Introduction

Rheumatoid arthritis (RA) is a systemic autoimmune disease of the chronic form. There exists a variation in severity between different patients and genders; however, females are in general more frequently affected as compared to males (Alam et al. 2017). The characteristic inflammatory changes in RA not only affect synovial tissue of joints but also bone and cartilage. Involvement of extra-articular regions is also reported. Clinically, these changes are manifested as redness, arthralgia, swelling, and limited mobility of symmetrical joints. One of the most distinctive features of RA is synovial hyperplasia which leads to the formation of pannus. A complex involvement of adaptive and innate immune responses to evoke inflammatory responses ensued. Formation of autoantibodies like rheumatoid factors (RF), ACPA, and hyperactive macrophage/ monocyte system in the target tissues give rise to a plethora of clinical and histomorphological features of the disease. Arachidonic acid metabolites and inflammatory cytokines have been identified as causing systemic manifestations of RA. The role of pro-inflammatory cytokines has been well established in causing joint damage during RA development (Liu et al. 2019). The destructive process of active RA is dominated by macrophages and fibroblasts (Scherer et al. 2020). The progression and severity of RA follow multiple interlinked mechanisms of inflammation, oxidative stress, and generation of reactive oxygen species (Shahmohamadnejad et al. 2015). Free radicals lead to molecular damage and are believed to trigger transcription of IL-6, TNF-alpha, and IL-1β associated with NF-κB ultimately resulting in marked synovitis and destruction of cartilage (Quiñonez-Flores et al. 2016).

The main goal of available conventional drug therapy (non-steroidal anti-inflammatory drugs, biological therapies, disease-modifying anti-rheumatic drugs, and glucocorticoids) is to mitigate RA symptoms but due to the possible occurrence of deleterious body system side effects, their use is being limited (Smolen and Aletaha 2015). A need to develop novel remedies with better effectiveness and lesser risk of adverse effects has diverted the current trend of therapy toward the use of herbal alternatives. To limit progression and control the severity of RA, natural plants possessing anti-inflammatory potential have been extensively studied (Zhang et al. 2019). Cinnamomum cassia (Lauraceae), Ligusticum chuanxiong (Umbelliferae), Aconitum kusnezoffii (Ranunculaceae), Tripterygium wilfordii (Celastraceae), and Curcumae Longae (Zingiberaceae) are some well-known plants, that significantly regulate immunological activity and inflammatory signaling pathways showing potential therapeutic potential against RA (Zhao et al. 2021).

Solanum nigrum L. or “Black Nightshade/ Makoi” is an annual/perennial member of the Solanaceae family. It generally grows as a dicot weed in damp environments in a variety of soils ranging from dry or stony to shallow and deep. It is indigenous to Eurasia; the tropical and subtropical regions are ideal for its cultivation during April and May. Therapeutically, it has been employed for the management of several ailments, namely toothache, pneumonia, tonsillitis, stomach pain, inflammation, fever, pain, tumors as an antioxidant, anti-inflammatory, diuretic, antipyretic, and hepatoprotective (Kuete 2014). In traditional Indian practice, infusions are used for dysentery, tuberculosis, stomach complaints, and fever. The juice is effective for skin diseases, ulcers, and whooping cough. The fruits are useful for laxative action, asthma and work well as an appetite stimulant. In oriental medicinal practice, its use as an antioxidant, anti-tumorigenic, hepatoprotective, anti-inflammatory, and diuretic is extensively reported (Goel et al. 2022). Several active phytochemicals like phenols, terpenes, flavonoids, sesquiterpenes, carotenoids, and lactones have been linked to the potential antioxidant and anti-inflammatory activities, making them a suitable treatment approach in RA (Singh et al. 2020).

The anti-arthritic potential of the aqueous methanolic extract of Solanum nigrum L. berries against the complete Freund's adjuvant (CFA) arthritis rat model has been explored (Alamgeer et al. 2019). Ethyl alcohol extract from Solanum nigrum L. leaves (100 and 200 mg/kg) significantly decreased pro-inflammatory cytokines (IL-1, IL-2, IL-6, TNF-α, and PGE2) in CFA-induced arthritis (ur Rasheed 2022). The current study evaluated the in vitro anti-arthritic and in vivo analgesic and anti-inflammatory potential of four different extracts of Solanum nigrum L. leaves in an acute study. To further explore the basis of the molecular mechanism of inflammation underlying rheumatoid arthritis and for appraisal of anti-arthritic action along with oxidative stress modulation, the most active extracts (Chloroform and aqueous) were selected for the sub-acute study using complete Freund/s adjuvant-induced arthritis model.

Materials and methods

Preparation of extracts

Solanum nigrum L. leaves were collected from their natural habitat from the area of District Jhang, South Punjab, and identified by the taxonomist from the Department of Botany, Government College University (GCU)—Lahore, Pakistan. The plant was issued authentication voucher numbers GC. Herb. Bot. 33,358 and deposited in the herbarium for future reference. The sample was well-cleaned, shade-dried, and powdered to a fine size. The n-hexane, methanol, chloroform (Sigma Aldrich), and distilled water were used separately to soak the powder in 1:5 w/v for maceration twice. The semisolid mass of all extracts was formed in a rotary evaporator at a temperature of 40 °C under reduced pressure.

