Effect of Saffron Extract, Astaxanthin, and Carnosic Acid on the Levels of Matrix Metalloproteinase-9 and on Body Weight Changes in Arthritis Experiments

Matrix metalloproteinase-9 (MMP-9), also known as gelatinase B, is a member of a family of matrix metalloproteinases that are zinc-dependent endopeptidases (Chakraborti et al., 2003). They are often found in extracellular space in an inflammatory type of diseases where they degrade all major components of an extracellular matrix (Konttinen et al., 1999; Ram et al., 2006). MMP-9 is also responsible for the formulation of a new bone after fracture by remodelling of the cartilage created in response to healed fractured bones (Colnot et al., 2003). This effect of MMP-9 is crucial to the process of bone healing and formation and reduction of cartilage callus (Colnot et al., 2003), but it is not desired for inflammatory diseases such as rheumatoid arthritis, where MMP-9 causes cartilage destruction (Tchetverikov et al., 2003).

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease that has a prevalence of 0.5% in the population, where women are affected more frequently than men, in a ratio of 1:3 (Ahlmen et al., 2010; Almutairi et al., 2021). Progression of RA usually lead to reduction in patients’ movement, which can lead to the reduction of productive age approximately 10 years from a diagnosis of RA and other socio-economic aspects (Burton et al., 2006). In the last two decades biological treatment of RA has become more and more common, but, it is still usually quite an expensive procedure following failure of the standard treatment, which is represented by methotrexate, M (Smolen et al., 2020). Currently approved therapeutic options for RA possess many limitations, such as resistance to treatment and side effects commonly associated with one of the DMARDs representatives, M (Wang et al., 2018).

We hypothesise that the investigation of natural substances administered alone and in combination with M on experimentally induced arthritis may lead to a better course of the treatment together with stabilisation of the disease. For this purpose, we investigated the effect of selected natural compounds on the two important parameters described below. RA is very often associated with cachexia. Cachexia occurs in RA as a loss of muscle mass and with respect to various criteria it has prevalence of 15%–33% (Santo et al., 2018). It is one of the main comorbidities that affect patients’ quality of life by reducing mobility, which can lead to the deterioration of RA (Ollewagen et al., 2021). The role of free radicals in rheumatoid cachexia has been described as well (Poništ et al., 2020b). Also, in animal models of RA, adjuvant arthritis cachexia is a prominent pathological condition (Roubenoff et al., 1997). Animal model of adjuvant arthritis (AA) in LEWIS rats has several similarities with RA, such as the production of inflammatory cytokines, swollen joints, and changes in body weight as a marker of cachexia (Bessis et al., 2017; Bendele, 2001; Choudhary et al., 2018).

With the administration of different natural substances, we focused on the possible anti-inflammatory effect, which will be represented in this paper as the capability of decreasing the levels of MMP-9 in plasma in AA animals. Carnosic acid is an active substance of Rosmarinus officinalis, and it is used in wide range of inflammatory diseases (Borges et al., 2019, 2018; Maione et al., 2017). Astaxanthin is a carotenoid from frequently occurring in the microalgae Haematococcus pluvialis, and it is a potent antioxidant that also has an anti-inflammatory effect in cardiovascular diseases (Pereira et al., 2021). Crocus sativus (Saffron) is plant well known for many beneficial properties; one of them is the anti-inflammatory effect described for several conditions (Li et al., 2015; Zeinali et al., 2019).

Therefore, we studied the effect of the three natural substances mentioned above in an AA model. We were interested not only in the MMP-9 plasma level mentioned above, but also in a biometric level such as the change of body weight in experimental animals.

Untreated animals, AA, had significantly lower weight gain compared with the healthy control group (Figure 1a). Animals treated with methotrexate had significantly higher weight gain compared with AA animals (p ≤ 0.001, AA-M vs. AA). The combination of methotrexate and saffron extract in different doses was also significant in terms of body weight gain (p ≤ 0.001, AA-SF1-M, AA-SF2-M vs. AA), but there was not any significant difference between the two groups and methotrexate itself (Figure 1a). Similarly, saffron extract in monotherapy significantly increased the weight of animal in both doses (Figure 1a) (p ≤ 0.05, AA-SF1, AA-SF2 vs. AA).

