Anti-inflammatory and analgesic activities of black cumin (BC, Nigella sativa L.) extracts in in vivo model systems

Black cumin (Nigella sativa) is a widely used ingredient of traditional medicine for its broad-spectrum pharmacological actions, including analgesic, bronchial asthma, anti-inflammatory properties, and others. We sought to evaluate BC extracts' efficacy for the anti-inflammatory and analgesic properties using a comprehensive in vivo and in silico experimental setup. To investigate whether BC extract has anti-inflammatory and analgesic therapeutic potentials in vivo anti-inflammatory activity by carrageenan-induced rat paw edema, analgesic activity by acetic acid-induced writhing test and ingenuity analysis of the BC extracts in inflammation control. The acetic acid-induced writhing test had shown a dose-dependent reduction of writhing number following BC administration. Rat paw edema test showed the dose-dependent reduction of paw edema volume following BC administration. Ingenuity Pathway Analysis (IPA) suggested BC extracts containing ferulic acid, p-coumaric acid, kaempferol, and quercetin can inhibit inflammation. This study suggests that bioactive compounds in BC extract act as an anti-inflammatory and analgesic agent by regulating several downstream and upstream inflammation pathways.


Background
Inflammation is a fundamental part of the body's physiological defense mechanisms against pathogenic infections and toxic substances (Medzhitov 2008). It is involved in the body's response to both the initial cause and the consequences of an injury. Often, however, inflammations can be triggered inappropriately, leading to tissue destruction, which in turn can result in a range of inflammatory disorders, including rheumatoid and gouty arthritis, psoriasis, and Crohn's disease (Okin and Medzhitov 2012). Many studies suggest that a persistent inflammatory condition can be pervasive and develop into more clinically severe afflictions, including cardiovascular disease and cancer, often with fatal outcomes (Roifman et al. 2011;Okin and Medzhitov 2012;Laveti et al. 2013).
Current management strategies for inflammatory diseases include medications, relaxation, exercise, and surgery to correct joint damage. The medications used to treat inflammatory diseases, such as non-steroidal anti-inflammatory drugs, corticosteroids, cyclophosphamide, hydroxy-chloroquine, and biologic drugs, possibly minimize disease progression by lowering joint pain and swelling and the inflammation itself (Diaz-Borjon et al. 2006;Cheng 2012;De Mattos et al. 2015;Cavaillon and Singer 2018). The management strategy is contingent on many factors, including the patient's age, medical background and comorbidity, immunity status, and the severity of inflammatory disease symptoms (Cheng Page 2 of 8 Shaheen et al. Bulletin of the National Research Centre (2022) 46:26 2012; Cavaillon and Singer 2018). However, the efficacy of these management strategies can be questionable, and even if the efficacy is satisfactory, many of these strategies are often not suitable for all patients because of the associated side effects (Li et al. 2017). Both steroidal and nonsteroidal inflammatory drugs are associated with a high range of adverse effects in the long term (Hanania et al. 1995;Gor and Saksena 2011). There is thus a constant push toward developing an effective and curative therapeutic strategy to treat inflammatory diseases (Schwartz et al. 2017).
Medicinal plants are an essential source of bioactive compounds with potential therapeutic efficacy (Atanasov et al. 2015). Pharmacological investigations of medicinal plants can yield primers for the effective treatment of inflammatory diseases (Atanasov et al. 2015). Black Cumin (BC, Nigella sativa L.) is a well-known medicinal plant used extensively in Unani, Ayurvedic, and Siddhi medicine for centuries (Houghton et al. 1995). This herb, endemic to South Asian and Mediterranean countries, is rich in bioactive compounds, including tocopherols, alkaloids, saponins, and vitamins A and C, all of which contribute to its biological functionality (Butt and Sultan 2010). Overwhelming evidence indicates the presence of bioactive ingredients in BC that can counteract the underlying pathophysiology of many diseases, including cancers, inflammatory conditions, cardiovascular defects, autoimmune disorders (Butt and Sultan 2010;Dajani et al. 2016;Tavakkoli et al. 2017). Previous studies have highlighted BC's potential anti-inflammatory and analgesic activities (Pise and Padwal 2017;Ikhsan et al. 2018). However, only a few studies have revealed BC's anti-inflammatory effects on subacute and chronic models of inflammation (Pise and Padwal 2017).
In this study, we sought to investigate the doseresponse effects of the anti-inflammatory activity of BC in rats and mice with in vivo anti-inflammatory, and analgesic activities. In addition, the present study attempted to develop a probable mechanism of action for Nigella sativa through Ingenuity pathway analysis.