Experimental animals

The approval for the experiment was acquired from the Animal Ethical Committee, Punjab University College of Pharmacy, Lahore vide no. D/ 241/ FIMS dated June 1st, 2022. The animal experiments were conducted in accordance with the guidelines of NIH publication #85–23, revised in 1985. Healthy male Wistar rats with an average weight of 180–200 g were housed under appropriate environmental temperature and humidity conditions (28 ± 2 °C and 55 ± 2%, respectively). Proper access to water and food was made possible throughout the study period.

Qualitative and quantitative phytochemical study

Phytochemical analysis of all four extracts was performed to identify primary and secondary metabolites according to previously reported methods (Aduwamai et al. 2018). Crude extracts were subjected to quantitative analysis of total phenol content (TPC) by the following method with slight modifications (Veerapagu et al. 2018).

Total phenol content

The total phenol content of all extracts was found through Folin–Ciocalteu (FC) method in triplicate. 1 ml of each Solanum nigrum L. leaves extract (1 mg/ml) was mixed with 5 ml of dilute FC reagent and 4 ml of 1 M Na2CO3. For half an hour, the mixture was kept until color developed at standard temperature. The absorbance was recorded in a UV–Vis Spectrophotometer at 765 nm. For the preparation of the standard curve; 10, 20, 30, 40, and 50 µg/ml concentrations of Gallic acid were prepared in methanol, and the absorbance was measured by mixing 1 ml of each concentration (1 mg/ml) as described above.

Total flavonoid content

0.5 ml of each extract (1 mg/ml) was mixed with ethanol 1.5 ml, 10% ALCL3 0.1 ml, 1 M potassium acetate 0.1 ml, and distilled water 2.8 ml to make a reaction mixture. This was incubated at room temperature of 37 °C for 30 min. The absorbance was read at 415 nm using a spectrophotometer. To prepare a standard curve, different concs.50, 100, 150, 200, and 250 mcg/ml of Quercetin stock solution in ethanol (1 mg/ml) were prepared and the absorbance was measured by mixing 0.5 ml of each conc (1 mg/ml) as described with extracts (Nancy and Ashlesha 2015).

High-pressure liquid chromatography analysis (HPLC)

Different phenols and flavonoids in all plant extracts were estimated by performing HPLC analysis. 20 µl of the sample was carefully injected into HPLC (Shimadzu, Japan) provided with a UV–visible detector. Phytochemical detection was performed by Gradient HPLC with Shim-Pack CLC-ODS (C18) column. The mobile phases used were (94:6) aqua-acetic acid and acetonitrile. Both the phenols and flavonoids were analyzed by their peaks and retention times. Their quantities were analyzed by comparing them with standard chromatograms (Saleem et al. 2020).

In vitro anti-arthritic activity

Protein denaturation inhibition assay

Test solution (TS) comprised 0.05 ml of 100, 250, and 500 mcg/ml test concentrations of plant extracts and 0.45 ml Bovine Serum Albumin (BSA). The Test Control (TC) contained 0.05 ml of DW in 0.45 ml of BSA (5% w/v). Likewise, the positive control (PC) consisted of 0.45 ml dist. water with 0.05 ml of extract concentrations. BSA (0.45 mL) and different concentrations of diclofenac sodium (0.05 ml) served as a standard solution. Following incubation for 20 min at 37 °C, the test temperature was raised to 57 °C for 180 secs. Afterward, to all solutions, 2.5 ml of 6.3 pH phosphate buffer was added after cooling. The absorbance of all solutions was recorded at 660 nm wavelength. The assay was completed in triplicate to calculate the percentage of protein inhibition (Bashir and Niazi 2020) as follows:

$${\text{Percentage Inhibition }} = \frac{{100 - \left( {{\text{absorbance of TS }}{-}{\text{ absorbance of PC}}} \right)}}{{\text{absorbance of TC}}} \times {1}00.$$

Analgesic activity

The analgesic activity of all extracts was evaluated using the previously described hot plate method by Khodabakhsh et al. 2015. Fifty-six rats were randomly divided into 14 groups (n = 4 each). Group 1 was kept as control and received only dist. water PO; group 2 served as positive control and was administered diclofenac Na 10 mg/kg b.wt. Treatment groups received 100, 200, and 300 mg/kg b.wt PO of different extracts as follows. Treatment groups 3, 4, and 5 received n-hexane extract of Solanum nigrum L. (HESN). Groups 6, 7, and 8 rats were given methanol extract of Solanum nigrum L. (MESN); Groups 9, 10, and 11 received chloroform extract (CESN) and groups 12, 13, and 14 were treated with aqueous extract (AESN). Test animals were exposed to noxious pain stimuli on a hot plate kept at 55 ± 2 °C. Latency time was recorded in seconds at 0, 15, 30, 45, and 60 min following drug administration as rats jumped or started licking of paws following one or both paws. To avoid tissue damage to the paw, 45-s cut-off time was set.

