Anti-inflammatory potential of 1-O-methyl chrysophanol from Amycolatopsis thermoflava ICTA 103: an exploratory study

Abstract

The present study was designed to investigate the anti-inflammatory potential of Amycolatopsis thermoflava producing 1-O-methyl chrysophanol (OMC), a member of the hydroxyanthraquinone family. The anti-inflammatory potential was evaluated initially through in silico analysis against tumor necrosis factor- α and cyclooxygenase-2. The same activity was further confirmed based on the in vitro protein denaturation method as well as in vivo by a carrageenan-induced paw edema model in rats. The OMC compound was isolated, purified, and characterized from the fermentation broth of Amycoloptosis thermoflava. In vitro data revealed that the OMC possesses significant protein denaturation properties with an IC₅₀ of 63.50±2.19 µg/ml higher than the standard drug, with an IC₅₀ value of 71.42±0.715 µg/ml. The percentage of inhibition in paw swelling was observed to be 40.03±5.5 in OMC-treated group, which is comparable to the standard group (52.8±4.7). The histopathological evaluation and immunohistochemistry revealed the anti-inflammatory potential of OMC.

Introduction

Highlights

• 1-O-methyl chrysophanol (OMC), an hydroxyanthraquinone, from Amycolatopsis thermoflava has been evaluated for its anti-inflammatory potential.

• The OMC revealed significant binding affinity against cyclooxygenase-2 and tumor necrosis factor- α.

• It exhibited significant in vitro protein denaturation property with the IC₅₀ of 63 µg/ml.

• The OMC also denoted prominent in vivo inhibition of the paw edema volume (>40%)

• Histopathological (edema and inflammatory cells score) and immunohistochemistry studies (CD80, 31 and 68, and arginase-I levels) confirmed the anti-inflammatory potential of OMC.

Chronic inflammation has been considered the world’s leading cause of death in various fatal diseases like cancer, ischemic heart disease, stroke, type-2 diabetes mellitus, chronic kidney disease, non-alcoholic fatty liver disease, autoimmune and neurodegenerative conditions including coronavirus disease 2019^1,2. Physiologically, inflammation is the primary acute innate immune response that protects the body from infections, tissue injury, and cell death. It accompanies homeostatic coordination between regulatory mechanisms that involve a switch from proinflammatory mediators (e.g.: cytokines, interleukins (IL), and prostaglandins) to anti-inflammatory mediators (IL-10, prostanoids, and corticosteroids) lymphocytes, and resolution-inducing lipoxins, which finally leads to tissue repair by removing inflammatory inducers at the affected site^3,4. However, the failure in the elimination of inflammatory inducer results in the development of chronic inflammation in infections such as unrepaired tissue damage, persistent allergens, indigestible foreign particles, and endogenous monosodium urate crystals⁵. Furthermore, the oxidative stress developed to protect against the inflammation process and to remove infectious agents causes the denaturation of immune proteins, which develops deleterious effects on the host. Inflammatory diseases can be grouped into either acute or chronic inflammatory conditions. Acute inflammatory diseases such as acute respiratory distress syndrome and gouty arthritis could be characterized by pain, redness, and edema, which may ultimately progress to death in acute respiratory distress syndrome and other associated airway diseases. Whereas, chronic inflammation leads to tissue damage due to a higher influx of macrophages, which is the underlying cause of the majority of diseases^6,7. On the other hand, gout complex inflammatory arthritis is caused by the accumulation of monosodium urate crystals in the joints that develops an acute inflammatory reaction which is later progressed into a chronic inflammation⁸. The association of inflammation with these life-threatening diseases challenged researchers to explore a cellular, molecular, and systemic mechanism of inflammatory responses that maintain the homeostatic balance between regulatory mechanisms, which can selectively regulate the deleterious effects of different target tissue responses without affecting the overall strength or duration of the inflammatory response at the molecular level. Tumor necrosis factor (TNF-α) is a strong proinflammatory cytokine that plays a key role during inflammation and infection by producing vasodilation and edema formation as well as leukocyte adhesion⁹. Furthermore. TNF- α also stimulates the expression of Cyclooxygenase-2 (COX-2) mRNA at the gene level¹⁰. On the contrary, the inducible enzyme COX-2 and antigen-presenting cell co-stimulatory molecule CD80 are the major proinflammatory sensors that affect the production and regulation of prostaglandins (PGE₂) and cytokines (IL-6, NO, and TNF-α), respectively, during the inflammation process^11–13. Whereas, anti-inflammatory signals act in a component-specific manner by inhibiting 10–15% of TLR-inducible inflammatory target genes rather than completely switching off the inflammatory process. Therefore, polyphenolic therapeutic compounds that may regulate the balance between proinflammatory and anti-inflammatory mediators are effective strategies to reduce inflammation¹⁴. However, the severe adverse effects (gastric ulceration and liver damage) associated with currently using nonsteroidal anti-inflammatory drugs (NSAID) limit their usage for inflammatory conditions of several diseases¹⁵.