Collection and preparation of black cumin samples
Trained food sample collectors collected the BC seed samples from Newmarket Kancha Bazar, Dhaka, Bangladesh. The BC seed samples were then critically checked by the expert faculty member of the Department of Botany, University of Dhaka. Preparation and processing of the collected samples were done using standard operating procedures (Bashir et al. 2015). Processing involved drying the fresh samples at 25-30 °C, grinding them by an electric blender, and preserving them in an airtight container until analysis.

Experimental animal
Swiss Albino mice (Scientific name: Mus musculus) (5-6 weeks of age; 20-30 g) and Long-Evans rats (Scientific name: Rattus norvegicus) (7-8 weeks of age; 100-130 g) were collected from the Animal Research Branch, International Centre for Diarrheal Disease Research, Bangladesh (ICDDR, B). The animals were housed in polyvinyl cages for seven days and maintained under standard laboratory conditions (temperature 25 ± 2 °C) and 12 h light-12 h dark cycles. The animals were fed on a standard laboratory animal diet formulated by ICDDR, B, and water ad libitum. To keep the hydration rate constant, food and water were stopped 12 h before the experiments. The Ethical and Animal Care Committee of the Institute of Nutrition and Food Sciences, University of Dhaka, Bangladesh, critically reviewed and approved this study involving in vivo models. The procedures described in this study were conducted in accordance with the Bangladesh Biosafety and Biosecurity guidelines 2019 and institutional oversight performed by qualified veterinarians (2019).

Assessment of anti-inflammatory activity by carrageenan-induced rat paw edema
Evaluation of anti-inflammatory activity was conducted in vivo in the Long Evans rat model. At first, 200 mg of samples (i.e., dried AWA extract) were mixed with 5.0 mL of DMSO in a shaking incubator at 130 rpm overnight. Then the mixture was centrifuged at 3000 rpm for 5.0 min. Then the supernatant was collected and pipetted 500 µL of aliquots to store at 25 °C.
To determine anti-inflammatory activity, edema was induced by the phlogistic agent carrageenan (Winter et al. 1962b). The control group (n = 3) received normal saline (p.o.) (10 mL/kg), while group 2 received a standard drug, Diclofenac sodium (50 mg/kg, p.o.). The rest of the groups were given 800 (n = 3), 400 (n = 3), and 200 mg/kg body weight (n = 3) p.o. of extract preparations. Thirty minutes after administering sample extracts, each rat received 0.1 mL of 1% (w/v) carrageenan (injected into the sub-plantar region of the right hind paw subcutaneously) (Sigma Aldrich, # C-1013, Germany). Paw volumes of the right hind paw of each rat were measured by plethysmometer before and 1, 2, 3, and 4 h after carrageenan injection to determine the edema volume. The hind paw volume was evaluated for anti-inflammatory activity and expressed as % inhibition of the hind paw volume, which was calculated by the following equation (Winter et al. 1962b): Here, Vc = average paw volume of the control group, and Vt = Average paw volume of the treated group.

Assessment of analgesic activity by acetic acid-induced writhing test
Peripheral analgesic activity of the extracts was determined by the acetic-acid-induced writhing inhibition method in mice (Whittle 1964). The control group (n = 3) (group 1) received normal saline (10 mL/kg, p.o.). Group 2 received a reference drug, Diclofenac sodium (50 mg/ kg, p.o.). The rest of the groups received 800 (n = 3), 400 (n = 3), and 200 mg/kg body weight (n = 3) p.o. of dried AWA sample extracts. Each mouse was administered intraperitoneally after 30 min of treatment with 0.6% acetic acid (10 mL/kg). Later, the writhing numbers of each mouse were observed for 10 min. To evaluate the level of analgesia, writhing numbers of the sample treated mice were compared with the writhing numbers of the control groups. Percent-inhibition of writhing was calculated using the following equation: Here, Wc = Average number of the writhing of the control group, and Wt = Average number of the writhing of the treated group.

