DMEM media containing 10 %v/v FBS, 100 U/mL peni- cillin, and 100 mg/mL streptomycin was used to culture RAW 264.7 macrophage cells. The culture was incubated in a humidified incubator set at 5 % CO2 and 37 ℃ with daily replacement of the culture media. The cells were then seeded at 1 × 106 cells/well in a 96-well microtiter plate. After overnight incubation, LPS (1 μg/mL) with or without the isolated compounds from PDH (Uridine, Ethyl-3,4-dihydroxy benzoate, (+) – Pinoresinol, Daucosterol, Vomifoliol, 2-(Hydroxymethyl)− M.B. Oppong et al. Phytomedicine Plus 4 (2024) 100533 4 5‑hydroxy pyridine, E – Caffeic acid, 1-O-Caffeoyl-β-d-glucopyranoside, 1-(β-d-Ribofuranosyl)− 1H-1,2,4,-triazole, Tiliroside (Kaempherol-3-O- β− 6’’-p-hydroxycoumaroylglucose), 3-Cinnamoyltribuloside, β-Adeno- sine, 3,4-Dihydroxybenzoic acid (Protocatechuic acid), Falandin B, Z- Caffeic acid, Murratetra C, Uracil, p‑hydroxy benzoic acid, 5-hydroxy- methyl-3-furoic acid, β-Sitosterol, 2-Furoic acid) serially diluted from 0 to 100 μM were then added and incubated further for 24 h. MTT re- agent was then added to each well and incubated at 37 ◦C for further for 2.5 h. The formazan crystals formed in each well were sonicated for 15 min in 150 μL DMSO. Finally, a microplate reader was used to estimate absorbance at 490 nm (Huang et al., 2011). Dexamethasone was used as the control drug. The cell inhibition percentage was estimated using the formula; Percentage cell inhibition = 100 − {(At − Ab) / (Ac − Ab)} × 100 Where At = Absorbance of test compound Ab = Absorbance of blank Ac = Absorbance of control 2.11.2. Nitric oxide assay RAW 264.7 macrophage cells were treated with test compounds or dexamethasone as described in Section 2.11.1. 100 μL of Griess reagent was added to 100 μL of the cell culture supernatant. The mixture was incubated at room temperature for 10 min, and the absorbance measured at 540 nm. NaNO2 solutions with concentrations of 10, 20, 40, 60, 80, and 100 μM were prepared, and their corresponding absorbance values at 540 nm were measured. A standard calibration curve of con- centration vs absorbance was plotted. The concentrations of nitrite in the treated RAW 264.7 cells were calculated using the standard calibration curve (Huang et al., 2011). 2.12. Data processing, statistical analysis and molecular docking Origin Lab software (2018) (OriginLab, USA) was used to analyze the data. Experimental data were reported as the mean value ± SD. Differ- ences between groups (One-way ANOVA) at a P<0.05 were considered significant. Four inflammatory targets namely, Cyclooxygenase-2 (COX-2, Uni- ProtID: Q05769), Phospholipase A2 (PLA2, UniProtID: P00624), Interleukin-1 receptor-associated kinase-4 (IRAK-4, UniProtID: Q9NWZ3) and NF-κB–inducing kinase (NIK, UniProtID: Q99558) were obtained from RSCB PDB. Where the targets were ligand bound, the coordinates of the ligand were removed. The structures were cleaned using Discovery Studio version 21.1.0 (BIOVIA, San Diego). The following compounds showing high anti-inflammatory activity, namely, (+) – pinoresinol, tiliroside, Z-caffeic acid, and 3,4-dihydroxybenzoic acid (protocatechuic acid) were virtually screened using Python Pre- scription Virtual Screening tool (PyRx 0.8, AutoDock Vina module). The interactions between the protein-ligand were analyzed using Discovery Studio version 21.1.0 (BIOVIA, San Diego). As a positive control, the binding interactions of known ligands for each of the four targets were also evaluated. 3. Results 3.1. Effect of aqueous and ethanol extracts of PDH on xylene–induced ear edema in mice The mice in the negative control group showed significant edema after xylene application for the aqueous extracts. Compared with the negative control group, the dexamethasone treated group significantly inhibited ear edema induced by xylene (##P < 0.01), which showed that the experimental model was appropriately designed. The low and me- dium doses of PDH extract had no significant inhibitory effect. However, the high dose had a significant inhibitory effect equivalent to dexa- methasone (**P < 0.01). The effects of the aqueous extracts are pre- sented in Fig. 1a. The ethanolic extracts also exhibited similar results as the aqueous extract. All the tested doses of the ethanol extract inhibited ear edema