Hydroxy anthraquinones (HAQ) belong to a group of polyphenols that have been studied for their significant anti-inflammatory and TNF-α as well as COX-2 antagonist activity for a long ago¹⁶. The promising anti-inflammatory activity exhibited by emodin derivatives (aloe-emodin, chrysazine, and chrysophanol) and their little side effects compared to NSAIDs attracts the attention of researchers as anti-inflammatory agents^17,18. At present, microbial sources have gained increased attention as a source of bioactive compounds since they are safe and potent low molecular weight compounds compared to inhibitors of plants and animal origin¹⁹. Furthermore, the easier extraction and purification of compounds from microbial sources can improve the bioactive potential of isolated compounds²⁰. Actinobacteria are considered a rich repertoire of unique HAQ with novel bioactivities. A novel HAQ, 1-O-methyl chrysophanol (OMC) (a derivative of chrysophanol) isolated from rare actinobacteria, Amycolatopsis thermoflava has shown significant antioxidant, antiproliferative, and antidiabetic properties^21,22. It is evident from earlier reports that compounds with antioxidant activity can reduce the oxidative stress generated during the inflammation process and would be effective to treat inflammatory diseases²³. Based on the reported antioxidant and anti-inflammatory potential of other HAQ as well as its proven antioxidant activity, the present attempt has been made to study the in vitro anti-inflammatory activity of OMC and its effect on alleviating inflammation of carrageenan-induced rat paw edema. Furthermore, molecular docking studies of OMC have been carried out to study the binding efficiency against COX-2 and TNF-α for supporting the anti-inflammatory response against the diseases.

Materials and methods

Indomethacin was obtained from Merck. CD80 (B7-1) Monoclonal antibody 16-10A1e was from Bioscience^TM (Invitrogen). CD31 Monoclonal Mouse antihuman was obtained from Dako. CD68 was from Abcam, Arginase -I Monoclonal Mouse were purchased from PathnSitu biotechnologies and Carrageenan, Bovine Serum Albumin (BSA), Diclofenac Sodium, Dimethyl Sulfoxide (DMSO), Coloring solution Diaminobenzidine-tetrahydrochloride (DAB) 0.5% Hydrogen Peroxide (H₂O₂) in 1X PBS buffer at P^H (7.6) were procured from Sigma.

Experimental animals

Adult male Wistar rats (160–200 g) used in the present study were housed in polypropylene cages under controlled temperature (22±2°C) with standard laboratory food, water ad libitum, and light-controlled conditions (12 h each in the dark and light cycle) throughout the experiment. Animals were quarantined for one week before the experiment to acclimatize under laboratory conditions. All the animal experiments were carried out as per the National Institutes of Health Guidelines for the care and use of Laboratory Animals at the Indian Institute of Chemical Technology (IICT) (Ethics No: IICT/IAEC/017/2019). The animals were used only once and then subjected to euthanization.

