Introduction

Glycyrrhiza glabra L. (Leguminosae) is an approximately one-meter tall perennial herb commonly known as sweet wood and mulethi, and native in Western Asia, Eurasia (Fiore et al. 2005; Shah et al. 2018). The roots of the Glycyrrhiza species are commonly known as licorice. Mulaithi root has numerous pharmacological properties like anti-viral, anti-cancer, anti-diabetic, anti-microbial and anti-inflammatory properties in addition to cardioprotective, hepatoprotective and immunomodulatory effects and its stem is used for the treatment of tuberculosis (Arseculeratne et al. 1985; Asl and Hosseinzadeh 2008). It is used for the treatment of mouth ulcers, alimentary tract disorders, aphrodisiac and also used to cough-relieving treatment (Anil and Jyotsna 2012; Saxena 2005; Sedighinia and Afshar 2012). Licorice mainly contains pentacyclic triterpenoid saponins and phenolics. This includes glycyrrhizin, glycyrrhetinic acid, flavonoids, isoflavonoids, chalcones, coumarins, stilbenoids, etc. Some major phenolics present in licorice are liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin, glabridin, formononetin, glabrene, hispaglabridin A, hispaglabridin B, glabrol etc. (Chin et al. 2007; Mamedov and Egamberdieva 2019; Nomura et al. 2002). Liquiritigenin (LTG) and its structurally related bio-precursor, Isoliquiritigenin (ISL), are two majorly explored bioactive secondary metabolites that possess diverse pharmacological activities like anti-inflammatory, anti-oxidant, anti-cancer, hepatoprotective and cardioprotective activities (Peng et al. 2015; Ramalingam et al. 2018). Isoliquiritigenin reduced osteoclastogenesis by reducing the expression of RANKL-RANK-TRAF6 pathways, thereby suppressing abnormal bone formation (Ji et al. 2018). Further, it also suppresses the activation of NF-κB and the production of MMPs induced by IL-1β in chondrocytes (Zhang et al. 2018). The earlier reports suggest that phenolics present in G. glabra showed significant anti-inflammatory potential; therefore present study aims to develop phenolics (ISL & LTG) rich extract of G. glabra coded as IVT-21 and explore their anti-arthritic activity in the collagen-induced arthritis model.

Inflammation is an important host defense mechanism against various stimuli such as infections, physical injury and chemical injury. Inflammation is initiated by migration of immune cells from blood vessels and release of mediators at the damage site and release of pro-inflammatory cytokines, reactive oxygen species and reactive nitrogen species to eliminate the infection and restore the injured tissues. Normal inflammation resolves quickly and self-limits, but abnormal resolution and continued release of inflammatory mediators result in chronic inflammation. Chronic inflammation is not a disease but a mechanistic process. The diseases associated with chronic inflammation are multiple and include cardio-vascular diseases, neurological disorders, diabetes, auto-immune disease, chronic hepatic and renal diseases.(Roe 2021). Rheumatoid arthritis (RA) is a progressive auto-immune joint disease caused by environmental and genetic factors (Meyer et al. 2019). The yearly occurrence of RA is approximately three in ten thousand people, increasing with age and peaking between 35 and 50 years (Gabriel 2001). Inflammation, cartilage destruction, synovial tissue hyperplasia and vasculitis are the common characteristics of RA. While the mechanism of RA is unknown, evidence from biological, genetic and histopathological studies both in clinical research and in-vivo models shows that inflammation and joint destruction are linked with stromal tissue dysregulation and immune-mediated etiologies (McInnes and Schett 2007). Inflammatory markers (TNF-α and IL-6) play a crucial role in RA diseases, so it is important to maintain a balance between the anti-inflammatory and pro-inflammatory markers (Alam et al. 2017). Production of TNF-α, induces several inflammatory markers like chemokines, cytokines,matrix metalloproteinases (MMPs), which finally leads to synovial inflammation and joint destruction (McInnes and Schett 2007). Nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMRADs), immunosuppressive, and hormone-based drugs are use for the treatment of rheumatoid arthritis but regular and long time use of these drugs have several side effects in RA patients. So, new medicines for RA management with a low occurrence of side effects and high effectiveness are urgently required (Kun et al. 2020). Medicinal and aromatic plants are being used for thousands of years in the traditional systems of medicine for the treatment of several diseases. Natural compounds like flavonoids, steroids, lignans, polyphenols, coumarins, terpenes, alkaloids etc. are scientifically verified to alleviate inflammation, pain and fever (Conforti et al. 2009; Singh et al. 2014). The experimental data in this study showed that treatment of IVT-21, a Isoliquiritigenin, Liquiritigenin rich root extract of Glycyrrhiza glabra is able to reduce the production of pro-inflammatory cytokines from activated macrophages as well as its oral treatment is able to attenuate collagen-induced arthritis in Rats. These results suggested that IVT-21 would be a promising therapeutic agent for additional research into the treatment of arthritis and other chronic inflammatory illnesses.

