Potential role of embelin in the prevention of Freund’s adjuvant induced inflammation and ROS

Abstract

Inflammation is a complex biological response involving immune cells to an infection creating injury to the normal tissues. The anti-inflammatory efficacy of embelin, a benzoquinone from the plant Embelia ribes, was screened for antioxidant and anti-inflammatory activity in carrageenan and Freund’s adjuvant-induced inflammation models. Embelin exhibited significant dose-dependent antioxidant potential. In carrageenan-induced inflammation, embelin (20 mg/kg) showed an inhibition of oedema by 71.01 ± 0.12% and 81.91 ± 0.67% in Freund’s adjuvant-treated chronic inflammation model and resulted in a noticeable increase in adrenal size and restoration of the weight of spleen. Embelin also demonstrated cytoprotective effects on HEK-293 cells under induced oxidative stress. In silico analysis, embelin demonstrated binding energy of − 7.7 kcal/Mol and − 7.0 kcal/Mol with COX1 and COX2 with two hydrogen bonds. These results further prove that embelin could be a promising anti-inflammatory agent and supports the traditional use of Embelia ribes for rheumatism.

Introduction

Rheumatoid diseases are the inflammatory conditions that possibly lead to more complex disability than other group of diseases (Guo et al. 2018). In normal conditions, inflammatory and reparative progressions advance easily from injury towards healing (Claes et al. 2012). Under acute inflammatory conditions, eosinophils concentrate in large number in the tissues leading to restoration. Whereas, chronic inflammatory conditions of indefinite aetiology affect organ systems causing tissue damage rather than repair (Jacobsen et al. 2007; Medzhitov 2008). Rheumatoid arthritis (RA) is characterized by chronic inflammatory changes of the synovial tissue of joints, cartilage and bone. The emergence of RA is mostly based on genetic as well as epigenetic effects, but also other factors as tobacco smoke, dust exposure could lead to this inflammatory condition (Scherer et al. 2020).

Free radicals can damage cellular components, and evidence suggests that they may play a role in inflammatory joint disease. Reactive oxygen species (ROS) can directly or indirectly damage basic articular components, leading to the clinical presentation of inflammatory arthritis. Adjuvant-induced arthritis in rats using carrageenan and Freund's adjuvant closely resembles the clinical and pathologic features of human inflammatory arthritis. Reactive oxygen species play a direct role in the pathophysiology of inflammatory arthritis, and scavenging free radicals can help prevent the onset or progression of the disease.

Many anti-inflammatory drugs lead to diminution in inflammation and pain resulting from it (Crofford 2013). In allopathic medical care, both steroidal and non-steroidal anti-inflammatory drugs are used to relieve arthritic symptoms (Choi and Hwang 2003) Anti-inflammatories and analgesics help recover from pain and stiffness but do not avert joint damage or slowdown the disease progression. However, they induce only temporary relief and also produce severe side effects such as nausea, gastritis, alexia etc., (Begum and Sadique 1988). Plants are the chief sources for the discovery of novel pharmacologically bioactive compounds, with a lot of drugs derived directly or indirectly from them.

Embelin, a para benzoquinone from Embelia ribes has been reported to possess pharmacological activities (Chitra et al. 2003; Swamy et al. 2007; Singh et al. 2009; Mahendran et al. 2011). In this objective investigation, an attempt has made to understand the therapeutic effect of embelin against expression regulation of the inflammation signals in carrageenan-induced acute and complete Freund’s adjuvant-treated arthritic rat models, including the in vitro, in vivo antioxidant potential of the active principle.

Materials and methods

Materials

All chemicals, reagents and solvents used in the present study were of analytical grade. 2, 2-diphenyl-1-picrylhydrazyl (DPPH), Quercetin and Gallic acid were procured from Sigma Co. (USA). Hydrogen Peroxide, Dulbecco’s MEM, Fetal Bovine Serum (FBS), Trypsin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dimethyl sulphoxide (DMSO), Carrageenan, Freund’s adjuvant was procured from HiMedia Laboratories, Mumbai.