Culture collection and maintenance

The glycerol stocks of rare actinobacteria A. thermoflava ICTA 103 from IICT were revived using Actinomycetes Isolation Broth (AIB) medium for ready use. The strain was further maintained on Actinomycetes Isolation Agar (AIA) agar plates at 4^oC for future studies.

Fermentation and extraction of crude metabolite

The fermentation studies of A. thermoflava were performed as per the method described by Ganesh Kumar et al., 2017. The strain was grown in the Asparagine Dextrose Salts media at pH 7.0 and incubated at 40°C with an agitation speed of 250 rpm for 5 days. The fermentation medium was subjected to centrifugation (REMI C-24BL) at 10 000×g to separate cell biomass. The cell free-supernatant was treated with Diaion HP-20 (2%) resin to bind the compound. The resin was washed and dried at room temperature to extract the compound by using methanol. Finally, the extract was concentrated under reduced pressure using a rotary vacuum evaporator (Heidolph Rotacool, Germany)²¹. The crude fractions obtained were analyzed qualitatively by thin-layer chromatography with a methanol and chloroform solvent ratio (10 : 90) as the mobile phase and visualized under a UV chamber.

Purification of crude extract

The concentrated crude fractions were collected from silica gel column chromatography (100–200 mesh size) with the increased polarity of the chloroform and methanol solvent mixture. The purest form of the metabolite was eluted with a methanol-chloroform system (5 : 95 v/v) and the process was continued until the complete elution of the compound. Finally, the compound was dried and appeared in a golden yellow waxy color.

Structural characterization of the pure compound

The structure of the isolated compound was elucidated by FT-IR (Thermo-Nicolet Nexus 670 FT-IR spectrophotometer, Thermo Fisher Scientific Inc.), 1D NMR spectra (Bruker Avance 300 and 600 MHz NMR spectrometers), and ESI-QTOF mass spectrophotometer (QSTAR XL Hybrid ESI-Q TOF mass spectrometer, Applied Biosystems Inc.).

In silico molecular docking studies

The in silico molecular docking procedures were performed to predict the binding mode and orientation of OMC and Indomethacin (Pub chem Id: 3715) at the active sites of the anti-inflammatory mediators, TNF- α and COX-2 using Molegro Virtual Docker (installed on an Intel Centrino Machine, Intel Corporation). The free energy of interaction was studied and expressed as a Mol Dock score. The pre-downloaded PDB structure of target proteins TNF- α (PDB ID: 2AZ5.pdb) and COX-2 (PDB ID: 4COX.pdb) were imported to the workspace. All parameters were kept in default. The binding site on the receptor was defined as extending in X=18.03, Y=166.10, and Z=196.29 directions around the active molecule with a radius of ~15.00 A˚. All the ligand structures were constructed using Chem 3D ultra 8.0 software, and then these structures were energetically minimized by using MOPAC (semi-empirical quantum mechanics), Jop Type with 100 iterations, and a minimum RMS gradient of 0.01, and saved as protein data bank (.pdb) format. The Mol Dock optimization search algorithm with a maximum of ten runs was used through the calculations, with all other parameters kept as defaults. One pose per run was retained based on root mean square division clustering using a heavy atom threshold set at 1.0 A˚ and an energy penalty of 100. All the poses were examined manually and the best poses were retained. The results were analyzed in terms of mole dock and rerank score. Furthermore, the hydrogen bonding interactions between the ligands (OMC, Indomethacin) and the active site amino acid residues of the enzyme were also established using 2D-Ligplot analysis.