Materials and methods

Chemicals

Collagen, Complete Freund’s Adjuvant, Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Sigma-Aldrich. Dexamethasone was procured from Qualigens Fine Chemicals, India. C-RP kit (ERBA diagnostics kit), IL-17A ELISA kit (KRISHGEN Biosystem), Rat-specific MMP-1 and MMP-9 ELISA kits (Elabscience) Mouse-specific (TNF-α, IL-6) and rat-specific (TNF-α, IL-6) ELISA Kit was procured from BD Biosciences, USA. NFkB-p65 primary antibodies were procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA), N-(1-Naphthyl) ethylene-diamine dihydrochloride (NED) SD fine chem limited, Secondary antibody (Jackson immune).

Plant material

G. glabra roots were obtained from Dabur India and authenticated by Dr. Narendra Kumar, taxonomist of the institute.

Extract preparation

The 2 kg of G. glabra roots were cut into small parts, dried under a shaded environment, and powdered with a mechanical grinder. The powder roots were extracted with 4 L of the ethanol–water mixture (1:1) at room temperature for 6 h. This process was repeated for three times (4 L X  3 times). After filtration, the combined hydroalcoholic solution was evaporated on the rota evaporator, giving 376 g of hydroalcoholic extract. This extract was subjected to acid hydrolysis in the presence of 5% HCl solution, and the resulting reaction mixture was extracted with ethyl acetate which gave 81.6 g of ethyl acetate extract obtained by evaporation in a rotary evaporator. The ethyl acetate extract was fractionated on silica gel (60–120 mesh size) chromatography using a mixture of ethyl acetate and chloroform solvent system as an eluent. The solvent system changes as a gradient mode from 100 CHCl3 to 25% Ethyl acetate: CHCl3. First, elution of column from CHCl3 and 5% ethyl acetate: CHCl3 solvent systems provided fractions containing an oily mixture. After that, other non-polar compounds came out at around 8–10% ethyl acetate: CHCl3 solvent system. Then isoliquiritigenin followed by liquiritigenin came out around 12–15% ethyl acetate: CHCl3 solvent system. Other polar compounds were eluted from 20 to 25% ethyl acetate: CHCl3 solvent system. Discarding the first oily fraction and last polar compounds containing fractions, all the middle fractions eluted between 8 and 15% ethyl acetate: CHCl3 solvent systems were combined and concentrated on a rota evaporator to provide 29.1 g of ISL and LTG enrich final extract, which was coded as IVT-21. The schematic representation of enrich extract preparation is given in Fig. 1.

Fig. 1
figure 1

Schematic presentation of the preparation of standardized enrich extract

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Chemical characterization of IVT-21

Authentic Standard Compounds (ISL and LTG) were procured from Sigma-Aldrich. The HPLC analyses were carried out on Waters Alliance 2695 separation module equipped with a 2998 photodiode array detector (Waters, Milford, MA, USA). Separation was carried out using a Phenomenex C18 Luna® column (5 um; 250 × 4.6 mm). The TLC analyses were performed on silica gel TLC plates of Merck, India, with the fluorescent indicator (F254), and TLC plates were visualized by ultraviolet light, and spots were developed by vanillin–sulphuric acid spraying reagent. Column Chromatography was performed on 60–120 mesh size silica gel (Merck, India).