Acute anti-inflammatory activity

Carrageenan-induced rat paw oedema model was employed to evaluate the acute anti-inflammatory potential of Solanum nigrum L. leaves extracts (Ijaz et al. 2021). A total of 84 rats were assigned different treatments (n = 6 rats in each group). A digital vernier caliper was used to measure the paw size of rats in mm. One h before 0.1 ml injection of 1% carrageenan solution (w/v) in the left hind paw, group 1 rats were given distilled water orally; diclofenac Na (10 mg/kg) b.wt. was administered intraperitoneally to group 2 rats; groups 3, 4, and 5 rats received HESN; groups 6, 7, and 8 rats received MESN; groups 9, 10, and 11 were treated with CESN; and groups 12, 13, and 14 received AESN at 100, 200, and 300 mg/kg b. wt., orally, respectively. The rat paw size was measured at intervals of 0, 1, 2, 3, 4, and 5 h after carrageenan injection. The percentage inhibition in paw oedema was measured according to the following formula:

$$\% {\text{ paw oedema inhibition }} = \frac{{\left( {{\text{E}}_{{\text{t}}} - {\text{E}}_{{\text{o}}} } \right)_{{\text{control group}}} - \, \left( {{\text{E}}_{{\text{t}}} - {\text{E}}_{{\text{o}}} } \right)_{{\text{treated group}}} }}{{\left( {{\text{E}}_{{\text{t}}} - {\text{E}}_{{\text{o}}} } \right)_{{\text{control group}}} }} \times { 1}00,$$

where Eo is the paw size at 0 h (before carrageenan injection), Et shows the paw size at each corresponding hour, and (Et–Eo) represents the paw oedema.

Sub-acute anti-arthritic activity

The sub-acute study was carried out on chloroform and aqueous extracts (300 mg/kg b.wt) of Solanum nigrum L. leaves in complete Freund's adjuvant-induced arthritis model.

Complete Freund’s adjuvant (CFA)-induced arthritis

CFA prompts an immunopathological condition resembling features of rheumatoid arthritis in humans. Edematous joints with infiltrates of neutrophils and lymphocytes in the synovium, followed by bone destruction and loss of cartilage ending up in dysfunctional joints are all similar characteristics in CFA-induced animal models to human RA (Choudhary et al. 2018).

Rats were randomly divided into five groups (n = 5). Arthritis in all experimental rats (except the control group) was induced by inoculation of 0.1 mL of CFA comprising of heat-killed M. tuberculosis (10 mg/mL) into the sub-plantar area of the left hind paw. The administration of standard and extracts started from the 12th day and continued till 28 days. (1) Control group: received distilled water PO along with standard pellet food. (2) Disease control group: arthritis-induced rats. (3) Drug control group: given diclofenac sodium (5 mg/ kg) PO. (4) Chloroform extract of Solanum nigrum L. (CESN)-treated group: 300 mg/ kg of CESN was given PO. (5) Aqueous extract of Solanum nigrum L. (AESN)-treated group: 300 mg/ kg of AESN (PO).

Macroscopic arthritic changes induced by CFA

Paw size and ankle joint diameter (mm) in CFA-induced arthritic rats were determined with the help of a digital vernier caliper on day 0 and then after every 4th day up to the 28th day (Gul et al. 2023).

Assessment of pain response in the CFA model of arthritis

Pain response was assessed in terms of latency time in seconds by the hotplate method. Using a cut-off time of 45 secs, the temperature was kept at 53 ± 2 °C, and the time to withdraw paw or jump was recorded (Mahdi et al. 2018).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Real-time polymerase chain reaction was performed to quantify pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, and IFN-γ), anti-inflammatory cytokines (IL-4 and IL-10), and other important mediators of inflammation (NF-ĸB, COX-2, and PGES-2). On the last day of the experiment, the rat blood samples were collected from anesthetized rats in EDTA tubes. RNA extraction was performed using Pure-Link RNA Mini Kit by Invitrogen (Catalogue No. 12183018A). RNA samples were measured and equalized by Nanodrop Quantification by testing samples in replicas. RNA samples were reverse transcribed into cDNA using RevertAid First-Strand cDNA Synthesis Kit (Catalogue No. K1622) by ThermoFisher Scientific using a minimum input of 10 ng/µL of RNA. The expression of targeted genes was performed using SYBR Select Master Mix (Catalogue No. 4472903) by having cDNA as the template with appropriate primers in duplicates. The relevant CTs of samples were compared with controls and control samples having housekeeping genes (GAPDH). Concisely, a reaction mixture of 0.02 mL composed of master mix (0.01 mL), forward and reverse primers (150–400 nM), complementary DNA template + RNase-free H2O was added to a microplate and placed in qPCR instrument (Thermo Fisher Scientific®, USA). The specifications for the thermal cycler were kept as: 40 circles of 2 min of denaturation at 90 °C, annealing at 60 °C (15 secs), and extension at 72 °C (1 min). Finally, the threshold cycles (CT) and relative quantification (ΔΔCT) were computed (Saleem et al. 2020). The primers used in the study are presented in Table 1