Experimental animals

Animals were procured from Central animal house, National Pharmacy College, Shimoga, and the experiments were carried out with the approval from Institutional Ethical Committee (Reg. No. 144/1999/CPCSEA/SMG). Animals were housed in cages under standard conditions and fed with standard pellets (Hindustan Unilever Ltd, India) and water ad libitum.

Preparation of drug

Embelin was isolated, purified from Embelia ribes and characterised in our previous work (Swamy et al. 2007). For oral administration, a suspension of embelin was dissolved in 0.9% saline, stored at 4 °C and warmed to room temperature before oral feeding. The drug formulations were prepared every fourth day and the drugs were administered orally by an oral gavage. A concentrated stock solution of diclofenac was prepared by dissolving the 100 mg tablet in the isotonic saline and further diluted prior to the experiment.

Acute toxicity test

Acute toxicity of Embelin was determined according to OECD guidelines 425. Mice weighing 20–25gm were divided into five groups (n = 6) were used to determine the safer dose. Administration of the drug was performed orally at doses of 100, 200, 300, 400 and 500 mg/kg bw. The observations for toxic symptoms during 48 h of drug administration and the rate of morbidity or mortality were recorded. The LD₅₀ of embelin was estimated within a set time-frame.

In vitro antioxidant activities

The method modified by Brand-Williams et al. 1995 was employed to investigate the DPPH free radical scavenging activity of embelin. Superoxide anion scavenging activity was measured based on the method of Nishikimi et al. (1972). Decreased absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. Hydroxyl radical (OH) Scavenging activity is done by measuring the hydroxyl radical generated by the Fenton reaction was measured according to the modified method of Kim and Minamikawa (1997).

Protective effects of Embelin on induced oxidative stress in HEK-293 cells

HEK-293 cells were procured from NCCS, Pune, India. The cells were cultured in DMEM media containing 10% FBS. MTT assay was used to evaluate the cytotoxic effects of Embelin at different concentrations (0.15–10 µM). Further the protective effects of Embelin on H₂O₂ treated (400 µM) HEK-293 cells for 2 h and the viability was determined using MTT assay (Mosmann 1983).

Intracellular reactive oxygen species (ROS) assay was performed according to the method of LeBel et al. (1992). HEK-293 cells were pretreated for 24 h with different concentrations of embelin (0.31–2.5 µM) followed with 400 µM H₂O₂ treatment for 2 h. Cells were further stained with Dichloro-dihydro-fluorescein diacetate (DCFH-DA) and fluorescence was observed with fluorescent microscope. Acridine orange/Ethidium bromide (AO/EtBr) double staining was performed on H₂O₂-induced oxidative damage in HEK-293 cells and cells were observed under fluorescent microscope (Kasibhatla et al. 2006).

In vivo antioxidant activity

The animals were grouped into three groups containing six animals in each group. The first group served as control, the second group was administered carbon tetrachloride (negative control), and the third group was administered with embelin. The extract was suspended in 0.5% sodium carboxymethylcellulose and was fed to third group rats via an oral route at 20 mg/kg body weight for 14 days, saline was administered to the animals in the first and second groups until the 7th day.

A single oral dose of carbon tetrachloride (1:1 in liquid paraffin) was administered to animals in the second and third groups at 1.25 mL/kg body weight 6 h after the last dose of extract/saline was administered on the 7th day. Rats were sacrificed after 24 h and the liver homogenate was prepared. Five percent homogenate was made in 0.15 M KCl and it was centrifuged for 10 min at 800 g. The supernatant was, thus, evaluated for lipid peroxidation (Buege and Aust 1978), peroxidase (Alexander 1962) catalase (Aebi 1984) and superoxide dismutase (SOD) (Beauchamp and Fridovich 1971).