Chromatographic method

Samples and the mobile phase were filtered through a 0.22 µm PTFE membrane and HPLC column thermostated at 35 °C. The optimum separation obtained with a gradient mobile phase consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). Gradient conditions were 0–27 min, 15–45% B; 27–37 min, 45–80% B; the column was kept at 80% B till 50 min. Column reconditioning was achieved with 15% B for 10 min using equilibration between sample runs. The flow rate is set at 1.0 mL/min. System control and chromatographic data processing were performed by Waters Empower 3 Chromatography Software. Data acquisition in the range of 200–400 nm done to monitor possible interference of co-elution/neighbouring peaks of the sample matrix. The detector was set at 254 nm wavelength for simultaneous quantitation because of the optimum chromatographic signal response of the targeted phytochemicals.

In-vitro study

Primary peritoneal macrophages isolation from healthy mice

Swiss albino mice (24 ± 2) were used for the primary macrophage cells isolation Institutional Animal Ethics Committee (IAEC) approved protocols (CIMAP/IAEC/2020–23/10). 1.0 mL peptone (1%) in saline was injected in peritoneal cavity of animals. Cells were isolated by phosphate buffer saline (PBS), pH-7.4 from mice in intraperitoneal region. Cell viability was checked by trypan blue exclusion and the viable macrophages at the concentration of 1.0 × 106 live cells/mL were used for the experimentation (Singh et al. 2014).

Pro-inflammatory cytokines inhibitory potential of IVT-21 and ISL, LTG

For the analysis of anti-inflammatory profile macrophages were seeded in 48 cell culture plate (0.5 million cells /well) in DMEM in 5% CO2 incubator at 37 °C. Cultured primary macrophages were treated with IVT-21, LTG and ISL at dose of 3, 10 and 30 μg/mL and dexamethasone (used as a standard drug) at a dose of 1 μg/ml. After 30 min of treatment, primary macrophages were stimulated with LPS at dose of 0.1 μg/mL. After six hours LPS stimulation, supernatants were collected and kept at − 80ºC for the quantification of pro-inflammatory markers like TNF-α and IL-6 through ELISA (BD Biosciences, USA). Percentage (%) inhibition of pro-inflammatory markers was calculated as given equation:

$$\frac{{\% {\text{inhibition = }}({\text{Concentration of vehicle control}}-{\text{Concentration of test treatment) }}}}{{{\text{Concentration of vehicle control}}}} \times 100$$

Cell toxicity

Mice peritoneal primary macrophages were seeded in 96 cell culture plate (0.2 million cells/well) in DMEM (Dulbecco modified Eagle medium, Sigma) supplemented with 10% fetal bovine serum with 1 × stabilized antibiotic–antimycotic solution (Sigma) in 5% CO2 incubator at 37 °C. Cell viability assay of IVT-21 was studied by MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium) test. Briefly, after culture of peritoneal macrophages, cells were treated with IVT-21 (10, 30 & 100 µg/mL) for 24 h. Macrophages were incubated with MTT dye for four hours. The media was changed with 100 µL DMSO and kept for 10 min and measurement of absorbance taken at 550 nm (Spectramax; Molecular Devices, USA). Percentage (%) of survival = (average experimental absorbance/average control absorbance)  X 100.

In-vivo study

Wister’s rats (180–200 g) were used in this study. All rats were obtained from institute animal house and acclimatized to experimental room for one week before experiment. Experimental animals were fed pellet animal feed and given free access to water and maintained under standard environmental temperature (22–24 °C) and a 12 h dark–light cycle. Animal experiments were carried out as per the appropriate procedure by the institutional animal ethics committee (IAEC) approved protocols (CIMAP/IAEC/2020–23/08) followed by the committee for the purpose of control and supervision of experimental animals (CPCSEA), Government of India (Registration no: 400/01/AB/CPCSEA).