In vitro anti-inflammatory activity

In vitro anti-inflammatory activity of the pure compound was performed by the protein denaturation method as described by Mizushima and Kobayashi 1968 with slight modification²⁴. The assay was performed using Bovine Serum Albumin (BSA) as a protein. In detail 1% BSA was solubilized in 50 mM sodium phosphate buffer (pH 6.4). The various test concentrations of 25, 50, 75, and 100 μg/ml were prepared using DMSO as a solvent. The reaction mixture consists of 0.1 ml of a test sample, and 0.2 ml of BSA and the final volume of the reaction mixture was makeup to 5 ml with buffer. The reaction mixture was incubated at 37°C for 20 min followed by heating at 95°C for 20 min. At room temperature, the turbidity of the resulting solution was measured at 660 nm using UV–Visible spectrophotometer (Model Spectrostar^Nano BMG LABTECH) whereas, negative control (without sample) and a positive control (indomethacin) have also been included in the assay. All these experiments were performed in triplicate and the results were expressed as Mean±SEM. The percentage inhibition of protein denaturation was calculated using the following formula to calculate the IC_50.

$$\mathrm{Percentage}\mathrm{inhibition}=\frac{(\mathrm{Abs}\mathrm{control}-\mathrm{Abs}\mathrm{sample})}{\mathrm{Abs}\mathrm{control}}\times 100$$

In vivo anti-inflammatory activity by carrageenan-induced paw edema model

The in vivo study was performed under ARRIVE criteria²⁵, Supplemental Digital Content 2, http://links.lww.com/MS9/A87 using the method of Carr-induced edema in the sub-plantar region of the right hind paw of the rats. Edema was induced by administering a subcutaneous injection of 0.1 ml of 1% freshly prepared suspension of carrageenan in the sub planar region of the rat’s right hind paw. Carrageenan induces inflammation by the release of various inflammatory mediators resulting from plasma extravasation, increased tissue water, and plasma protein exudation along with neutrophil extravasation, due to the metabolism of arachidonic acid. The anti-inflammatory activity was measured after 4 h to assess the potency of the test compound. In addition, inhibition of Carr-induced inflammation is highly predictive of anti-inflammatory drug activity in human inflammatory diseases and the dose of NSAIDs in this model correlates with the effective dose that should be administered to patients.

Furthermore, the anti-inflammatory effect of OMC was compared with the standard indomethacin. Being a basic method with minimal equipment, it was chosen over other methods but requires much practice to perform accurately.

Experimental design

The anti-inflammatory activity of the test compound was evaluated in Wistar rats by employing the method of Winter et al.²⁶. Animals fasted overnight and the study was performed under similar conditions using 8 rats per group having a total of 32 and continued for 6 h. Animals were divided into four groups, Group I (vehicle control), Group- II (positive control (1% Carrageenan), Group- III (Carrageenan + indomethacin), and Group IV (Carrageenan + OMC). The test compound OMC was administered by the oral route as gum acacia suspension (2%w/v) at the dose of 100 mg/kg. Animals in the standard group have received indomethacin at the dose of 10 mg/kg by oral route. Rats in the vehicle control group received the gum acacia suspension only. Finally, Groups II-IV were challenged with 0.1 ml of 1% carrageenan in the sub-plantar region of the right hind paw after one hour of drug administration. Paw volumes were measured before and after 4 h of carrageenan induction using a digital Plethysmometer (Ugo Basile). The disease induction (percent increase in paw thickness) was measured based on the paw volume difference between the paws of the carrageenan-induced and the control groups. The percent of inhibition of paw volume for treated groups was calculated by comparing it with the mean paw volume of the control group. The

$$\mathrm{Percentile}\mathrm{inhibition}=[1-(V_T/V_0)]\times 100.$$

$V_T$ - represents the edema volume in the drug-treated group.

$V_0$ - represents the edema volume in the control group.

Histopathology examination of the hind paw

The tissue samples of rat hind paws were collected after 5 h of intraplanetary injection of carrageenan followed by fixation of the tissue samples in a 10% neutral buffered formalin solution, embedded in paraffin wax. The 5-micron thick paw tissues were stained with hematoxylin-eosin (H&E) stain to observe pathological and morphological changes. Images of all samples were captured using a MOTIC BA310 microscope at ×10 magnification.