in mice induced by xylene. The low dose and high dose exhibited signifi- cant inhibition of edema comparable to the dexamethasone group (**P < 0.01). The medium dose, however, did not show significant inhibition. The effects of the ethanol extracts are presented in Fig. 1d. Fig. 1. : a. Effect of dexamethasone and aqueous extracts of PDH on xylene-induced ear edema in mice. b. Effect of Pangdahai aqueous extract on carrageenan- induced paw edema in rats. c. Effect of aqueous extracts of Pangdahai on vascular permeability induced by acetic acid. d. Effect of dexamethasone and ethanol extracts of Pangdahai on xylene-induced ear edema in mice. e. Effect of Pangdahai ethanol extract on carrageenan-induced paw edema in rats. f. Effect of ethanol extracts of Pangdahai on vascular permeability induced by acetic acid. ** and ## Statistically significant at P < 0.01, *** statistically significant at P < 0.001. M.B. Oppong et al. Phytomedicine Plus 4 (2024) 100533 5 3.2. Effect of aqueous and ethanol extracts of PDH on carrageenan- induced rat paw edema With the aqueous extract, the rats in the negative control group showed increasing paw edema at 1, 2, and 4 h, while the positive control (Dexamethasone) group significantly inhibited rat paw edema induced by carrageenan (##P < 0.01). This indicated the appropriateness of the experimental design. All tested doses of PDH aqueous extract signifi- cantly inhibited rat paw edema induced by carrageenan at 1 h after compared to the negative control group (**P < 0.01), but the effect was not obvious at 2 h. However, the high dose demonstrated a significant (**P < 0.01) inhibitory effect compared with the negative control group, and the effect was most obvious at 4 h. Nevertheless, the degree of this inhibitory effect was lower than that of the dexamethasone group. The paw edema/swelling index of the experimental groups is shown in Fig. 1b. The ethanolic extracts also exhibited similar results as the aqueous extract. For all the tested doses, the ethanol extract exhibited some de- gree of inhibition of rat paw edema induced by carrageenan. The low and medium doses exhibited significant inhibition of edema comparable to the dexamethasone group (**P < 0.01) at 1 h. The high dose, how- ever, showed no significant inhibitory effects. The paw edema/swelling index of the experimental groups is shown in Fig. 1e. 3.3. Effect of aqueous and ethanol extract of PDH on acetic acid-induced mice vascular permeability The vascular permeability was measured by the OD which repre- sented the amount of Evans blue exuded into the peritoneal cavity. In both aqueous - and ethanol-treated groups, the OD values of the negative control groups significantly increased following treatment with acetic acid. Compared with the model group, the aqueous extract exhibited a non-significant reduction in the OD values at all tested doses (Fig. 1c). The ethanol extracts, however, demonstrated a significant (**P < 0.01) but dose-independent reduction of the OD values (Fig. 1f). Fig. 2. Pathological changes in mice ears treated with Pangdahai ethanol extract (HE staining, ×100). M.B. Oppong et al. Phytomedicine Plus 4 (2024) 100533 6 3.4. Histo-pathological study of sections of mice edematous ear induced by xylene HE staining was used to observe and confirm the changes in the inflamed cells induced by xylene. The normal group showed normal tissues. Compared with the normal group, the negative control group exhibited high degree of swelling which was marked by blistering of the epithelial and conjunctival tissues, red-stained mesh-like collagen fibers, and significant infiltrated inflammatory cells. Contrary to the xylene groups previously given dexamethasone (positive control group) or PDH extracts (low and medium dose groups), there was a reduction in edema (slight edema), amount of red-stained mesh-like collagen fibers and infiltration of inflammatory cells (Fig. 2). These results collectively indicate that ethanol extracts of PDH inhibited xylene-induced ear edema and infiltration of inflammatory cells. 