 Collagen-induced arthritis in rats

Healthy and acclimatized animals were separated into 6 groups [n = 6 (male = 3 and female = 3)]. One of the groups served as normal control whereas, the other 5 groups were subjected to collagen-induced arthritis in rats CIA induction. CIA was induced by the subcutaneous injection of 0.2 mL mixer of collagen and Freund’s complete adjuvant (1:1 ratio) at the base of tail (Except normal group) on days 1. Collagen was dissolved in 0.05 M acetic acid (4 mg/mL) and emulsified with Freund’s complete adjuvant in equal volume. Booster dose was inserted in to the base of the tail (subcutaneous) avoiding the primary injection sites after 7 days of the primary immunization. The development of arthritis symptoms was detected after the 7–10 days of second immunization, paw and knee joint inflammation is the main visible sign of arthritis. After 3 weeks of the primary immunization animals were treated with test extract and standard drug for 21 days. Group- 1 (Normal control), group- 2 (CIA + Vehicle control) treated with 1 mL 0.7% CMC, group 3–5 orally treated with IVT-21 at dose of 30,100,300 mg/kg respectably and group 6 received dexamethasone (0.1 mg/kg) used as a standard drug.

Assessment of arthritis index and arthritis score

Thickness of the joint and paw was measured by screw gauge on day 0,21,28,35 and 42 and arthritis index (AI) was calculated by given equation: AI (%) = [(Thickness of paw and joints on day x—Thickness of paw and joints on day 0)/ Thickness of paw and joints on day 0]  X 100. Visual monitoring of the arthritis score were performed to measure the severity of arthritis in all animals. The Arthritis scores ranged from 0 to 4 per paw, and were noted once a week for 21 days after the treatment. Scores were added and highest arthritis score was 16 per rat, with the highest arthritis score of 4 for each paw. Score 0 = no sign of arthritis, 1 = a low inflammation and/or redness, 2 = moderate inflammation and redness in joint and paw, 3 = extraordinary edema with limited joint activity, and 4 = excessive edema with difficulty in walking.

Mechanical pain thresholds measurement

Pressure application measurement (PAM) instrument (ugobasile, Italy; Model No.-38,500) was used for the measurement of knee joint withdrawal threshold in gram force. It has a force transducer, fitted on thumb and placed on rat’s knee joint. Mechanical force (300 g force/second) was gradually increased and applied by the operator. To avoid the tissue damage mechanical force was applied only for 5 s. An attempt to withdraw the joint from operator’s thumb and forefinger was taken an indication of the test end point. The limb withdrawal threshold (LWT) measurement was repeated for 3 times and mean LWT was calculated (Barton et al. 2007).

Quantification of inflammatory markers

Six weeks after the first immunization, blood was collected by orbital plexus of experimental animals, and separated the serum for quantifying the inflammatory markers TNF-α, IL-6, IL-17A, MMP-1, MMP-9, C-RP, NO as per manufacture's instruction.

Histopathology and immunohistochemistry study 

On day 42 (end of experiment day) animals were anesthetized and sacrificed, the knee joints were cut carefully, skin was removed and fixed in 10% formalin buffer. After the fixation and decalcification, the samples were processed and sectioned into 4 µm and stained with hematoxylin and eosin (H and E) staining (Suvarna et al. 2018).

Immunohistochemistry was used to measure NFкB-p65 in the knee joint tissue of experimental rats. Knee joint tissue section of five mm thickness were cut and taken on the poly-l-lysine coated slides. Tissue section were dewaxed and dehydrated then antigen retrieval and quenching step by EDTA and PBS + H2O2 (4:1) respectably. After that section were blocked with 5% BSA for 10 min and then incubated with NFкB-p65 primary antibodies (Santa Cruz, CA, USA) for 1.5 h at room temperature. After primary antibody incubation tissue section were washed with PBS and HRP-conjugated secondary antibody (Jackson immune) was overlaid and incubated for 30 min at room temperature. Tissue section washed with PBS and overlaid with DAB (3´3—diaminobenzidine) to detect antibody binding. Sections were counterstained using hematoxylin solution. The stained sections were dehydrated, mounted and microscopic analysis were carried out (Sereika et al. 2018). Staining intensity was assessed on a semi quantitative five-point scale (0 = no expression, 1 = very low expression, 2 = low expression, 3 = high expression, and 4 = very high expression) by percentage of stained cells and synovial membrane damaged area (Choy et al. 2002). Immunohistochemical/ NFkB-p65 protein expression assessment was evaluated in a blinded manner by Olympus CX21iLED light microscope.