Histopathological studies

Histopathological sections of liver in all the groups of rats were fixed in ten percentage of neutral formalin solution for 24 h and dehydrated in a sequence with solutions of ethanol-xylene series (Mukherjee 2013). Further, inflicted material was fixed with paraffin at 40–60 °C. Microtome sections were taken at 10 μm thickness. Processed sections were thus stained with haematoxylin–eosin and observed under the microscope to evaluate the protective effect of embelin against the toxic effects of CCl₄.

Carrageenan-induced hind paw oedema

Male Swiss albino rats (150–200 g) were used for this study. The animals were divided into five groups each containing eight animals. Group one served as a control, group two served as standard drug diclofenac (20 mg/kg bw) and group three, four and five animals were administered with three different doses (10 mg/kg bw, 20 mg/kg bw and 30 mg/kg bw) of test drug embelin orally 30 min before carrageenan injection. Paw oedema was induced through intradermal injection of Carrageenan (0.1% in normal saline solution) to the plantar surface of the right hind paw of the rats with a volume of 0.1 ml. Hind paw volumes were determined using plethysmograph, acute inflammation was measured by the increase in volume of the treated paw 3 h after carrageenan injection.

Freund’s adjuvant-induced chronic inflammation

Male Swiss albino rats weighing 150–200 g were used for this study. The animals were divided into five groups of eight each. Group one served as adjuvant control, group two served as standard and group three, four and five were group test. All the five groups of animals were injected with Freund’s complete adjuvant (0.5 mg/0.1 ml killed Mycobacterium butyricum, Bangalore Genei, Bangalore), into the left hind paw. The body weight was recorded at the beginning of the experiment and subsequently at weekly intervals. The adjuvant control group received vehicle only (0.9% saline), the second group was treated with 20 mg/kg bw of standard drug Diclofenac (Novarties India Ltd., Pune) for 28 days. The third, fourth and fifth group of animals received a daily dose of test drug embelin at the concentration of 10 mg/kg bw, 20 mg/kg bw and 30 mg/kg bw. Chronic inflammation was estimated by the attenuation in volume of the treated and untreated paw 28 days after administration of adjuvant injection using a plethysmograph. The following formula was used to calculate the inhibition of acute and chronic inflammation (Talat et al. 2015).

$$\text{Paw oedema}=\frac{\text{Final paw volume}-\text{Initial paw volume}\times 100}{\text{Initial paw volume}}.$$

On 7th, 14th, 21st and 28th day, each rat in all the groups were placed separately on an experimental table, gait test was performed and allowed to move freely. Two observers unaware of the drug treatment and their assessment were taken into consideration to score the use of untreated right paw as follows; No use of paw was scored as 0, submissive use to support the body scored as 1; active use of the right paw is scored as 2. Due to major damage of the treated paw, rats with a score of ‘0’ exhibited a creeping behaviour (moving on the two forelegs, dragging the two hind paws). Scores for each animal was also added collectively to attain a group score.

Radiological studies

Radiological studies were performed according the method of (Uttra and Hasan 2017). On 28th day after adjuvant injection, the rats were anaesthetized with ether and radiographs were taken in a lateral-medial direction of the treated and untreated hind paw. An experienced X-ray technician not aware of the drug treatment observed the condition of tibiotarsal joints.

Erythrocyte sedimentation rate (ESR) estimation

On day 28, all the animals were sacrificed by cervical dislocation and blood samples were collected separately into a dry test tube by cardiac puncture. The ESR was estimated by mixing 5 ml of blood with 5% sodium-citrate solution, subsequently transferring the mixture into a glass ESR pipette, wherein the sedimentation rate was measured (in mm) after 1 h. The spleen and adrenals from the sacrificed animals were removed and wet weights of these organs were recorded.