Table 1 Primers list
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Biochemical and hematological analysis

Using mild ether anesthesia, rat blood was obtained by cardiac puncture and collected in ethylenediaminetetraacetic acid (EDTA) tubes on the 28th day of the study. Serum samples were prepared to analyze antioxidant enzymes superoxide dismutase (SOD), reduced glutathione (GSH), and catalase (CAT) using mouse super oxidase dismutase (SOD), mouse reduced glutathione (GSH), and mouse catalase (CAT) ELISA Kits by following instructions of the manufacturer (Zokeyo, China). An advanced immune analyzer, ichromα reader (KR) was used to determine C-reactive protein (CRP) conc. Hematological parameters were analyzed on a Sysmex XP- 300 Hematology analyzer.

GC–MS analysis

The specifications of GC–MS (Shimadzu®, Japan, model: QP 2010 plus) for the plant extracts analysis were: use of capillary column, DB-5 MS (film thickness of 30 m × 0.25 mm × 0.25 µm) as stationary phase; helium (99.9%) with a flow rate of 1 ml/min as a carrier gas; a split-less injection method and temperature of 80 °C later increased to 220 °C for 5 min. The temperature of MS was kept at 280 °C. The total run time of the sample was 30 min (Uroos et al. 2017).

Radiographical analysis

On the 28th day, radiographs of the hind paws of rats were taken. The X-ray films were obtained and observed for swelling of tissue joints, narrowing among joint spaces, and bony erosions (Ijaz et al. 2021).

Statistics

Results are presented as Mean ± SEM. Unpaired t-test (where relevant) and one-way ANOVA followed by Dunnett's test were used for the assessment of all numerical variables and a p-value < 0.05 was considered statistically significant.

Results

The percentage yield of n-hexane, methanol, chloroform, and aqueous Solanum nigrum L. extracts was found to be 1.74, 12.7, 4.65, and 15%, respectively.

Qualitative phytochemical analysis

The qualitative analysis for the identification of various primary and secondary plant metabolites showed that n-hexane extract contained phenols, flavonoids, protein, and sterols. In methanol, chloroform, and aqueous extracts, the presence of alkaloids, terpenoids, flavonoids, phenols, and proteins was noticed. Results are given in Table 2.

Table 2 Phytochemical analysis of Solanum nigrum L. extracts
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Quantitative phytochemical analysis

Total flavonoid content

The total flavonoid content of all extracts is presented in Table 3. It is expressed as mg quercetin equivalents/g using the calibration curve y = 0.0012x + 0.7994, R2 = 0.9896, where x represents the absorbance and y is the quercetin equivalent. The maximum total flavonoid content was found in CESN (109.6 ± 0.38 mg QE /g) and aqueous (75.81 ± 0.52 mg QE /g) extract.

Table 3 Quantitative phytochemical content of Solanum nigrum L. leaves extracts
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Total phenolic content

Table 3 represents the total phenolic contents of all extracts. The maximum total phenolics were present in CESN (64.36 ± 0.50 mg GA/g) and AESN (81.29 ± 0.55 mg GA/g). It was calculated using the calibration curve: Y = 0.0009x + 0.052, R2 = 0.9837, where x is the absorbance and y represents the gallic acid equivalent.

HPLC analysis

The HPLC quantitative estimation of Solanum nigrum L. leaves extracts revealed that in all extracts, quercetin was present. The maximum conc. of quercetin was found in an aqueous extract of Solanum nigrum L. leaves (13.90 mcg/g). Higher amounts of phenolic and flavonoids were present in chloroform and aqueous extracts as compared to methanol and n-hexane extracts. Different quantities of phytochemicals detected in various Solanum nigrum L. extracts are displayed in Table 4, Figs. (1, 2, 3, 4).

Table 4 HPLC analysis of various extracts of Solanum nigrum L. leaves
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Fig. 1
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HPLC chromatogram of n-HESN

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Fig. 2
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HPLC chromatogram of MESN

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Fig. 3
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HPLC chromatogram of CESN

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Fig. 4
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HPLC chromatogram of AESN

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Effect of Solanum nigrum L. on protein denaturation inhibition

Chloroform and aqueous extracts of Solanum nigrum L. showed maximum inhibition of protein denaturation (87.93% and 82.44%) followed by n-hexane (80.68%) and methanol extracts (74.44%) as compared to the standard diclofenac sodium (83.97%) as shown in Table 5

Table 5 Effect of Solanum nigrum L. leaves extracts on protein denaturation inhibition
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Effect of Solanum nigrum L. on pain response in hot plate test

A significant increase in latency time (p < 0.05) was observed with AESN at all dose levels. However, the variable response was observed in HESN, MESN, and CESN 100 mg/kg, 200 mg/kg, and 300 mg/kg treated rats. HESN at 200 mg/kg dose showed a significant increase in LT up to one hour; MESN 200 and 300 mg/kg showed a significant increase after 30 min. CESN 300 mg/kg significantly increased latency time from 30 to 60 min compared to normal control as shown in Table 6.