RNA isolation and RT-PCR analysis

After anesthetized and cervical dislocation hind paw of experimental rats were cut, claws and skin were dissected, then paw tissues were directly frozen in liquid nitrogen then stored at − 80 ˚C. Paw tissues were used for quantitative RT- PCR analysis. Approximately 45–50 mg tissue was cut from frozen paw and homogenized in 1 mL triazole regent by tissue homogenizer and isolate the total RNA with TRI-reagent (Sigma). Purity of RNA was checked by ration of A 260 /A280 nm using Nano Drop (Thermo Scientific). The RNA sample showing A 260/A280 ratio 1.95–2.05 and A260/A230 ratio > 1.5 were used for synthesis of cDNA using commercially available cDNA synthesis kit (Applied Biosystems) according to manufacturer’s protocol. The Real time PCR reaction was performed with 12.5 μL of Power SYBR Green PCR master mix (Applied Biosystems) 0.8 μL of forward and reverse primers and 1.0 μL of sample cDNA. PCR reaction efficiency was optimized, and the final concentration of each primer was 0.5 μM. Real time PCR was carried out in Applied Biosystems QuantStudio™ 5 System using PCR master mix (SYBER green) the relative expression of TNFα, IL-6, IL-1 β, MMP-9, IL-17A mRNA was normalized to the amount of GADPH in the same cDNA using the relative quantification 2 − ΔΔCt method. The fold change in target gene cDNA relative to the GADPH internal control was determined using the following formula: Fold change = 2−ΔΔCt, where [ΔΔ Ct = (Ct,target gene–Ct,GAPDH) treatment − (Ct,target gene–Ct,GAPDH) Control]. Primer sequences for analysis of TNFα, IL-6, IL-1 β, COX-2, MMP-9, IL-17A and GAPDH are described in Table 1.

Table 1 Real-time PCR primers used in the experiment
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Acute toxicity study in swiss albino mice

Safety evaluation of IVT-21 was done as per reported method (Babu et al. 2021) at single acute oral doses at 2000 mg/kg.

Statistical analysis

Results are expressed as the means ± standard error of mean (SEM) and compared the statistical significance of vehicle verses treatment groups by ANOVA. Differences with a P value < 0.05 were considered significant.

Results

HPLC quantitative analysis

The HPLC analysis suggested that the enrich extract (IVT-21) contained 6.65% of LTG and 3.13% of ISL (Fig. 2).

Fig. 2
figure 2

HPLC Chromatograms of IVT-21

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In-vitro study

Anti-inflammatory activity of IVT-21 and LTG, ISL present in IVT-21

IVT-21, LTG and ISL compounds were assessed for their anti-inflammatory activity against the production of pro-inflammatory markers (TNF-α and IL-6) using the ELISA method in LPS-induced inflammation in primary macrophages at the concentrations 3,10 and 30 μg/mL. Pro-inflammatory markers production was significantly (p < 0.05) increased in LPS-stimulated macrophages when compared with normal macrophages. Cells treated with IVT-21, LTG and ISL significantly (p < 0.05) reduce the production of pro-inflammatory markers (TNF-α and IL-6) in a dose-dependent manner (Table 2).

Table 2 Dose dependent response of IVT-21,LTG and ISL on production of pro-inflammatory cytokines (TNF-α, IL-6,IL-1β) from LPS-activated primary macrophages
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Cytotoxicity profile of IVT-21

To examine the direct effect of IVT-21 on the cells, we performed the cell viability test using the MTT assay as shown in Fig. 3. IVT-21 at the doses of 10, 30 and 100 µg/mL did not affect the cells ‘ viability, suggesting these doses of IVT-21 do not have a direct toxic effect on the cells when compared with non-treated cells.

Fig. 3
figure 3

Cell viability assay of IVT-21 at different concentrations in primary peritoneal macrophages, n = 3. Data are expressed as mean ± SEM: *p < 0.05; normal vs treatment

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In-vivo study

Effect of IVT-21 on arthritis score and arthritis index in CIA animals

Development of CIA was noticed with the swelling and redness of the joints after 2 weeks of the primary immunization. A plateau of the peak of CIA response was maintained from day 21–42. Oral treatment of IVT-21 at a dose of (100,300 mg/kg) and dexamethasone (0.1 mg/kg) from day 21–42 significantly (P < 0.05) reduced the arthritis index of knee and elbow joint in CIA animals (Fig. 4 A-C). Figure 4 E shows the arthritis score of all groups of experimental rats. Arthritis score was decreasing during the treatment period (days 21–42) in a dose-dependent manner. Arthritis score was 11 ± 0.68, 9.66 ± 0.80 and 9 ± 0.68 in animals treated with IVT-21 at a dose of 30, 100,300 mg/kg respectively, animals treated with dexamethasone at a dose of 0.1 mg/kg show the arthritis score 8.66 ± 0.42 and non-treated CIA + vehicle group animals show the arthritis score was 14.33 ± 0.80.