Ingenuity analysis of the BC extracts in inflammation control
We evaluated the effects of the compounds extracted from BC using the knowledge-based path explorer feature of the Ingenuity Pathway Analysis (IPA) tool (Chindelevitch et al. 2012;Krämer et al. 2014). We explored new ingenuity-based pathways that showed the relationship between the genes involved in the biosynthetic pathways of inflammation-related compounds (e.g., arachidonic acids, prostaglandins, and leukotrienes) and compounds in the BC extracts, which includes ferulic acid, quercetin, p-coumaric acid, and kaempferol. The shortest possible pathways were generated individually for each compound where the compounds in the BC extracts were set as the initial compound, and inflammation-related compounds were selected as the target.

Statistical analysis
The data obtained were expressed as mean ± SEM. Statistically significant differences between groups were calculated by the application of analysis of variance (one-way ANOVA) followed by Dunnett's test. P values less than 0.05 (p < 0.05) were used as the significance level.

In vivo anti-inflammatory activity
4 h after carrageenan injection, the maximum edema volume in control Long Evans adult rat models was 1.18 ± 0.03 ml. Another group of rats pretreated with Diclofenac sodium (reference drug; 50 mg/kg, p.o.) showed significantly reduced (p < 0.05) edema in the paw. Dose-response analysis of DMSO extract of BC produced a significant inhibition of paw edema in Long Evans adult rats in a dose-dependent manner at 400, and 800 mg/kg, p.o., administration of carrageenan (p < 0.05) ( Table 1).
The maximum inhibition of rat paw edema by BC was noted 4 h after carrageenan administration at a dose of 800 mg/kg, p.o., as compared to the control but less than the reference drug. Figure 1 shows the changes of percent inhibition of carrageenan-induced paw edema volume at 1, 2, 3, and 4 h at the doses of 800 mg/kg, p.o., by the DMSO extract of BC when compared to the control. We found a slightly increasing trend from hour 1 to hour 2, a slightly decreasing trend in hour 2 to hour 3, and a relatively increasing trend from hour 3 to hour 4.

In vivo analgesic activity
The effects of DMSO extract of BC on 0.6% acetic acidinduced writhing in Swiss albino mice are summarized in Table 2. A dose-dependent and significant (p < 0.05) reduction in the number of abdominal constrictions induced by intraperitoneal administration of 0.6% acetic acid was observed with oral administration of BC, at the doses of 400, and 800 mg/kg, p.o., when compared to the control. Among the different doses, the DMSO extract of BC at the dose of 800 mg/kg exhibited the maximum inhibition of the number of writhing compared to control and followed a dose-dependent inhibition trend in two other doses, 200 and 400 mg/kg, p.o.
The maximum inhibition of rat paw edema by BC was noted 4 h after carrageenan administration at a dose of 800 mg/kg, p.o. Compared to the control but less than the reference drug. Figure 1 shows the percent decrease in carrageenan-induced paw edema volume changes at

In silico analysis provides insights into the mechanism of how BC extracts reduce inflammation
The in-silico analysis of the bioactive compounds of BC illustrates a facet reactome depicting potential models of how BC extracts reduce inflammation in humans (Fig. 2, Additional file 1: Table S1). Both prostaglandin and leukotriene synthesize from the arachidonic acid. Quercetin, p-coumaric acid, and kaempferol reduce the expression of myc, MAPK, EGFR, and TNF, all of which are involved in the upregulation of the expression of arachidonic acid. Consequently, quercetin, p-coumaric acid, and kaempferol downregulate the expression of arachidonic acids. Ferulic acid, another component in the BC extract, negatively induces the synthesis of phospholipids. Phospholipid biosynthesis is critically important for the synthesis of arachidonic acids. Also, ferulic acids upregulate the expression of HMOX and nrf2, resulting in the reduction of prostaglandin E2 (PGE2). Moreover, all the compounds of the Nigella sativa extract directly reduce the expression of cox1 and cox2, two essential factors for the synthesis of PGE2. Collectively, ferulic acid, quercetin, p-coumaric acid, and kaempferol help reduce inflammation by interfering with the extended arachidonic acid pathway ( Fig. 2; Additional file 1: Table S1).