Immunohistochemistry studies (IHC)

The IHC analysis of paw sections was performed as described previously²⁷. The formalin-fixed and paraffin-embedded tissue sections (~4–5 µm thickness) were pretreated and incubated with anti-CD68, anti-CD 31, anti-CD80, and antiarginase-I primary antibodies overnight at 4⁰C and followed by pretreatment of tissues using EnVision^TM FLEX Target retrieval solution (Dako, Glostrup). Thereafter, tissue sections were stained using a solution containing diaminobenzidine-tetrahydrochloride and 0.5% H₂O₂ in 1X PBS (pH 7.6). Finally, all the slides were counterstained with hematoxylin and mounted. IHC evaluation of rat joints was performed in a blind fashion using a semi-quantitative score of 0–4 (0—no staining; 1–0–25 staining; 2–25–50 staining; 3–50–75 staining; 4—more than 75% staining). Images were acquired using a MOTIC BA310 microscope equipped with a color camera.

Statistical analysis

Statistical analysis was performed by one-way ANOVA (Dunnet´s Multiple Comparison Tests) using Graph Pad Prism Version 8 (Graph Pad). The statistically significant result was observed at P less than 0.001.

Results and discussion

Isolation, purification, and characterization of crude metabolite

The yield of the crude metabolite from the fermentation studies was found to be 2 g/l. The thin-layer chromatography results of the crude extract had shown a UV active metabolite as a single spot under UV. The crude extract was further purified by using column chromatography and the yield of the purified metabolite was assessed as 37 mg/l and appeared as a golden yellow waxy color substance. This purified compound was characterized by various analytical spectral data (NMR, FT-IR, HRMS, and HPLC) and data suggested that the purified compound is similar to that reported by Kumar et al., 2017, hence confirming that the present purified XO inhibitor belongs to the HAQ family and structure was identified as 1,8-Dihydroxy-3-methylanthracene-9,10-dione²¹ (Figure S1–S5, Supplemental Digital Content 1, http://links.lww.com/MS9/A86)²¹.

Docking studies

The Molegro Virtual Docker program successfully docked OMC into the active site of COX-2 and TNF- α and the results were expressed as Mol Dock score and Rerank score (Tables 1 and 2). The hydrogen bond interactions between active site amino acids of protein (COX-2, TNF- α) and ligand OMC, (Figs. 1 and 2), and indomethacin (Figs. 3 and 4) were represented diagrammatically.

Table 1.

Ligand-receptor interaction studies of OMC with TNF-α

Compound	Mol dock score	Rerank score	Number of H-bond interactions	Protein-Ligand interactions
OMC	−88.07	−69.26	0	–
Indomethacin	−117.365	−88.73	1	Ser 60 [B]-Oxygen [2.60 Ao]

Table 2.

Ligand-receptor interaction studies of OMC with COX-2

Compound	Mol dock score	Rerank score	Number of H-bond interactions	Protein-Ligand interactions
OMC	−99.99	−87.50	2	Ser530 [A]-Oxygen [3.17 Ao] Ser530 [A]-Oxygen [3.10 Ao]
Indomethacin	−136.411	−105.748	3	Ser530 [A]-Oxygen [3.10 Ao] Arg120 [A]-Oxygen [3.00 Ao ] Tyr530 [A]-Oxygen [3.09 Ao]

Figure 1.

Interaction of TNF-α with OMC (A) 3D structure (B) Ligplot structure.

Figure 2.

Interaction of TNF-α with Indomethacin (A) 3D structure (B) Ligplot structure.

Figure 3.

Interaction of COX-2 with Indomethacin (A) 3D structure (B) Ligplot structure.

Figure 4.

Interaction of COX-2 with OMC (A) 3D structure (B) Ligplot structure.