Fig. 4
figure 4

(A,B,C) Arthritis Index of Elbow, Knee Joint and Paw respectively. (D) Limb withdrawal threshold (LWT) measured by Pressure Application Measurement (PAM) in gram force. Data are expressed as mean ± SEM: *p < 0.05; vehicle vs treatment; #vehicle vs normal; (Tukey’s multiple comparison test); n = 6. (E) Arthritis Score, Data are expressed as mean ± SEM: p < 0.05; # normal vs vehicle; using one-way ANOVA followed by a non-parametric Kruskal–Wallis test; n = 6

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Effect of IVT-21 on mechanical pain threshold in CIA animals

To determine the effect of IVT-21 on pain induced by mechanical force, the limb withdrawal threshold (LWT) in gram force was measured on days 21, 28, 35, and 42 using pressure application measurement(PAM) apparatus. LWT was significantly (P < 0.05) increased in IVT-21 treated animals when compared with the CIA + vehicle group of animals in a dose-dependent manner ( Fig. 4 D).

Effect of IVT-21 on pro-inflammatory markers in CIA animals

The serum concentrations of inflammatory markers (TNF-α, IL-6, IL-17A, MMP-1, MMP-9, C-RP, NO) level were significantly increased in CIA animals when compared with normal group animals. The serum concentration of above mentions inflammatory markers were significantly (P < 0.05) decreased in animals treated with IVT-21 at a dose-dependent manner when compared with non-treated CIA + vehicle group animals (Fig. 5A-G).

Fig. 5
figure 5

Effect of IVT-21 treatment on the Inflammatory markers production in the serum of CIA rats. A TNF-α B IL-6 C IL-17A, D MMP-1, E MMP-9, F CRP, G Nitric oxide. Data are expressed as mean ± SEM: *p < 0.05; vehicle vs treatment; # vehicle vs normal; (Tukey’s multiple comparison test); n = 6

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Effect of IVT-21 on histopathology and immunohistochemistry (IHC) of ankle joint after CIA injection

On day 42 after primary immunization, the histological features were observed. The knee joint of CIA animals showed the typical signs of arthritis with inflammatory cells and inflammatory materials infiltration, cartilage degradation and synovial membrane disruption. In contrast, animals treated with IVT-21 and dexamethasone all of the above mention’s symptoms were alleviated in a dose-dependent manner. In the case of a normal control group of animals show that, synovial membrane was a smooth and had regular cellular arrangement, maintain the synovial space, without any inflammatory cell/ material infiltration, and no any erosion and disruption of cartilage tissue and synovial membrane when compared with CIA group of rats (Fig. 6). To further validate the NFкB-p65 protein expression by immunohistochemistry staining of the knee joint tissue of experimental animals. The results of immunohistochemistry show that the expression (brown color indicates) of NFкB-p65 protein was significantly (P < 0.05) increased in the non-treated vehicle group of animals compared to a normal control group of rats. In contrast, animals treated with IVT-21 and dexamethasone significantly reduce the expression of NFкB-p65 protein in a dose-dependent manner compare with non-treated vehicle group of animals. whereas the normal group shows no or very low NF-kB p65 protein expression in the synovial joint tissue (Fig. 7).