Discussion
In this study, we demonstrate the in vivo dose-response of the AWA extracts with DMSO to evaluate the antiinflammatory and analgesic activities in the experimental models (Winter et al. 1962a). Carrageenan-induced paw edema in rat models is increasingly being employed to determine anti-inflammatory effects in animal models. In response to carrageenan injection, edema formation occurs in a biphasic process. Mast cells around the damaged tissue released prostaglandin with histamine and serotonin mediate the first phase (1-2 h) of the carrageenan model. Bradykinin, leukotrienes, polymorphonuclear cells, and continuous secretion of prostaglandins from macrophages tissue mediate the second phase (3-5 h) of the carrageenan model (Winter et al. 1962b;Rosa et al. 1971). Our study indicates that the DMSO extracts of BC exhibited significant (p < 0.05) inhibition of paw edema in rats at the doses of 400, and 800 mg/ kg, p.o., in the second phases of inflammatory response. DMSO extracts of BC at the 800 mg/kg dose produced the maximum inhibition of carrageenan-induced paw edema volume and inhibition is also seen in other doses in a dose-dependent manner. The present study showed the maximum inhibition of carrageenan-induced paw edema volume in rats by BC in the second phase of post carrageenan injection at 3-5 h, maybe due to the modulatory principles acting with the prostaglandin alley. Moreover, the present study supports the previous findings that BC seed polyphenol plays a crucial role as a protective factor against inflammation (Shaheen et al. 2016).
The "acetic acid-induced writhing experiment" is a well-known protocol for assessing the analgesic potency of medicinal products (Afzal et al. 2012). Intraperitoneal injection of acetic acid that triggers pain sensation is due to the prostaglandins and lipoxygenase products from arachidonic acid liberated from phospholipids by cyclooxygenase (Fish et al. 2008). Thus, the significant (p < 0.05) reduction of writhing in this study by DMSO extracts of BC at the doses of 400, and 800 mg/kg, p.o., suggest analgesic activity peripherally mediated through inhibition of prostaglandins and other endogenous pain mediators. The highest concentration (800 mg/kg) of the test samples exhibited a peak of analgesic effect significantly (p < 0.05) in the acetic acid-induced writhing test. For other doses, reduction of writhing were found in a dose-dependent manner. These results indicate the Fig. 2 Possible mechanism of action of BC extracts to reduce inflammation in humans. In the BC extracts, phenols and polyphenols, e.g., ferulic acid, p-coumaric acid, kaempferol, and quercetin, inhibit the signaling molecules, stimulation of which may directly or indirectly result in inflammation. Proteins linked to inflammation participate in several canonical pathways underlying a range of biological activities. Ingenuity pathway analysis (IPA) identified that the BC extracts' components could target many of these proteins (Additional file 1: Table S1) and control inflammation. Arrows (↓) indicate direct stimulatory modification, whereas light-up signs (┬) indicate an inhibitory modification. The names of the proteins followed the Hugo nomenclature, NCBI Entrez, UniProtKB/Swiss-Prot system Page 6 of 8 Shaheen et al. Bulletin of the National Research Centre (2022) 46:26 peripheral analgesic potential of the DMSO extracts of the test samples, which could be exhibited due to the suppression of peritoneal surface receptors through inhibited cyclooxygenase activity. The current study findings concordance with previous findings that BC seed polyphenol inhibits acetic acid-induced writhing in the mouse model (Ghannadi et al. 2005). We also note how to present findings that confirm previous work in the field by examining the literature. Specifically, the in vivo experiments performed in this study are in good agreement with those of prior experiments on BC's antiinflammatory activity, lending credence to the idea that BC is a potent medicinal plant for therapeutic uses.