In silico analysis

The in vitro, in vivo protective and anti-inflammatory activities of embelin were validated using in silico molecular docking simulation studies. 2D structure of embelin and standard diclofenac was retrieved in SDF format from the Pubchem database and the energy minimization was performed using Dundee prodrg server (Schüttelkopf and Van Aalten 2004; Bolton et al. 2008). The ligands were further converted into PDBQT with the help of ‘MGL tools’ graphical interface program (Dallakyan and Olson 2015). 3D coordinates of the crystal structure of COX1 (PDB ID: 5WBE) and COX2 (PDB ID: IPXX) and was obtained from the Protein Data Bank (PDB) (Berman et al. 2003) ‘MGL tools’, was used to adjust the gridbox to run docking simulations. AutoDockVina was used to explore the best docked conformation of ligand with protein and binding energy was determined. LigPlot + and PyMol were used to infer the pictorial depiction of interaction between the ligands and the target protein (DeLano 2002; Laskowski et al. 2011).

Results and discussion

In vitro antioxidant activity

An active constituent embelin at the concentration of 18.69 ± 0.39 µg/ml showed 50% inhibition in DPPH scavenging activity and increasing antioxidant activity of the molecule in different concentrations. The markable (50%) superoxide anion scavenging activity was reported in embelin (161.5 ± 3.74 µg/mL) when compared with the standard Butylated hydroxytoluene (BHT) (90.2 ± 0.55 µg/mL). The decrease in the absorbance at 560 nm with the active constituent embelin, thus, indicated the consumption of superoxide anion in the reaction mixture. Hydroxyl radical scavenging of embelin exhibited IC₅₀ of 144.01 ± 0.85 µg/mL which is similar to that of BHT (IC₅₀ of 150.16 ± 0.66 µg/ml). The embelin at the concentration of 250 µg/ml showed maximum of 82.82 ± 0.58% of inhibition (Table 1). According to these results the radical scavenging activity is attributed to phenolic nature of the compound.

Table 1.

Percentage of inhibition and IC₅₀ values of embelin in DPPH radical scavenging activity, superoxide anion scavenging activity and hydroxyl radical scavenging activity

Activity	Embelin (μg/ml)	% inhibition	IC50 (µg/ml)	IC50 Standard (BHT) (µg/ml)
DPPH Radical Scavenging activity	10	8.5 ± 0.11	18.69 ± 0.39	3.61 ± 0.17
	20	19.98 ± 0.89
	30	37.8 ± 0.02
	40	62.18 ± 0.53
	50	83.22 ± 1.60
Superoxide anion scavenging activity	50	46.34 ± 0.75	161.5 ± 3.74	90.2 ± 0.55
	100	45.95 ± 0.02
	150	48.84 ± 0.26
	200	52.08 ± 0.20
	250	54.21 ± 0.15
Hydroxyl radical scavenging activity	50	19.48 ± 0.28	144.01 ± 0.85	150.16 ± 0.66
	100	33.59 ± 0.34
	150	52.15 ± 0.19
	200	73.67 ± 0.25
	250	82.82 ± 0.33

Results are expressed as mean ± SE

Protective effects of Embelin on induced oxidative stress in HEK-293 cells

Embelin at various concentrations did not show any cytotoxicity in the cell proliferation over 24 h. Embelin showed more than 70% cell viability in all the concentrations ranging from 0.5–10 µM in H₂O₂-induced oxidative stress (Fig. 1). Embelin inhibited intracellular accumulation of ROS in comparison to H₂O₂ treated negative control as showed in the (Fig. 2). Similar results were observed wherein icarin ameliorated cisplatin induced oxidative stress in HEK-293 cells and reduction in ROS during intracellular ROS assay (Zhou et al. 2019). Previous reports also showed the embelin prevented UVB damage in lymphocytes through intracellular reduction in ROS. (Radhakrishnan et al. 2012).

Fig. 1.

Protective effects of embelin against H₂O₂-induced oxidative damage in Human Embryonic Kidney-293 cells assessed by MTT assay

Fig. 2.