Fig. 6
figure 6

Rat: knee joint: representative histopathology images: normal control group of rats showed no erosion and disruption of synovial membrane (Sm), no cartilage (C) damage, maintained joint cavity (Jc) and bone marrow (Bm). In vehicle treated rats, indicate the synovial membrane disruption/erosion (smd) (blue arrow), cartilage damage (cd) (red arrow), and inflammatory cells (ICs) and inflammatory material infiltration(s) (IMIs) (black arrow) which indicate the presence of arthritis, Animals treated with GHEE-248 at dose of 100 and 300 mg/kg and dexamethasone at dose of 0.1 mg/kg, decrease the inflammation, maintain the synovial membrane and no erosion/damage of cartilage tissue as compare to vehicle group—H & E staining- 200 X

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Fig. 7
figure 7

Effect of IVT-21 on the expression level of NFkB-p65 proteins was detected by immunohistochemistry. Representative photomicrographs of IHC staining demonstrating the expression of NFkB-p65 on the knee joint surface and synovial membrane of the experimental rats–IHC-200 X. Data are expressed as mean ± SEM: *P < 0.05; Vehicle vs treatment; #Vehicle vs normal; n = 5

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Effect of IVT-21 on mRNA level of inflammatory cytokines in CIA rat hind paws

We measured pro-inflammatory cytokines gene expression in CIA group animals at the end of the treatment period (day 42). Paw tissue of CIA rats exhibited, significantly higher TNF-α, IL-6, IL-1 β, COX-2, MMP-9 and IL-17A gene expression compared with the normal group of rats. Oral treatment of IVT-21, significantly reduced the mRNA gene expression in a dose- dependent manner compare to CIA + vehicle group of rats (Fig. 8).

Fig. 8
figure 8

Effect of IVT-21 on mRNA expression of inflammatory mediators (TNF-α, IL-6, IL-1 β,IL-17A,MMP-9) in paw tissue isolated from CIA rats. Data are mean ± SEM; n = 3. *Vehicle versus treatment, #normal versus vehicle (ANOVA; Tukey test), p < 0.05

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In-vivo safety study of IVT-21

The result of this study exhibited that one-time oral treatment of IVT-21 (2000 mg/kg) did not produce any mortality, behavioral changes in treated animals compared to non-treated animals. Similarly, no any significant changes were observed in biochemical as well as hematological parameters of the treated animals when compared with control group animals (Table 3).

Table 3 Effect of IVT-21 on acute toxicity at 2000 mg/kg as a single oral dose in Swiss albino mice
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Discusion