Figure 1

MMP-9 significantly increased in the plasma of the AA animal group compared with HC (Figure 1b) (p ≤ 0.001, AA vs. HC). All groups tested had significantly decreased concentrations of MMP-9 in plasma except the SF2 group, which had no significant effect compared with the AA group (Figure 1b) (p ≤ 0.05, AA-SF1, AA-SF2-M vs. AA, p ≤ 0.01AA-M vs. AA, p ≤ 0.001 AA-SF1-M vs. AA). The combination of SF1 and methotrexate had greater significance than methotrexate alone; moreover, the level of MMP-9 was lower than M monotherapy (Figure 1b) (p ≤ 0.01AA-M vs. AA, p ≤ 0.001 AA-SF1-M vs. AA).

Body weight gain was significantly lower in AA group compared to HC group (p ≤ 0.001, AA vs. HC; Figure 2a). Methotrexate and both doses of astaxanthin had significantly increased body weight compared with AA group (p ≤ 0.001, AA-M vs. AA; p ≤ 0.05, AA-AS1, AA-AS2 vs. AA; Figure 2a).

Figure 2

MMP-9 had been significantly increased in AA group compared to HC group (p ≤ 0.05, AA vs. HC; Figure 2b). Methotrexate and astaxanthin in a lower dose significantly decreased the levels of plasmatic MMP-9 compared with those of the AA group (p ≤ 0.05, AA-AS1 vs. AA; p ≤ 0.05, AA-M vs. AA; Figure 2b). However, the higher dose of AS had no significant effect compared to AA group (Figure 2b).

Increase of body weight was significantly lower in AA group compared to HC group (Fig 3a). Carnosic acid in monotherapy did not show a significant difference compared with the AA group, but methotrexate and a combination of methotrexate and carnosic acid significantly increased body weight at same level (p ≤ 0.001, AA vs. HC, AA-M vs. AA, AA-C-M vs. AA; Figure 3a).

Figure 3

MMP-9 was significantly increased in AA group compared to HC group (Figure 3b) (p ≤ 0.001, AA vs. HC). Also, in the case of body weight change carnosic acid had no significant effect in monotherapy, but the combination of carnosic acid and methotrexate had higher significance compared with methotrexate alone, and moreover the level of MMP-9 was lower than M monotherapy (p ≤ 0.01, AA-C-M vs. AA; p ≤ 0.01 AA-M vs. AA; Figure 3b).

Modulation of inflammation with the use of medicinal plant extracts and their main substances serve as a complementary tool for conventional therapeutic strategies for numerous ailments, particularly when the suppression of inflammation is expected (Gallotti et al., 2020). In up-to-date literature, several species of medicinal plants have been shown substantial anti-inflammatory and immunomodulatory actions including inhibitory effects on the suppression of cellular and humoral immunity, lymphocyte activation, and the propagation of apoptosis (Tasneem et al., 2019; Zhao et al., 2021).

Combined therapy is very common in RA. Beside conventionally used and recommended therapeutic regiments for RA, research has shown promising result in AA also for atypical combinations, such as the combination of M and losartan. This combination has shown better results than M and losartan alone (Refaat et al., 2013). In our previous experiments we focused on combination therapy of M with natural substances and extracts, which has shown significant improvement of several inflammatory markers and biometrical parameters (Tsiklauri et al., 2019, 2021).

In this article we have focused on potential beneficial effect of the combination therapy of M with saffron extract and carnosic acid. Astaxanthin was evaluated only in monotherapy design. In all experiments described here plasmatic levels of MMP-9 and change of body weight were monitored. MMP-9 is produced by inflammatory cell types such as synovial fibroblasts. MMP-9 causes inflammation, bone, and cartilage erosion by stimulation of TNF-α and IL-6 which are both determinate factors in RA (Xue et al., 2014; Ram et al., 2006). Saffron extract has significantly reduced levels of MMP-9, but only in lower doses. We can observe the same trend in the combination of saffron extract and methotrexate, where a lower dose has more profound effect as M itself (Figure 1b). The relationship between the drug dosage and clinical outcomes has not been established for many medications, which are used to treat chronic diseases such as RA. Dimmitt and Stampfer (2009) claim there is evidence that chronic diseases could be treated effectively with low doses of saffron extract and methotrexate (Dimmitt & Stampfer, 2009). Similar effects of saffron extract in decreasing levels of MMP-9 have been presented in different studies (Zeinali et al., 2019). None of them have been performed on a rat AA model, which makes our results original. Some studies suggest that saffron can target Toll-like receptors that can regulate various transcription factor such as nuclear factor-κ B (NF-κB), which is related to the production of MMP-9 (Zeinali et al., 2019). Therefore, this immunomodulatory effect of saffron can be responsible for decreasing levels of MMP-9 in our experiment. Astaxanthin has a very similar effect to that of saffron in terms of the dose (Figures 2b and 1b). A lower dose has very a similar effect to that of M alone, and only a lower dose has a significant effect on the reduction of plasma levels of MMP-9. A decrease of MMP-9 levels by astaxanthin (5–10 μM) was also described by Kishimoto et al. (2010) on macrophages. The reduction of MMP-9 in the brain after intracerebroventricular administration of astaxanthin (20 μl of 0.1 mmol) correlated with decreased levels of IL-1β, TNF-α (Zhang et al., 2015).