Intracellular ROS assay to determine the ROS with DCFH-DA probe. (A) Control cells (B) Cells treated with 400 µM H₂O₂. (C). Cells treated with 0.31 µM embelin with 400 µM H₂O₂ (D). Cells treated with 0.62 µM embelin with 400 µM H₂O₂ (E). Cells treated with 1.25 µM embelin with 400 µM H₂O₂ (F). Cells treated with 2.5 µM embelin with 400 µM H₂O₂

Acridine orange/ethidium bromide (AO/EtBr) staining was used to visualize nuclear changes and apoptotic body formation that are characteristics of apoptosis. Cells pretreated with embelin showed better cell viability compared to H₂O₂ treated negative control which showed more number of apoptotic cells (Fig. 3). Previous results showed an inhibition in oxidative stress in buccal cells wherein cytoprotective effects of Marmelosin was established through the dual staining method (Pynam and Dharmesh 2018).

Fig. 3.

Cytoprotective effects of embelin in HEK-293 cells with AO/EtBr double staining. (A) Control cells (B) Cells treated with 400 µM H₂O₂. (C) Cells treated with 0.3125 µM embelin and 400 µM H₂O₂ (D) Cells treated with 0.625 µM embelin and 400 µM H₂O₂ (E) Cells treated with 1.25 µM embelin and 400 µM H₂O₂ (F) Cells treated with 2.5 µM embelin and 400 µM H₂O₂ 

Acute toxicity

Oral treatment of mice with the embelin up to 500 mg/kg showed obvious behavioural changes after administration. Lethal dose (LD₅₀) could be evaluated as the highest dose tested that resulted in mortality. All groups exhibited hypersensitivity effects within 2 h of drug administration. Dose of 300 mg/kg resulted in the death of one-third, 34% within 2 h, while 100% death was observed with a dose of 500 mg/kg. These implied that the LD50 was 300. In comparison with the toxicity-rating chart the embelin could be classified as non-toxic substance when administered by oral route (Gosselin et al. 1984).

In vivo antioxidant activity

The studies of the oxidative stress markers implied that the chronic administration of embelin decreased the levels of LPO, SOD and CAT and peroxidases. Chemopreventive effect of embelin was apparent against CCl₄-induced liver cirrhosis. The treatment of rats with a single dose of CCl₄ at 1.25 ml/kg of body weight significantly reduced the activities of catalase, SOD by 1.60 ± 0.09 IU/min/mg of tissue, 0.14 ± 0.03 IU/min/mg of tissue, and increase in the activity LPO and peroxidase by 8.40 ± 0.33 nano mol of MDA/mg weight of tissue and 9.13 ± 0.45 IU/min/mg of tissue, respectively. The lipid peroxidation activity increased to twofold in comparison with the control owing to CCl₄ treatment. However, pretreatment of the rats with the embelin at 50 mg/kg caused restoration of catalase activity, SOD and inhibition of LPO and peroxidase activities by 3.33 ± 0.09 IU/min/mg of tissue, 0.33 ± 0.05 IU/min/mg of tissue and 4.65 ± 0.16 nano mol of MDA/mg and 4.78 ± 0.13 IU/min/mg of tissue of the control values, respectively. This showed the protection provided by feeding of embelin to the rats by protecting and maintaining the activities of these enzymes even after CCl₄ treatment. The effect of treatment of rats with embelin followed with administration of carbon tetrachloride on the effects of various enzymes and the lipid peroxidation; catalase; Super Oxide Dismutase; peroxidase is summarized in Table 2. The bioactivity of the constituent was attributed to the hydrogen-donating ability (Shimada et al. 1992). The antioxidants are known to interrupt the free radicals and donate hydrogen from the hydroxyl groups, resulting in the formation of a stable product. Administration of embelin reduced the effects of hydrogen peroxide and superoxide ions, resulting in the reduction of the lipid peroxidation; thus, enabling enzyme activity.

Table 2.