3.5. Effect of PDH ethanol extract on the levels of inflammatory cytokines The levels of TNF-α and IL-1β were significantly (p < 0.01) inhibited by dexamethasone in both in vivo acute inflammation models. The ethanol extracts of PDH exhibited significant (p < 0.01) dose -depen- dent inhibition of the expression of TNF-α and IL-1β in both models (as shown in Fig. 3a–d). These results indicate that the anti-inflammatory properties of PDH ethanol extract were cognate to the inhibition of TNF-α and IL-1β. 3.6. In-vitro anti-inflammatory effects of some isolated compounds from PDH The results show that all tested compounds showed inhibition of NO production with no obvious cytotoxicity at 100 μM. Among these com- pounds, (+) - pinoresinol, tiliroside, 3-cinnamoyltribuloside, 3,4-dihy- droxybenzoic acid, Z-caffeic acid, and 2-furoic acid showed significant inhibition (P < 0.05) of NO-production in LPS stimulated RAW cells with percentage inhibitions greater than 70 %. The percentage inhibitions and IC50 are shown in Table 1. 3.7. Binding affinities of selected compounds The binding interactions between the four COX-2, PLA2, IRAK-4 and NIK, which are known mediators of anti-inflammatory process and four of the isolated compounds were evaluated. As a positive control, known ligands of these targets were used positive control. COX-2 and PLA2 were evaluated with celecoxib and Niflumic acid, respectively. IRAK-4 and NIK on the other hand were evaluated using 1-(3-Hydroxypropyl)- 2-[(3-Nitrobenzoyl) amino]-1h-Benzimidazol-5-Yl Pivalate and Cdk1/2 Inhibitor III, respectively. The results of these studies are shown in Table 2. 4. Discussion The current study ascertained and validated the traditional or folk- loric use of PDH as an anti-inflammatory agent. Thus, we report the anti- inflammatory properties of the aqueous and ethanol extracts and some isolated secondary metabolites of PDH. Data from this study showed that extracts of PDH (ethanol and aqueous) exhibited an inhibitory effect on xylene-induced ear edema in mice. The aqueous extracts demonstrated a dose–independent inhibi- tion, while the low and high doses of ethanol extracts significantly inhibited ear edema in mice induced by xylene. Xylene induces acute neurogenic edema (Singsai et al., 2020) and cause swelling by increasing vasodilation and vascular permeability when applied (Zhao et al., 2018). Furthermore, this study showed that the extracts of PDH inhibited paw edema in rats induced by carrageenan. Carrageenan induces edema in two phases with several mediators including histamine, serotonin, 5- hydroxytryptamine, prostaglandins, bradykinin, cyclooxygenase, TNF- α, IL-1 and IL-6 involved (Umare et al., 2014; Karim et al., 2019; Patil et al., 2019). This study suggests that the ethanol extracts markedly attenuated acetic acid-induced vascular permeability in a dose-independent manner (Fig. 1c and f). Acute inflammation is characterized by vasodi- latation, exudation of plasma, increase in vascular permeability, and Fig. 3. a. Effect of Pangdahai ethanol extract on the levels of TNF-α in paw tissues of rats induced by carrageenan. b. Effect of Pangdahai ethanol extract on the levels of IL-β in paw tissues of rats induced by carrageenan. c. Effect of Pangdahai ethanol extract on the levels of TNF-α in ear tissues of rats induced by xylene. d. Effect of Pangdahai ethanol extract on the levels of IL-1β in ear tissues of rats induced by xylene. ##Compared with blank group p < 0.01; *Compared with model group p < 0.05; **Compared with model group p < 0.01. M.B. Oppong et al. Phytomedicine Plus 4 (2024) 100533 7 neutrophil migration into the site of inflammation (Chen et al., 2018). Exudation is a direct consequence of increased vascular permeability. In acetic acid-induced vascular permeability assay, acetic acid causes the level of mediators such as prostaglandins, serotonin, and histamine in peritoneal fluids to increase consequently, resulting in dilation of the capillary vessels and an increase in vascular permeability (Dantas et al., 2020; Rezq et al., 2021). Data from this study showed that the anti-inflammatory effects of PDH ethanol extract could be related to TNF-α and IL-1β inhibition. Though several cytokines are involved in inflammation (Delgado et al., 2003), TNF-α is the most significant cytokine associated with local and/or systemic inflammation (Cuzzocrea et al., 1999). TNF-α stimu- lates T cells and macrophages. It elevates levels of kinins and leukotri- enes (Yun et al., 2008; Huang et al., 2011). One of the of aims of