In addition to the wet-lab analyses, ingenuity pathway analysis provides evidence for the role of the Nigella sativa extracts in reducing inflammation. Earlier studies revealed that some molecules, including myc, MAPK, EGFR, TNF, are involved in the cellular responses for inducing the inflammation (Kaminska 2005;Bradley 2008;Sipos et al. 2016;Rayego-Mateos et al. 2018). We have found that compounds in the Nigella sativa extracts, including ferulic acid, quercetin, p-coumaric acid, and kaempferol, downregulate the inflammation-inducing signaling molecules in humans (Fig. 2). Many research groups have reported the potential role of activating pKA, HMOX, and nrf2 pathways, leading to anti-inflammatory effects (Alcaraz et al. 2005;Campo et al. 2012bCampo et al. , 2012aTu et al. 2019). Our analysis uncovered that compounds in the Nigella sativa extracts upregulate the pKA, HMOX, and nrf2 (Fig. 2).
Furthermore, we have shown the potential aspects of the anti-inflammatory mechanism of BC that were previously only speculated upon. For example, Bordoni et al. (2019) found BC to reduce only the acute inflammation suggested that BC's mechanism of action is tied to interleukins, TNF, and prostaglandins (Cinzia et al. 2013). In the ingenuity pathway analysis, we have shown that various components of BC act through IL32, IL8, IL1, TNF, and PGE2 to reduce inflammation. In together, ferulic acid, quercetin, p-coumaric acid, and kaempferol help reducing inflammation by interfering with the extended arachidonic acid pathway. In another study, researchers discovered that BC could decrease IL-6 levels or IL-1B levels, depending on storage conditions (Bordoni et al. 2019). These findings establish a connection between BC and interleukins in BC's anti-inflammatory activity, which we have at least partially outlined in Fig. 2.
Similarly, other researchers speculated that BC WSE inhibits COX2 and that BC inhibits prostaglandin synthesis through interaction with COX-1 and COX-2 (Babar et al. 2019). Both hypotheses are supported by the results of our Ingenuity pathway analysis, as depicted in Fig. 2. Furthermore, Babar et al. suggested that phenols/polyphenols in BC might be responsible for its anti-inflammatory activity (Babar et al. 2019). Our pathway analysis found that these phenols and polyphenols are ferulic acids, p-coumaric acid, kaempferol, and quercetin. It is speculated that the inhibitory effect on the secretion of leukotrienes and prostaglandins by thymoquinone may be responsible for BC's anti-inflammatory activity (Bashir et al. 2015). We have shown that this is the case, although, instead of thymoquinone, quercetin and ferulic acid were found to be the components of BC responsible for this aspect of its anti-inflammatory mechanism. In another study, Nigella sativa showed superior activity in screening Nigella species for in vitro inhibition of PGE2 production catalyzed by COX1 and COX2 (Landa et al. 2009). This lends credence to the mechanistic schematic we have depicted in Fig. 2; as shown in that diagram, several components of BC act to inhibit COX1/COX2 activity, which is necessary to produce PGE2. In yet another study, researchers discovered that levels of erk are decreased upon Nigella sativa treatment of damaged rat lungs (Aslan et al. 2015). The erk repression discussed here is supported by our mechanistic analysis, which has found that kaempferol, a component of Nigella sativa, inhibits ERK1/2 activity. As evidenced by these examples from the literature, the results we have found build on previous research to provide crucial insight into the medicinal properties of a common natural product.
Together with the in vivo studies and in silico analysis, this study provides strong evidence that the bioactive compounds in BC extract act as an anti-inflammatory agent by regulating several downstream and upstream inflammation pathways.

Conclusions
In summary, the present study's findings support that BC extract has anti-inflammatory, analgesic activity and in silico analyses suggested that the phenolic compounds in BC extracts may play a key role in inhibiting human inflammatory reactions. Thus, in understanding how to present findings related to prior work in the field, we have gained insight into the vitality of this research for future efforts to study the anti-inflammatory effects of Nigella sativa. Future studies should focus on elucidating the mechanistic pathways through which compounds in BC extracts exert such pharmacologic effects to prevent inflammation.