In vivo antioxidant activity of embelin against CCl₄-induced oxidative stress

Treatment	Catalase (IU/min/mg of tissue)	SOD (unit/min/mg of tissue)	LPO (n mol of MDA/mg of tissue)	Peroxidase (unit/min/mg of tissue)
	Liver	Liver	Liver	Liver
Normal	3.83 ± 0.19	0.37 ± 0.05	3.90 ± 0.13	3.90 ± 0.24
Control (CCl4)	1.60 ± 0.09	0.14 ± 0.03	8.40 ± 0.33	9.13 ± 0.45
Silymarin + CCl4	3.53 ± 0.07	0.35 ± 0.05	4.55 ± 0.15	4.75 ± 0.18
Embelin + CCl4	3.33 ± 0.09	0.33 ± 0.05	4.65 ± 0.16	4.78 ± 0.13
F value	54.2	4.15	72.0	53.4

Values are mean ± SE. n = 6 in each group

Histopathology of liver tissue in control group showed normal hepatocytes with portal triad, veins, visible hepatic artery and vein Fig. 4A. CCl₄-treated rats showed marked destruction of hepatic architecture accompanied by degeneration, areas of haemorrhage, inflammatory infiltrations and necrosis Fig. 4B. In the group pretreated with embelin followed by CCl₄, the liver retained the normal hepatic architecture with few areas of haemorrhage indicating the protective effect of embelin Fig. 4C. The results strongly indicate the protection provided by the constituent embelin. Similar results were previously reported wherein embelin treated at 25 mg/kg body weight showed peroxidative damage which was negligible in hepatic tissue showing potent effects in CCl₄-treated rats (Singh et al. 2009; Poojari et al. 2010). Other results also showed that embelin at a concentration of (25 mg/kg bw) showed reduction in oxidative stress markers such as SOD, MDA, catalase, inflammatory cytokines and and protein expression of NF-kB, p38 MAPK under paraquat induced damage in wistar rat model (SreeHarsha 2020).

Fig. 4.

(A) Sections of the liver tissue of the Control group showing hepatocytes with normal architecture and portal triad, portal veins, and hepatic artery and vein. Vein (V), and artery (A) (H&E 100X). (B) Section of the liver tissue of the CCl₄-treated rats showing total loss of hepatic architecture, areas of hemorrhage, necrosis (N), hepatic duct (D), vein (V), fatty vacuoles (F) and Artery (A) (H&E 100X). (C) Section of the liver tissue of rats pretreated with embelin followed by exposure to CCl₄, the liver is showing the normal hepatic architecture with few areas of hemorrhage between the columns of hepatocytes. Hepatic duct (D), vein (V), and artery (A) (H&E 100X)

Carrageenan-induced hind paw oedema

Carrageenan-induced hind paw oedema model was employed for anti-inflammatory assessment, the embelin at the dose of 20 mg/kg bw exhibited statistically significant inhibition (71.01 ± 0.12%) whereas, at the dose of 30 mg/kg bw and 10 mg/kg bw inflammation inhibition was less significant. The results were quite comparable with the standard drug Diclofenac which showed significant inflammatory inhibition of 71.79 ± 03% as depicted in Table 3. It has been recorded that carrageenan-induced paw oedema is an appropriate in vivo model to anticipate the effects of anti-inflammatory drugs whose action is to inhibit the mediators of acute inflammation (Morebise et al. 2002). Recent study from Cui et al. 2020 showed that the chitosan nanoparticle loaded with embelin (25 and 50 mg/kg) showed reduction in adjuvant-induced arthritis through downregulation of NO, MDA, TNF-alpha, IL-6 and IL-1β.

Table 3.