this work was to assess the anti-inflammatory potentials of some isolated compounds from PDH. This was achieved by measuring the degree of inhibition of nitric oxide (NO) in RAW 264.7 macrophages treated with Lipopolysaccharides (LPS). LPS activates macrophages to release cytokines and inflammatory mediators. These include NO, cyclooxygenase-2, TNF-α and IL-6 (Saadat et al., 2019). NO plays a significant role in regulating physiological responses including inflammation (Doulias and Tenopoulou, 2020), and is used as a biomarker of inflammation in many biological samples (Rana, 2020). Therefore, the ability of a compound to inhibit the production of nitric oxide in LPS-stimulated RAW cells is indicative of its anti-inflammatory potentials (Rana, 2020). In addition, molecular docking studies performed using known me- diators of the inflammatory process, i.e. COX-2, PLA2, IRAK-4 and NIK demonstrated that (+) – pinoresinol, tiliroside, Z-caffeic acid and 3,4- dihydroxybenzoic acid demonstrated good binding affinities. This further supports our assertion that these agents could be the components responsible for the observed anti-inflammatory activity. It is interesting to note that amongst the top four compounds, tiliroside demonstrated binding affinities comparable and in some cases higher (PLA2 and NIK) that those of the positive control as shown in Table 2. Previous anti-inflammatory studies on some of the compounds iso- lated from PDH have shown that these compounds have anti- inflammatory properties. Tiliroside is reported to significantly inhibit mouse paw edema induced by phospholipase A2 and mouse ear edema inflammation induced by TPA (Sala et al., 2023). The work of Correa et al., 2018 and Luhata et al., 2017 have also demonstrated the anti-inflammatory effects of tiliroside. Several studies (both in vitro and in vivo) have established that protocatechuic acid possess anti-inflammatory effects (Semaming et al., 2015; Kakkar and Bais, 2014; Song et al., 2020; Hu et al., 2020). Anti-inflammatory effects of caffeic acid is also well demonstrated. Caffeic Acid is reported to significantly inhibit pro-inflammatory cytokines, downregulated mRNA expression of IL-1β, IL-6, and TNF-α (Gamaro et al., 2011; Wan et al., 2021; Ehtiati et al., 2023), lymphocytes, polymorphonuclear neutro- phils and, macrophages (Morones et al., 2016). Pinoresinol is reported to exert potent anti-inflammatory effects (Jang et al., 2022). Studies con- ducted by Jung et al., 2010 and During et al., 2012 have demonstrated that pinoresinol significantly inhibits NO, PGE(2), TNF- α, IL-1β and IL-6 and attenuates mRNA and protein levels of inducible nitric oxide syn- thase (iNOS), cyclooxygenase-2 (COX-2) and proinflammatory cyto- kines in LPS-activation. It is believed to exhibit the strongest anti-inflammatory properties by acting on the NF-κB signaling pathway (During et al., 2012). 5. Conclusions The aqueous and ethanol extracts of PDH have significant anti- inflammatory effects in the animals used. This study validates and provides scientific evidence for the traditional use of the aqueous de- coctions of PDH in the treatment of inflammatory-related conditions such as pharyngitis. Additionally, (+) – pinoresinol, tiliroside, 3- Table 1 Inhibitory activity of the compounds from Pangdahai on LPS-induced NO release in RAW 264.7 cells. Compound Concentration/ µM NO inhibition (%) IC50/ µM Uridine 100 46.53 >100 Ethyl-3,4-dihydroxy benzoate 100 32.29 >100 *(+) – Pinoresinol 100 84.68 16.36 ± 0.79 50 83.06 25 79.26 12.5 36.94 Daucosterol 100 36.84 >100 Vomifoliol 100 46.65 >100 2-(Hydroxymethyl)− 5‑hydroxy pyridine 100 43.61 >100 E – Caffeic acid 100 34.86 >100 1-O-Caffeoyl-β-d-glucopyranoside 100 14.49 >100 1-(β-d-Ribofuranosyl)− 1H-1,2,4,- triazole 100 47.11 >100 * Tiliroside (Kaempherol-3-O- β− 6′’-p- hydroxycoumaroylglucose) 100 89.47 17.58 ± 1.05 50 85.55 25 77.69 12.5 31.04 *#3-Cinnamoyltribuloside 100 102.65 27.78 ± 1.58 50 77.13 25 46.63 12.5 0.66 β-Adenosine 100 65.55 56.57 ± 2.55 50 48.27 25 23.24 12.5 18.98 *3,4-Dihydroxybenzoic acid (Protocatechuic acid) 100 100.79 18.55 ± 1.35 50 75.40 25 62.88 12.5 37.92 3,6-Dihydroxy-5,11-epoxy-7E- magastimaen-9-one (Falandin B) 100 43.34 >100 *Z-Caffeic acid 100 57.03 30.34 ± 2.00 50 56.46 25 48.27 12.5 26.98 