Xia at al. (2017) described the anti-inflammatory effect of carnosic acid on diabetic mice on a collagen-induced arthritis. This effect was provided by lowering the expression of reactive oxygen species and the suppression of receptor activator for NF-κB ligand. Carnosic acid was administrated intraperitoneally 30 mg/kg body weight and 60 mg/kg body weight.

In our experiment we administrated carnosic acid orally, and it had no significant effect alone on MMP-9 levels (Figure 3b). In combination therapy we observed a more pronounced effect of M alone. Hadad and Levy (2012) showed the synergy of carnosic acid with other natural compounds where carnosic acid itself has no effect. The combination of carnosic acid with other natural compounds has a significant anti-inflammatory effect by redox-based inhibition of NF-κB (Hadad & Levy, 2012).

Methotrexate in therapeutic doses does not affect cachexia or sarcopenia in RA (Mochizuki et al., 2019; Ollewagen et al., 2021). However, in AA we found that M in subtherapeutic doses was able to increase the body weight of AA animals (Figures 1a, 2a, and 3a). We had this finding also in our other studies with M (Poništ et al., 2020a). Jurcovicova et al. (2009) also found that a subtherapeutic dose of M significantly increased the body weight of animals. The improvement of clinical parameters was observed in clinical trials of saffron administration in patients with RA, which suggested the beneficial effect of saffron in RA in general (Hamidi et al., 2020). We observed the ability of saffron extract to significantly increase the biometric parameter change of body weight in both doses in monotherapy (Figure 3a). There were no significant differences between the M group and the combination with saffron extract (both doses). This finding implies that M has already reached the maximum effect. Therefore, in this case, the addition of saffron extract to M was not able to increase the body weight any further. Similar results with the combination therapy of methotrexate and saffron (100 mg saffron pill/day) were observed by Sahebari et al. (2021) in RA patients.

Kumar et al. (2020) described an improvement of the clinical parameters arthritic score and hind paw volume in an AA model with astaxanthin (50 and 100 mg/kg). In our experiment with astaxanthin, both doses had a therapeutic effect in the clinical parameter change of body weight (Figure 2a). It was not on the level of M, but it was significant, so further investigation with different doses will be needed as well as the use of a combination with M.

Carnosic acid (5 mg/kg body weight) has been described as a natural compound that can potentially have a positive effect on a change of body weight (Liu et al., 2018) in collagen-induced arthritis in rats. In our model we did not observe that the carnosic acid alone had any significant effect on this parameter (Figure 3a). This could be caused by the oral method of administration we use whereas Liu et al. (2018) used intraperitoneal administration. Finally, methotrexate alone and its combination with carnosic acid caused a significant improvement of this parameter. There was not any difference between the two groups. Methotrexate probably reached its maximum effect just as occurred in the saffron extract groups mentioned above (Figures 3a and 1a).

In summary, astaxanthin in monotherapy and saffron extract in monotherapy and in combined therapy with M have significantly decreased plasmatic levels of MMP-9 and increased body weight in animals suffering with AA. More efficient was the lower dose for both experiments: astaxanthin and saffron extract. Carnosic acid has no effect in monotherapy in both parameters, but a combination with M has a significant effect for the improvement of cachexia as well as the inhibition of inflammation. AA is a model of inflammatory cachexia; therefore, our results of selected natural compounds and extract can be considered as evidence of their anti-cachexic properties. The combination of methotrexate and natural compounds can attract awareness of a new therapeutic option for RA clinical trials.