The chemical profiling of G. glabra root extracts (IVT-21) using HPLC analysis showed the presence of isoliquiritigenin and liquiritigenin as the major active ingredients. Natural molecules derived from plants play a significant role in the development of anti-inflammatory drugs in the pharmaceutical industry which can serve as good lead molecules suitable for further modification during the drug development process (Saxena et al. 2016). In the present study, we examined the pro-inflammatory cytokines inhibitory potential of IVT-21, a LTG, ISL rich root extract of G. glabra as well as pure ISL and LTG against the LPS-induced inflammation in primary peritoneal macrophages. IVT-21 was further evaluated in the in-vivo condition against collagen-induced arthritis model in rats. Several biomarkers of inflammation are implicated in chronic disease like cytokines (TNF-α, IL-1, IL-6, IL-8) and other proteins like cyclooxygenase-2 (COX-2), C-reactive proteins, matrix metalloproteinases (MMP), etc. (Prasad et al. 2016). In-vitro anti-inflammatory studies of IVT-21, ISL, and LTG significantly reduce the pro-inflammatory cytokine (TNF-α, IL-6) level without any cytotoxic effects in a dose-dependent manner. This report is correlates with the previous finding that G.glabra and its natural compounds have shown anti-inflammatory activities (Yang et al. 2017). Liquiritigenin inhibits the NF-κB activation in macrophages thereby reducing the pro-inflammatory markers and iNOS production (Kim et al. 2008). Isoliquiritigenin decreases the inflammation and fibrosis in the kidney of the unilateral ureteral obstruction model and inhibited inflammatory and fibrotic responses in BMDM by blocking Mincle/Syk/NF-kappa B signaling pathway (Liao et al. 2020). We have further evaluated the therapeutic effect of IVT-21 in the in-vivo system using the collagen-induced arthritis model in rats. In-vivo study exhibit that, oral administration of IVT-21 shows anti-arthritis effects in a dose-dependent manner. Collagen-induced arthritis (CIA) model is the most commonly used animal model for rheumatoid arthritis (RA) because of similar pathological and arthritis appearances between RA patients and CIA animal model (Liu et al. 2018). For the treatment of RA disease-modifying anti-rheumatic drugs (DMARDs), non-steroidal anti-inflammatory agents (NSAIDs) and biologics have found some successes in improving the life quality of patients. But these drugs have several severe side effects and the high price of biologics, several people would favor choosing traditional herbal medicines as their long-term drug treatment approaches (Linghu et al. 2020). In-vivo study shows that oral administration of IVT-21 for 21 days significantly decreased the arthritis index, arthritis score, and significantly increase the limb withdrawal threshold (gf) of experimental rats using PAM apparatus in a dose-dependent manner, which detects the mechanical hypersensitivity of chronic inflammatory joint pain (Barton et al. 2007). In this experiment, we also found that the oral administration of IVT-21 significantly decreased the production of TNF-α, IL-6, IL-17A, MMP-1, MMP-9, Nitric oxide, and C-RP levels in a dose-dependent manner in CIA rat’s serum. Pro-inflammatory markers play important roles in RA development and are also important targets for the treatment of RA. Pro-inflammatory inhibitors are generally used for RA treatment (Chinese Rheumatology 2018). Histological estimation of the knee joint, CIA animals shows arthritis features like inflammatory cell infiltration, decrease synovial space, cartilage destruction, and disruption of the synovial membrane. Oral treatment of IVT-21 significantly alleviates inflammatory cell infiltration, reduces cartilage damage, and decreases the hyperplasia of the synovial membrane. Immunohistochemistry of the knee joint demonstrates that NFкB-p65 protein expression was significantly reduced in animals treated with IVT-21 and dexamethasone in a dose-dependent manner compared with a non-treated group of CIA rats. NFκB-p65 is an important transcriptional factor involved in the production of pro-inflammatory cytokines, protein, and adhesion molecules (Bureau et al. 2000). Oral treatment of IVT-21, significantly decrease mRNA gene transcription levels in the hind paws of CIA rats compare to the non-treated vehicle group of rats. mRNA levels of TNF-α, IL-6, IL-1 β, MMP-9, and IL-17A genes were significantly increased in the non-treated vehicle group of rats compared to the normal group of rats. Phenolics are present in several fruits, vegetables, herbs, spices and beverages which was used by humans for food and traditional medicine (Yoon and Baek 2005). Phenolic compounds have exhibited efficacy against several chronic diseases including RA because of their ability to control pro-inflammatory and pro-oxidant pathways and can control the production and action of inflammatory markers by modulating the action of other molecules involved in RA pathogenesis (Rosillo et al. 2016). Isoliquiritigenin is used for the treatment of osteoarthritis and reduced the IL-1β-induced MMPs production and NF-κB activation in in-vitro and in-vivo conditions (Zhang et al. 2018). ISL improved cisplatin-induced acute kidney injury by reducing Formyl peptide receptors 2 involved in macrophagic inflammation (Rui-Zhi et al. 2022), and it has a protective effect on chronic obstructive pulmonary disease induced by Cigarette smoke (Yu et al. 2018). LTG protects against hepatotoxicity induced by arsenic trioxide due to its antioxidant and anti-inflammatory activities (Zhang et al. 2021), and it reduce the IL-1β-induced inflammation and cartilage matrix degradation in rat chondrocytes (Tu et al. 2019). IVT-21 was safe and well tolerated by Swiss albino mice up to the 2000 mg/kg dose in acute oral toxicity. Safety is an essential component in the drug development process and is nowadays referred to as ‘Pharmacovigilance’. This finding resembles a previous report that the pharmacological effect of plant-derived leads without any toxic effect at higher doses (Saxena et al. 2016).

Conclusion

The result of this study shows that IVT-21, ISL, and LTG were able to reduce the level of pro-inflammatory cytokine in activated peritoneal macrophages. Oral treatment of IVT-21, a LTG, ISL rich root extract of G. glabra ameliorate the rheumatoid arthritis symptoms, reduce the inflammatory mediators in CIA rat’s serum and also reduce the mRNA gene expression in paw tissue of CIA rats. This finding confirms the suitability of IVT-21 as a drug-like candidate for further investigation in the management of rheumatoid arthritis.