2-Methoxy-benzoyl-β-d- glucopyranoside (Murratetra C) 100 33.34 >100 Uracil 100 51.20 94.85 ± 4.13 50 40.61 25 33.46 12.5 14.73 p‑hydroxy benzoic acid 100 40.96 >100 5-hydroxymethyl-3-furoic acid 100 25.64 >100 β-Sitosterol 100 19.46 >100 *#2-Furoic acid 100 101.25 25.49 ± 1.73 50 99.58 25 50.99 12.5 22.09 *Significant inhibitory effect on NO release at (P < 0.05). #Some degree of cytotoxicity. Table 2 Binding affinities of selected compounds with mediators of inflammation. Ligands Binding Affinities (kcal/mol) COX-2 PLA2 IRAK-4 NIK 3,4-dihydroxybenzoic acid -5.1 -5.5 -6.3 -5.4 Caffeic acid -5.8 -6.6 -6.6 -6.6 (+) – Pinoresinol -7.7 -5.8 -7.4 -6.5 Tiliroside -8.2 -9.1 -7.8 -9.6 Native Ligand -8.4a -8.4b -9.4c -9d Native Ligands: aCelecoxib; bNiflumic acid; c1-(3-Hydroxypropyl)-2-[(3-Nitro- benzoyl) amino]-1h-Benzimidazol-5-Yl Pivalate; dCdk1/2 Inhibitor III. Targets: COX-2 - Cyclooxygenase-2; PLA2 - Phospholipase A2; IRAK-4 - Interleukin-1 Receptor-Associated Kinase-4; NIK - NF-κB–Inducing Kinase. M.B. Oppong et al. Phytomedicine Plus 4 (2024) 100533 8 cinnamoyltribuloside and 3,4-dihydroxybenzoic acid (protocatechuic acid) isolated from PDH showed in vitro anti-inflammatory activities supported by molecular docking studies, which could be the anti- inflammatory constituents of PDH. Funding This work was supported by the National Key Research and Devel- opment Program of China (2019YFC1711000). CRediT authorship contribution statement Mahmood B. Oppong: Investigation, Formal analysis, Data cura- tion, Writing – original draft. Shijie Cao: Investigation, Formal analysis, Data curation. Shi-Ming Fang: Supervision. Seth K. Amponsah: Writing – review & editing, Formal analysis, Data curation. Paul O. Donkor: Writing – original draft, Formal analysis, Data curation. Michael Lartey: Writing – review & editing, Formal analysis, Data curation. Lawrence A. Adutwum: Writing – review & editing, Investi- gation, Formal analysis, Data curation. Kwabena F.M. Opuni: Writing – review & editing, Formal analysis, Data curation. Feng Zhao: Writing – review & editing, Validation, Supervision, Methodology. Qiu Feng: Supervision, Conceptualization, Project administration, Funding acquisition. 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Oppong et al. https://doi.org/10.1016/j.phrs.2020.105109 https://doi.org/10.1155/2020/3176391 https://doi.org/10.1155/2020/3176391 https://doi.org/10.1021/acsomega.1c04797 https://doi.org/10.1155/2014/385297 https://doi.org/10.1155/2014/385297 https://doi.org/10.3389/fmicb.2021.784211 https://doi.org/10.3389/fmicb.2021.784211 http://doi.org/10.1017/erm.2018.3 http://apps.who.int/bookorders https://doi.org/10.7150/ijbs.58695 https://doi.org/10.7150/ijbs.58695 https://doi.org/10.1016/j.intimp.2007.11.003 https://doi.org/10.1155/2018/7965306 https://doi.org/10.1080/10408398.2016.1251390 In-vitro and in-vivo anti-inflammatory properties of extracts and isolates of Pangdahai 1 Introduction 2 Methods 2.1 Chemicals and reagents 2.2 Preparation of PDH extracts 2.3 Acquisition of animals Xylene–induced ear edema in mice 2.5 Carrageenan-induced paw edema in rats 2.6 Acetic acid-induced vascular permeability in mice 2.7 Histo-pathological study of sections of mice edematous ear induced by xylene 2.8 Determination of the levels of TNF-α and IL-1β in the rat foot tissue 2.9 Determination of the levels of TNF-α and IL-1β in mouse ear tissue 2.10 Isolation and characterization of compounds from Pangdahai 2.11 In vitro anti-inflammatory screening of isolated compounds 2.11.1 Cell culture and MTT assay 2.11.2 Nitric oxide assay 2.12 Data processing, statistical analysis and molecular docking 3 Results 3.1 Effect of aqueous and ethanol extracts of PDH on xylene–induced ear edema in mice 3.2 Effect of aqueous and ethanol extracts of PDH on carrageenan-induced rat paw edema 3.3 Effect of aqueous and ethanol extract of PDH on acetic acid-induced mice vascular permeability 3.4 Histo-pathological study of sections of mice edematous ear induced by xylene 3.5 Effect of PDH ethanol extract on the levels of inflammatory cytokines 3.6 In-vitro anti-inflammatory effects of some isolated compounds from PDH 3.7 Binding affinities of selected compounds 4 Discussion 5 Conclusions Funding CRediT authorship contribution statement Declaration of competing interest References