Table 6 Effect of various extracts of Solanum nigrum L. leaves extracts on mean latency time (secs)a
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Effect of Solanum nigrum L. in Carrageenan-induced paw oedema

Significant reduction in rat paw size (p < 0.05) was shown by all the extracts at different dose levels as shown in Table 7. However, maximum percentage inhibition in paw oedema occurred with AESN and CESN as compared to HESN and MESN (Fig. 5).

Table 7 Effect of various extracts of Solanum nigrum L on paw size (mm) b
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Fig. 5
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Effect of n-hexane, methanol, chloroform, and aqueous extracts (100, 200, and 300 mg/kg) of Solanum nigrum L. leaves on % inhibition in paw oedema. HESN: Hexane extract of Solanum nigrum L., MESN Methanol extract of Solanum nigrum L., CESN Chloroform extract of Solanum nigrum L., and AESN Aqueous extract of Solanum nigrum L

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Effect of Solanum nigrum L. on paw oedema in CFA-induced arthritic rats

A significant increase (p < 0.0001) in paw size was observed after sub-plantar inoculation of CFA in the disease control group rats with respect to the control group throughout the study duration. Moreover, on the 20th day post initiation of treatment with plant extracts and drug control on day 12, a significant reduction (P < 0.05) in paw size was achieved with AESN at 300 mg/kg dose. Likewise, the subsequent reduction in paw size on day 24 and day 28 was also statistically significant (Fig. 6).

Fig. 6
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Effect of chloroform and aqueous extracts of Solanum nigrum L. leaves on paw size (mm). Values are expressed as Mean ± SEM (n = 5). *p < 0.05, **p < 0.01and *** p < 0.001 symbolizes a significant decrease in the paw size as compared to disease control after the start of treatment at day 12

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Effect of Solanum nigrum L. on ankle joint diameter in CFA-induced arthritic rats

At day 16, a significant reduction (P < 0.01) in joint diameter was achieved with AESN at 300 mg/kg dose which was retained till the 28th day (5.83 ± 0.16 mm). Similarly, CESN also showed a significant decrease (P < 0.01) in joint diameter (6.73 ± 0.10 mm) as compared to disease control (7.52 ± 0.34 mm) after 1 week post-treatment at day 20 till day 28 (Fig. 7).

Fig. 7
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Effect of chloroform and aqueous extracts of Solanum nigrum L. leaves on joint diameter (mm). Values are expressed as Mean ± SEM (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 symbolize a significant decrease in the paw size as compared to the disease control after the start of treatment at day 12

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Effect of Solanum nigrum L. on pain response in CFA-induced arthritic rats in hot plate test

The latency time to pain response is significantly increased (6.95 ± 0.58 and 6.92 ± 0.42 s) in rats treated with both chloroform and aqueous extracts of Solanum nigrum L. leaves compared to disease control group 2.85 ± 0.36 s (Fig. 8).

Fig. 8
figure 8

Effect of chloroform and aqueous extracts of Solanum nigrum L. leaves on latency time (Secs). Values are expressed as Mean ± SEM (n = 5). *p < 0.05, **p < 0.01, and ***p < 0.001 symbolize a significant decrease in the paw size as compared to the CFA-induced arthritic control after the start of treatment at day 12

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Effect of Solanum nigrum L. on mRNA Expression of Inflammatory Cytokines in arthritic rats

Quantitative real-time polymerase chain reaction (qRT- PCR) was used to determine the effect of chloroform and aqueous extracts of Solanum nigrum L. on mRNA expression of various pro-inflammatory and anti-inflammatory biomarkers (Fig. 9). Treatment with both extracts resulted in a significant decrease (P < 0.001) in IL-1β levels 4.61 ± 0.45 and 6.791 ± 0.15 as compared to disease control 12.87 ± 0.96. Following CFA induction, IL-6 levels increased significantly. However, treatment with both extracts resulted in a marked decline (0.34 ± 0.07 and 0.93 ± 0.01) when compared to disease control rats 1.64 ± 0.24. Both TNF-alpha and NF-kB genes were upregulated (1.48 ± 0.01 and 2.29 ± 0.21) as a result of adjuvant induction but post-treatment with 300 mg/kg of CESN and AESN significantly downregulated (P < 0.0001) the levels of these pro-inflammatory biomarkers as shown in Fig. 9.

Fig. 9
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Effect of chloroform and aqueous extracts of Solanum nigrum L. leaves on mRNA expression of inflammatory cytokines IL-1β: Interleukin-1 beta; IL-6: Interleukin-6; TNF-α: Tumor Necrosis Factor; NF-κB: nuclear-factor-kappa B; COX-2: Cyclo-Oxygenase-2; IFN-γ: Interferon Gamma; PGES-2: Prostaglandin E Synthase 2; IL-4: Interleukin-4 and IL-10: Interleukin-10. Results are presented as Mean ± SEM (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 indicate differences in treatment groups (CESN and AESN vs. disease control)

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The mRNA expression of COX-2 was also significantly downregulated (P < 0.001) after treatment with both extracts (0.56 ± 0.09 and 0.64 ± 0.07) in relation to disease control (4.72 ± 0.44). Increased levels of IFN- γ after CFA induction (5.98 ± 0.02) were significantly restored to normal following treatment with both chloroform and aqueous extracts (3.36 ± 0.10 and 3.72 ± 0.32). A similar decrease in PGES-2 expression with both extracts was determined which was significantly lower (P < 0.0001) than the disease control group.