The ligand-active site amino acid interactions were represented in the form of 3D and 2D docking structures. The binding affinity was represented as the Mol dock score, Rerank score, and the number of hydrogen bond interactions. Figures 1 & 2 represents the hydrogen bonding interaction of OMC with TNF-α and COX-2 (4COX), respectively. Furthermore, it gives information about the type of amino acid and atom of ligand that is involved in the interaction process along with their bond length.

From the results, it has been evident that there is a stable binding interaction between the ligands and the proteins, which was confirmed by negative docking energy and a higher bond length (> 2.60 A^o). However, the binding interaction of standard drug indomethacin and OMC is strong with COX-2 when compared to TNF- α, which was confirmed by the higher number of hydrogen bonds (Tables 1 and 2). Whereas, the binding interaction of ligands with TNF- α was very weak owing to Vander walls and hydrophobic interactions rather than hydrogen bonding effects. This selective inhibition towards COX-2 is due to the large binding pocket of COX-2, which can accommodate bulkier groups easily in its binding pocket. It is well known from previous studies that Arg 120, Tyr 355, Ala 527, and Ser 530 are the active site amino acids in COX-2; however, serine 530 amino acid interaction is the prerequisite for the inhibitory potential of COX inhibitors. It has been well studied by the binding interaction of aspirin with COX-1/COX-2²⁸. Similarly, in the present study, the oxygen’s of the carbonyl group and 8-OH group of OMC formed two hydrogen bonds with Ser 530 of COX-2 indicating the prominent anti-inflammatory potential of OMC by inhibition of COX-2. These results are in corroboration with in silico studies of its structural analog chrysophanol and other derivatives²⁹, which explained that bulky substitutions on hydroxyl groups of chrysophanol attributed to inhibition of COX-2.

In vitro anti-inflammatory activity

After studying the anti-inflammatory activity of OMC by protein denaturation, it was found that the compound effectively inhibits the heat-induced denaturation of albumin protein at different concentrations when compared with standard Indomethacin as shown in Table 3 and Fig. 5.

Table 3.

In vitro anti-inflammatory activity of OMC by protein denaturation method

	% Inhibition
Compounds	25 (µg/ml)	50 (µg/ml)	75 (µg/ml)	100 (µg/ml)	IC50 (µg/ml)
1-O-Methyl Chrysophanol	10.75±0.24*	32.51±0.32*	59.05±0.34*	77.03±0.28*	63.50±2.19
Indomethacin	27.3±0.44*	45.5±0.64*	52.4±1.06*	72.5±0.80*	71.42±0.715

^*Note: Results are expressed as Mean±SEM and at P<0.001level of statistical significance.

Figure 5.

In vitro effect of OMC on protein denaturation.

It is well known that protein denaturation is one of the effects of inflammation. Anti-inflammatory drugs such as diclofenac sodium, salicylic acid, flufenamic acid, phenylbutazone, etc., have shown dose-dependent inhibition of thermally induced protein denaturation. The ability of polyphenols in inhibiting protein denaturation was studied previously³⁰. However, the effect of HAQ belonging to the group of polyphenols on protein denaturation was limited to their crude extracts only^31–33. This is the first study of isolated HAQ on inhibition of protein denaturation.

OMC alleviates inflammation in carrageenan-induced rat paw edema

The studies revealed the significant inhibition of inflammation by OMC in the late phase of the carrageenan-induced inflammation model. This is a widely used method to assess the anti-inflammatory activity of natural and synthetic chemical entities and comprises two phases³⁴. The early phase is accompanied by a release of acute inflammatory mediators like histamine and serotonin in the first hour of induction³⁵ which is followed by the release of prostaglandins in the last phase and lasts within 3–5 h³⁶. The results concluded that OMC showed promising results in the inhibition of the paw edema volume in the late phase (40.03±5.5) compared to standard indomethacin (52.8±4.7). This could be due to the inhibition of COX-2, which prevents the synthesis of prostaglandins in the late phase of carrageenan induction.