Effect of embelin on acute and chronic inflammation

Groups	Acute Inflammation	Chronic inflammation				F value
	(% of inhibition)
	(After 3 h)	7th day	14th day	21st day	28th day
Control	–	14.02 ± 0.47	11.18 ± 0.54	8.16 ± 0.30	37.53 ± 6.26	18.1
Standard	71.79 ± 03*	44.60 ± 0.97	63.73 ± 0.77	72.29 ± 0.84	84.84 ± 0.48*	461.3
Embelin
10 mg/kg	68.44 ± 6.34	18.40 ± 0.57	25.73 ± 0.77	32.76 ± 0.68	39.62 ± 0.57	195.1
20 mg/kg	71.01 ± 0.12*	23.65 ± 0.73	40.28 ± 0.60	58.55 ± 0.64	81.91 ± 0.67*	936.3
30 mg/kg	69 ± 0.06	40.50 ± 0.32	62.80 ± 0.53	70.52 ± 0.48	62.53 ± 0.26	1152.0

Values are mean ± SE; n = 8 in each group

P < 0.01 when compared to control

Adjuvant-induced chronic inflammation

In adjuvant-induced chronic arthritic model, the rats developed chronic swelling at multiple joints with the impact of inflammatory cells, destruction of joint cartilage and bone. These inflammatory changes eventually result in the complete damage of joint integrity and functions in the affected rats. The untreated paw of the control animal showed chronic inflammation due to the effect of Freund’s adjuvant (Fig. 5A). The increase in size of the chronic inflammation was significantly reduced to normal size in the animals treated with 20 mg/kg bw embelin (Fig. 5E) as similar to that of standard drug diclofenac treated animals (Fig. 5C). In gait test, the gross malfunction of the paw was observed in the adjuvant control group which was apparent from the very low group score of 9 and the animals crawled with their forelimbs. Among the three doses of embelin tested, maximum improvement in gait was observed in the rats treated with 20 mg/kg bw embelin and the gait group score of all the animals is shown in Table 4. The adjuvant disease is often accompanied by an attenuation in adrenal corticosteroids and treatment with embelin resulted in a marked increase in adrenal size (Zurier and Weissmann 1972). Earlier reports from Dharmapatni et al. (2015) showed that embelin reduced the inflammation and bone erosion in collagen antibody induced arthritic mice. Our results are in correlation with this previous report wherein low-dose embelin at 20 mg/Kg was more effective in treating inflammation compared to the higher doses.

Fig. 5.

(A) The adjuvant control rat showing swollen paw which received daily oral administration of saline (0.9%). (B) X-ray of control animal taken in lateral-medial direction of the untreated hind paw involving arthritic lesions, tibiotarsal joint destruction. (C) The rat treated with the standard drug Diclofenac (20 mg/kg bw) showing reduced inflammation in the hind paw. (D) X-ray of the hind paw of the animal treated with standard drug Diclofenac showing reduction in arthritic lesions with no tibiotarsal joint destruction. (E) The embelin (20 mg/kg bw) treated rat paw showing completely reduced inflammation after 28 days. (F) X-ray of the hind paw of the animal treated with embelin showing reduction in arthritic lesions with no tibiotarsal joint destruction

Table 4.

Effect of embelin on gait of the arthritic rats

Groups	7th day	14th day	21st day	28th day	Total score
Control	0	1	1	7	9
Standard (diclofenac)	2	3	9	14	28
Embelin
10 mg/kg	2	4	8	3	17
20 mg/kg	3	5	8	14	29
30 mg/kg	2	4	6	10	22

Radiological study

The radiological studies of the tibiotarsal joints showed gross destruction of the joints of the untreated paw of the adjuvant control group (Fig. 5B). The test drug embelin and the standard drug diclofenac at the dose of 20 mg/kg bw each were most effective in preventing the tibiotarsal joint destruction of the animals (Fig. 5D, F). Reduction in chronic inflammation was accompanied by repression of arthritic lesions which was observed in the non-injected paw. The X-ray studies showed that the embelin almost completely countered the joint destruction. Chronic inflammation implicates the release of the number of inflammatory mediators like GM-CSF, cytokines (IL-IB and TNF-α), PGDF and interferons. These inflammatory mediators are responsible for causing pain, destruction of bone and cartilage that could result in severe disability (Eric and Dick 1996).