An increase in IL-4 levels post-treatment with both extracts occurred (1.26 ± 0.35 and 1.36 ± 0.20) in comparison to the CFA control (0.05 ± 0.03). Similarly, significant amplification in IL-10 was observed in CESN and AESN treated groups (0.55 ± 0.05 and 0.57 ± 0.12).

Effect of Solanum nigrum L. on hematological parameters in CFA-induced arthritis

Both CESN and AESN treatments resulted in significant improvement in hematological parameters in arthritic rats (Table 8). CRP showed an increase in conc. in the disease control group compared to normal control. However, treatment with both extracts significantly lowered CRP levels (3.28 ± 0.20 and 2.85 ± 0.10) mg/ L when compared with the disease control group as shown in Table 8. Likewise, restoration of ESR to normal values (23.00 ± 1.73 and 28.67 ± 0.88) mm/ 1st h occurred post-treatment with both extracts relative to arthritic rats.

Table 8 Effect of Solanum nigrum L. leaves extracts on hematological parameters in CFA-induced arthritis
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Hemoglobin levels also decreased in the disease control group (9.90 ± 0.15) g/dL and augmented as a result of treatment with both CESN and AESN 300 mg/kg (12.43 ± 0.27 and 12.70 ± 0.05) g/dL as presented in Table 8. Significant alterations in blood cell counts as depicted by decreased RBC count and increased WBCs and platelet count were recorded in arthritic rats. Both the extracts improved RBCs count (5.90 ± 0.11 and 6.20 ± 0.20) 106/µL, reduced the elevated levels of WBCs (6.08 ± 0.06 and 6.63 ± 0.05) 103/µL, and platelets (693.7 ± 1.85 and 740 ± 1.15) 103/µL.

Effect of Solanum nigrum L. on oxidative stress in CFA-induced arthritis

Serum levels of catalase, SOD, and GSH declined significantly (P < 0.0001) as a result of CFA induction (Table 9). However, CESN and AESN treated rats showed a marked reduction in oxidative stress with a significant increase in levels of catalase, SOD, and GSH.

Table 9 Effect of Solanum nigrum L. Leaves extracts on oxidative stress parameters in CFA-induced arthritis
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GC–MS analysis

Phytochemicals identified in the Chloroform and aqueous extracts included various esters, alcohols, fatty acids, ketones, and hydrocarbons (Fig. 10 and 11). The maximum conc. (%) in GC–MS spectrum was found to be of docosanoic acid—methyl ester, hexadecanoic acid, tetradecanoic acid, and hexanedioic acid, bis (2-ethylhexyl) ester. The phytochemicals detected by the GC–MS spectrum are listed in Tables 10 and 11.

Fig. 10
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GCMS chromatogram of chloroform extract of Solanum nigrum L. leaves

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Fig. 11
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GCMS chromatogram of aqueous extract of Solanum nigrum L. leaves

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Table 10 Phytochemicals identified in the GC–MS spectrum of the chloroform extract of Solanum nigrum L. leaves
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Table 11 Phytochemicals identified in the GC–MS spectrum of the aqueous extract of Solanum nigrum L. leaves
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Radiographic analysis

The disease control rats showed noticeable swelling of paw tissues representing inflammation. Also, narrowed joint spaces accompanying periarticular bone resorption were evident in radiological findings. On the other hand, rats treated with diclofenac Na, CESN, and AESN showed comparatively no remarkable changes (Fig. 12).

Fig. 12
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Radiographic findings of CFA-induced arthritis in rats

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Discussion

Solanum nigrum L. has been extensively used in traditional medical practice for the treatment of various inflammatory conditions, rheumatism, and joint pain. Plant-derived constituents show good medicinal properties when extracted using organic solvents (Madane et al. 2013) which formed the basis of using four different organic solvents for extraction. The current study aimed to investigate the effect of Solanum nigrum L. in regulating the expression of inflammatory cytokines and the modulation of oxidative stress biomarkers in acute and sub-acute arthritis models. The in vitro anti-arthritic, anti-nociceptive, and in vivo acute anti-inflammatory potential of different extracts of Solanum nigrum L. leaves was determined. Two extracts (chloroform and aqueous) were further evaluated for sub-acute anti-arthritic activity in CFA-induced arthritis rat model as depicted by the dose–response data.

A plethora of powerful phytoconstituents especially flavonoids is believed to not only possess antioxidant capability but also cause significant inhibition of cyclooxygenases, phospholipase A2, and protein tyrosine kinase and therefore have great biological importance. Phenolic compounds moreover, by reduction of ROS expression pathways, are important anti-inflammatory constituents (Owolabi et al. 2018). The qualitative and quantitative analysis of all Solanum nigrum L. leaves extracts showed the presence of bioactive phytochemicals responsible for analgesic, anti-inflammatory, and anti-arthritic effects.