HAQ are well known for its anti-inflammatory activity since ancient times. In the present study, the OMC belonging to the class of HAQ showed a high inhibition rate (Fig. 6 and Table 4) in carrageenan-induced paw edema than other HAQ, that is, chrysophanol³⁷ aloe-emodin¹⁷. It has been evident from the Structure-Activity Relationship of HAQ that the presence of hydrophobic groups and bulky groups on the branches might improve the anti-inflammatory potential of compounds^29,38. Therefore, the methoxy group of OMC could be responsible for the potent inhibition compared to other HAQ possessing the same parent structure (1, 8-dihydroxyanthraquinone).

Figure 6.

In vivo inhibitory effect of OMC on Carrageenan-induced rat paw edema.

Table 4.

Anti-inflammatory effect of OMC on Carrageenan-induced rat paw edema

	The volume of paw edema (ml)
Groups	1 hr	2 hr	3 hr	4 hr	% inhibition
Group I (1% Carragenan)	1.79±0.07*	1.8±0.07*	1.392±0.07*	1.4±0.15*	–
Group –II (Carrageenan + Indomethacin)	1.308±0.07*	0.854±0.08*	0.748±0.09*	0.587±0.09*	52.8±4.7
Group –III (Carrageenan + OMC)	1.28±0.07*	1.106±0.10*	0.858±0.06*	0.728±0.06*	40.03±5.5

^*Note: Results are expressed as Mean±SEM and at P<0.001level of statistical significance.

Effect of OMC on pathological consequences of carrageenan-induced rat paw edema

Figure 7 shows the histopathological changes in the carrageenan-induced paw edema model. The histopathological evaluation of rat paw tissues was evaluated by using a semi-quantitative score (1–5) represented in Fig. 8. The results revealed that rats treated with vehicle control showed normal skin tissue with layers of the epidermis, sub-epidermis, and dermis without edema and inflammatory cells. Whereas, the carrageenan control tissue section showed extensive dermal and subcutaneous tissue edema extending to the subepidermal region including muscles. In addition, it showed moderate infiltration of acute inflammatory cells like neutrophils admixed along with occasional lymphocytes. However, the rats pretreated with indomethacin (10 mg/kg) showed lesser edema compared to the carrageenan group with occasional small foci of inflammatory cells Whereas the OMC-treated group showed reduced neutrophils number in the inflammatory infiltrate which is comparable to the standard drug indomethacin (P<0.001 in disease control vs. treatment groups shown in Fig. 8).

Figure 7.

Histopathology of rat paw tissue (H&E stain - 10×magnification): (A) Vehicle control (B) 1% Carrageenan-induced (C) Carrageenan + indomethacin (D) Carragenan + OMC. Arrows indicate the infiltration of acute inflammatory cells.

Figure 8.

Effect of OMC on histopathology of carrageenan-induced rat paw tissue: (A) Inflammatory cells (B) Edema. *P<0.001 level of statistical significance.

COX-2 is a constitutive housekeeping enzyme usually expressed in the kidney, brain, and ovaries. However, its expression levels will be elevated during the inflammatory conditions which are provoked by proinflammatory molecules such as IL-1, TNF-a, LPS, and TPA³⁹.

It is reported from earlier studies that COX-2 presents mainly in the neutrophils of inflammatory infiltrate^40,41. Therefore, the reduction in the number of neutrophils will automatically reduce the expression level of COX-2. Furthermore, Grover et al. (2014) explained the structure-activity relationship studies of chrysophanol and its derivatives, which had been suggested that the substitution of 1-, 8-OH groups in chrysophanol might enhance the COX-2 inhibition. In the present study too, OMC which had a methoxy substitution at 1-OH of chrysophanol led to the suppression of COX-2. This additive effect may further potentiate the anti-inflammatory activity of OMC.

Immunohistochemical (IHC) evaluation

IHC is an important diagnostic method used to confirm the expression of target molecules in the disease process. In the present study, the effect of OMC on the levels of proinflammatory markers (CD80, CD 31, CD68) and anti-inflammatory markers (arginase-I) were represented in Fig. 9.