Erythrocyte sedimentation rate (ESR) estimation

The ESR value of the adjuvant control is 7.05 ± 0.09 mm/h. In embelin treated group, ESR value is significantly decreased to 2.02 ± 0.05 mm/h whereas, in standard drug Diclofenac-treated animals, the ESR values 5.01 ± 0.12 mm/h. The ESR count was upregulated in arthritic control group has been significantly countered by the standard drug diclofenac and the test drug embelin; thus, justifying its key role in arthritic conditions (William 1996). The loss of body weight and a decrease in weight of the spleen were noticed in the control adjuvant-treated group. Whereas, in embelin treated animals weight of the spleen was restored as similar to that of normal animals. The data of the difference in ESR value and the body weight of the different animals are shown in Table 5.

Table 5.

Effect of embelin on weight of adjuvant-induced arthritic rats

Groups	Initial body weight (g)	Final body weight (g)	Spleen weight (g)	Adrenal weight (mg)	ESR (mm/h)
Control (0.9% saline)	192.38 ± 3.97	189.38 ± 4.12	0.94 ± 0.02	0.06 ± 0.01	7.05 ± 0.09
Standard (diclofenac)	196.75 ± 3.17	202.75 ± 1.98*	0.70 ± 0.01*	0.04 ± 0.01	5.01 ± 0.12*
Embelin
10 mg/kg	190.62 ± 3.20	187.12 ± 2.67	0.83 ± 0.02	0.06 ± 0.01	5.88 ± 0.12
20 mg/kg	196.88 ± 1.62	201.62 ± 1.76*	0.70 ± 0.01*	0.03 ± 0.01	2.02 ± 0.05*
30 mg/kg	192.25 ± 2.58	190.38 ± 2.24	0.79 ± 0.01	0.04 ± 0.01	4.06 ± 0.09

Values are mean ± SE; n = 8 in each group

*P < 0.01 when compared to control

In silico analysis

The cyclooxygenases are the important class of enzymes which convert arachidonic acids to prostaglandins and these lipid mediators play a major role in pain and inflammation (Smith et al. 1998). Considering COX1 and COX2 as the target receptors automated docking studies with Embelin was conducted to evaluate the best in silico conformation using Autodock Vina. Docking of Embelin with the targets presented established bonds with one or more amino acids within the active pocket of the target receptor. Embelin showed binding affinities of − 7.0 and − 7.7 kcal/mol with two hydrogen bonds with COX1 and COX2, respectively. Diclofenac showed binding affinities of − 8.0 and − 8.8 kcal/mol with 2 hydrogen bonds with COX1 and COX2, respectively. Ligplot was used to visualize the 2D representation of the ligand–protein complex and pymol was used to visualize the 3D representation of the Embelin–protein complex as shown in Fig. 6.

Fig. 6.

In silico analysis of embelin and standard drug diclofenac with COX1 and COX2 proteins. (A) 2D representation of embelin and COX1 protein interaction (B) 3D representation of embelin and COX1 protein interaction (C) 2D representation of embelin and COX2 protein interaction (D) 3D representation of embelin and COX2 protein interaction. (E) 2D representation of diclofenac and COX1 protein interaction (F) 3D representation of diclofenac and COX1 protein interaction (G) 2D representation of diclofenac and COX2 protein interaction (H) 3D representation of diclofenac and COX2 protein interaction

Conclusion

The present investigation supports traditional claims of Embelia ribes Burm for relieving rheumatic inflammation and provides new perspectives for the therapeutic use of the active compound embelin isolated from Embelia ribes, which is safe, effective and better option as anti-inflammatory agents than synthetic ones. The current study on embelin from the Embelia ribess suggested that it could be a good candidate for pharmaceutical use.

Author contributions

KHM performed all the experiments and wrote the manuscript. Prof. VK supervised the whole work and edited the manuscript. SR helped in performing certain parts of the experiment. SND helped in compound characterization. MP conducted in silico analysis. SKJ and PN performed cell-based assays. All the authors contributed in editing the manuscript.

Declarations

Conflict of interest

Authors declare no conflict of interest.