The HPLC analysis of all Solanum nigrum L. leaves extracts showed the presence of different bioactive phytoconstituents including: quercetin, ferulic acid, chlorogenic acid, gallic acid, syringic acid, sinapic acid, caffeic acid, p- and m-coumaric acid, cinnamic acid, and vanillic acid having significant anti-inflammatory properties; quercetin is believed to decrease the level of potent inflammatory mediators like prostaglandins and leukotrienes (David et al. 2016). The highest conc. of quercetin determined in the aqueous extract of Solanum nigrum L. was 13.90 µg/g. Another phytoconstituent present in AESN was the ferulic acid (39.74 µg/g) which has been found to remarkably inhibit IL-17-mediated RA severity and bone erosions (Ganesan and Rasool 2019). An important polyphenol identified in different extracts is the chlorogenic acid that defends against inflammatory conditions brought on by oxidative stress and has anti-edematous properties (Han et al. 2019). Numerous investigations have shown that the anti-inflammatory effects of gallic acid occur by inhibiting the release of myeloperoxidase and the activities of polymorphonuclear leukocytes (PMNs) (Karimi-Khouzani et al. 2017). Syringic acid has an anti-inflammatory effect via the regulation of different genes involved in inflammation (Ham et al. 2016). Chemically, sinapic acid is a derivative of cinnamic acid. It mediates anti-inflammatory action by decreasing the expression of iNOS and COX-2 (Yun et al. 2008). Cinnamic acid, however, is itself a derivative of phenolic acid causes cyclooxygenases and lipoxygenases to be inhibited (Hadjipavlou-Litina and Pontiki 2015). Caffeic acid and its derivatives have been found to have both antioxidant and pro-oxidant effects. These effects have been attributed to a combination of mechanisms, including radical scavenging action, prevention of lipid peroxidation, and protection against LDL oxidation (Khan et al. 2016). It is known that p-coumaric acid has anti-inflammatory properties by reducing the production of inflammatory mediators and circulating immune complexes (Zhu et al. 2018). Moreover, It is reported that tumor necrosis factor (TNF) and interleukin (IL-6) production is inhibited by vanillic acid, which increases lipopolysaccharide levels (Kim et al. 2011).

Since protein denaturation has been reported as the leading cause of RA (Qasim et al. 2021) for investigation of in vitro anti-arthritic effect, its inhibition using BSA was determined. All the extracts decreased BSA denaturation in vitro. However, the chloroform and aqueous extracts showed maximum inhibition in % protein denaturation. This ability of Solanum nigrum L. to stabilize protein denaturation is suggestive of its potential as an anti-arthritic agent.

Different physiological mediators like prostaglandins, bradykinins, and substance-P play a crucial role in mediating the complex pain response (Nguyen et al. 2020). Debilitating pain in joints due to arthritis is one of the leading symptoms. The analgesic activity of Solanum nigrum L. leaves extracts was determined using the hot plate method. Significant analgesia was reported with aqueous extract of Solanum nigrum L. at all doses 100, 200, and 300 mg/kg as compared to n-hexane, methanol, and chloroform extracts.

The biphasic inflammation process induced by carrageenan is one of the most widely studied models of acute inflammation. Varieties of inflammatory mediators like histamine, bradykinin, 5-hydroxytryptamine, prostaglandins, cyclooxygenases, etc. are released during this process (Fatima et al. 2021). All the plants' extracts showed significant anti-inflammatory potential against carrageenan-induced rat paw oedema. Maximum inhibition in paw oedema was observed with chloroform and aqueous extracts of 300 mg/kg each.

The most extensively used animal model to study clinical and histopathological changes associated with human RA is CFA-induced polyarthritis. In the CFA model, the chronic inflammation is exhibited as an increase in the paw size after induction of adjuvant and this occurs in two phases. The initial irritant reaction of adjuvant is causative of oedema formation followed by a late-phase secondary reaction leading to an immunologic response (Ijaz et al. 2021). The chloroform and aqueous extracts of Solanum nigrum L. (300 mg/kg) leave significantly decreased the size of injected paws and ankle joint diameter recorded with the help of a digital vernier caliper.

Since heat is responsible for causing local tissue damage and further inducing the release of inflammatory mediators, along with nociceptors sensitization. It ends up in pain as a cumulative response (Angst and Clark 2006). The analgesic effect of both extracts owes to the modulation of pain response as well as the down-regulation of inflammatory mediators.

The pathogenesis of RA has been linked with an augmentation in levels of pro-inflammatory mediators namely TNFα, IL-6, and IL-1β along with increased cyclooxygenase and nuclear factor ĸB levels (Kim et al. 2016). The above mediators have a key role in an adjuvant-induced arthritis model (Choudhary et al. 2018). Not only the tissue regeneration reduced by IL-1 and IL-6, the IL-1 also stimulates numerous inflammatory genes both locally and systemically. Hence, a decrease in the cytokine IL-1 plays an essential part in improving inflammation (Etemadi et al. 2022). A similar effect was observed in both treated groups administered with Chloroform and aqueous extracts of Solanum nigrum L. leaves extracts.