Figure 9.

Immunohistochemical evaluation of rat paw tissue (A) CD80 (B)Arginase (C) CD 31 (D) CD68. Arrows indicate the staining area.

The results of IHC revealed the high expression levels of arginase-I, an enzyme that generates ornithine from arginine in Group IV. The production of ornithine promotes cell proliferation and repairs tissue damage in the repair type of M2 macrophages during inflammation and other associated disorders^42,43. This has been confirmed by the stimulatory effect of OMC for arginase-I, which promotes tissue repair mechanism during inflammation. Furthermore, CD 31 is an endothelial adhesion molecule that has been highly expressed during the inflammation process^44,45. The reduced levels of CD 31 in Group-III and Group IV compared to Group II confirm the anti-inflammatory potential of indomethacin and OMC, respectively. Similarly, there is a reduction in levels of CD68 in treatment groups (Groups III and IV), which is high in the inflamed group. CD68 is one of the members of the scavenging receptors of macrophages that can be significantly upregulated in response to inflammatory stimuli^46,47. Similarly, the levels of CD80 are high in Group II compared to treatment groups (III and IV). CD80 is the co-stimulatory molecule produced by T cell activation during inflammation/infection. The expression of high levels of CD80 leads to the production of inflammatory cytokines^13,48 (Fig. 9). These results explore the anti-inflammatory potential of OMC by down regulation of levels of inflammatory markers, that is, CD31, CD68, and CD80, and up-regulation of anti-inflammatory marker arginase-I. (Fig. 10).

Figure 10.

Effect of OMC on levels of (A) Arginase-I (B) CD80 (C) CD 31 (D) CD68 in rat paw tissue. *P<0.001 level of statistical significance.

Conclusion

In summary, the derivative of chrysophanol, OMC was isolated from A. thermoflava and showed significant anti-inflammatory activity both in in vitro and in vivo models. Furthermore, the compound showed in silico molecular interactions with TNF-α and COX-2. The anti-inflammatory activity of the compound was measured by denaturation of proteins (in vitro) and inhibition of carrageenan-induced inflammation in rat paw edema (in vivo). The activity is also confirmed by histopathological assessment. OMC reduced edema and neutrophils count in the inflammatory infiltrate in carrageenan-induced local acute inflammation. In addition, the compound upregulated the anti-inflammatory marker (arginase-I) and downregulated the inflammatory markers CD 31 (endothelial marker), CD68 (macrophage receptor), and CD80. Hence, it is further recommended to study the molecular mechanism of OMC against inflammatory mediators using in vitro cell line models, which could potentiate the further selective application of the compound in other inflammatory diseases such as gouty arthritis, rheumatoid arthritis, atherosclerosis, and coronavirus disease 2019.

Ethical approval

All the animal experiments were carried out as per the National Institutes of Health Guidelines for care and use of Laboratory Animals. Approval was granted by the institutional animal ethical committee of Indian Institute of Chemical Technology (IICT) under the Ethics No: IICT/IAEC/017/2019.

Consent

Not applicable.

Sources of funding

The authors declare that no financial and other support were received.

Conflicts of interest disclosure

The authors declared that there are no conflicts of interest.

Author contribution

All authors provided significant contribution for the study. U.R.B.: data collection, research work; Dr. J.R.S. and Dr P.R.S.: design of the work; Dr. N.P.: Design of docking and animal studies; Dr. C.C.: animal studies; Dr. K.M.: statistical analysis; Dr. S.M.V.: histophathology and IHC.

Guarantor

Dr. Joshna Rani Surapaneni, Dr. Prakasham Reddy Shetty.

Provenance and peer review

Not commissioned, externally peer-reviewed.

Supplementary Material

ms9-85-2617-s001.docx ^{(816.5KB, docx)}

ms9-85-2617-s002.doc ^{(65.5KB, doc)}