One of the prominent transcription factors in inflammatory response regulation is nuclear factor-kappa B, causing an upregulation of receptors necessary in immune cell functioning, as well as inducing transcription of various inflammatory cytokines like TNF-α, IL-6, IL-1β, and enzymes like COX-2 (Peng et al. 2012). The increased expression of TNF- alpha, IL-1, Il-6, and COX-2 results due to increased production of NF-κB leading to the development of polyarthritis. This role of NF- κB has also been established in the current study as well, shown by the upregulation of TNF-alpha, IL-6, and IL-1 in the diseased group. Pro-inflammatory cytokines-mediated upregulation of COX-2 leads to an indirect rise in PGE2 in an active RA disease (Fattahi and Mirshafiey 2012). PGES-2, a glutathione-independent microsomal subtype of PGE2, is remarkable in constitutively expressing and coupling with cyclooxygenases 1 and 2 to produce PGE2. The plant extracts downregulated levels of both COX-2 and PGES-2.

Another cytokine playing an important role in both innate and adaptive immune responses is interferon-gamma. Based upon its diverse role in physiological immunity, it has been hypothesized that IFN-gamma contributes to the development of RA. The current study also shows a linkage of increased IFN-gamma levels to the establishment of RA in CFA-induced animals, which were significantly improved after treatment with both extracts (Okada et al. 2014).

Interleukin-4 is a major anti-inflammatory cytokine and it impedes the formation of some very important pro-inflammatory cytokines like TNFα, IL-1β, and IL-6 by peripheral blood mononuclear cells, synovial tissue, and rheumatoid SF. Another anti-inflammatory cytokine named human cytokine synthesis inhibitory factor or interleukin-10 also decreases the formation of TNFα, IL-(1α, 1β, 6, 8, and 12), and GM-CSF (Mateen et al. 2016). Both of these anti-inflammatory cytokines were upregulated as a result of treatment with chloroform and aqueous extracts of Solanum nigrum L. leaves in CFA-induced arthritic rats.

The active inflammation in arthritis is evaluated by a convenient serum marker CRP which is an acute phase systemic reactant (Pepys 2003). CRP levels were increased in rats induced with CFA and subsequently decreased as a result of treatment with chloroform and aqueous extracts of Solanum nigrum L. leaves. Aggravated response to pathogenic stimuli results in a rise in WBCs and platelet count in CFA-induced arthritic rats (Perumal et al. 2017). Both plant extracts by modulation in immune response caused improvement in WBCs and platelet count. Low RBC count and resultant anemia have been previously reported widely in RA patients. Significant improvement in RBC count and hemoglobin conc. occurred after treatment with both extracts of Solanum nigrum L. leaves.

SOD, catalase, and GSH were estimated for oxidative stress in rat serum. Both extracts also exhibited antioxidant activities as apparent from the improvement of oxidative stress biomarkers. TNF-alpha is also one of the major cytokines and a strong inducer of pro-inflammatory cytokines causing a disturbance in the standard physiological equilibrium among pro- and anti-inflammatory mediators. Interleukin-6 carries out acute phase responses such as anemia, lipid-metabolism deregulation, and cognitive dysfunction through leukocyte activation and autoantibody production (Alam et al. 2017).

The GCMS analysis of chloroform extract showed the presence of 22 compounds and 18 compounds were determined in the aqueous extract. Docosanoic acid found in aqueous extract possesses high antioxidant activity as previously reported (Olukanni 2020). Hexadecanoic acid methyl ester possesses anti-arthritic effects as suggested by literature showing the potential of aqueous extract as an anti-inflammatory and anti-arthritic (Nabi et al. 2022). Tetradecanoic acid (lauric acid) presence in chloroform extract validates its antioxidant role since it has been reported to act as a lipid anchor in biological membranes (Obasi and Ogugua 2021).

Conclusion

The present results conclude that Solanum nigrum L. possesses analgesic, anti-inflammatory, and anti-arthritic potential. The chloroform and aqueous extracts have more marked anti-inflammatory and anti-arthritic activities as compared to n-hexane and methanol extracts. This owes to the presence of potent phenolics and flavonoids in Solanum nigrum L. that provide the basis for their use in RA. The chloroform and aqueous extracts of Solanum nigrum L. leaves showed promising anti-arthritic activity by regulating gene expression of different pro-inflammatory and anti-inflammatory cytokines namely, IL-1β, IL-6, NF-ĸB, IFN-γ, IL-4, and IL-10. Moreover, the modulation of biomarkers related to oxidative stress also shows the convincing role of both extracts in regulating the inflammatory process. The bioactive phytoconstituents, as revealed in GCMS analysis also validate its anti-arthritic potential making it a suitable choice as a rheumatoid arthritis remedy. Further experiments for the standardization of extracts and formulation of their safety profile are required.