**3.1 Natural substances isolated from plants**

The world of plants is an unlimited source of compounds with healing effects, including antiinflammtory, antioxidative and immunomodulating properties. We chose some of them for our experiments with AA. Figure 1 compares all these plant ingredients concerning their effect on the basic clinical parameter – change of hind paw volume (HPV), together with selected parameters of OS as plasmatic TBARS and GGT assessed in spleen and joint tissue homogenates. We compared the effect of *Boswellia serrata* extract (Bo), *Arctostaphylos uva-ursi* extract (UV), *Zingiber officinale* extract (Zg) in combination with two previous extracts (Bo-UV-Zg), sesame oil in combination with *Arctostaphylos uva-ursi* extract (Bo-So), arbutin (Ar), curcumin (CU), Pycnogenol® (PYC) and two stilbenoids – pinosylvin (PIN) and pterostilbene (PTE). The compounds and extracts were all given per os in a single dose immediately after induction of AA and were administered daily until the end of the experiment – experimental day 28. The experimental protocol was approved by the Ethics Committee of the Institute of Experimental Pharmacology and Toxicology and by the Slovak State Veterinary and Food Administration. AA was induced by a single intradermal injection of heat-inactivated *Mycobacterium butyricum* in incomplete Freund's adjuvant (Difco Laboratories, Detroit, MI, USA) to male Lewis rats. The injection was performed near the tail base. All experiments included healthy animals (HC), arthritic animals not treated (AA), arthritic animals treated with the compounds/extracts studied. The oral daily doses used were 30 mg/kg b.w for AA-PIN and AA-PTE, 10 mg /kg b.w for A-PYC, 50 mg/kg b.w for AA-UV, AA-Bo, AA-Bo-So, AA-Ar and AA-CU, 50+25+25 mg/kg b.w for the mixture AA-Bo-UV-Zg and 0.1 ml/kg b.w. for sesame oil. For statistical analysis of the obtained data the same procedure was applied in all experimental cases. The data for all parameters are expressed as arithmetic mean ± S.E.M. For significance calculations unpaired Student`s *t*-test was used with \**p*<0.05 (significant), \*\**p*<0.01 (very significant), \*\*\**p*<0.001 (extremely significant). The arthritis group was compared with healthy control animals (\* symbol). The treated arthritis groups were compared with untreated arthritis (+-symbol). In each experimental group 8–10 animals were used. In Figure 1 the reduction of HPV and OS parameters is illustrated in relation to untreated arthritic rats (100% represented by dot-anddash line). The situation for AA is complicated due to the dominant involvement of Th 1 driven autoimmune etiopathology. OS in this animal model occurs as a reaction to autoimmune processes. Under these conditions, control of OS is of secondary importance, although it could enhance immunomodulatory therapy of RA (Bauerova et al., 2011). Figure 1 clearly shows that plant-related treatment is not enough for successful improvement of

Modern Pharmacological Approaches to Therapies:

(Table 2) (Bauerova et al., 2010b).

**HPV** 

**AA** 33.68 ±5.22

**AA-MTX** 7.57 ± 0.87

**AA-PIN-MTX** 4.34 ± 0.79

compared to methotrexate monotherapy (AA-MTX).

\*\*\*

+++

+++ / ##

**3.1.1 Pinosylvin and methotrexate combination in AA** 

Substances Tested in Animal Models of Rheumatoid Arthritis 243

significant decrease of aortic endothelium-dependent relaxation. Administration of So resulted in mild, non-significant decrease of hind paw swelling and in significantly

PIN [3´,5´-dihydroxystilbene] and PTE [3,5-dimethoxy-4´-hydroxystilbene] used in our experiments were synthesized and purified by Šmidrkal et al. (2010) and Harmatha et al. (2011). PIN and PTE are natural substances from the stilbenoid group, wide-spread in a variety of plants. They are chemically related to resveratrol. Both substances studied inhibited significantly the chemiluminescence (CL) of whole human blood and the CL of isolated human neutrophils (Perečko et al. 2008). The new information on the inhibitory effect of PIN and PTE on HPV, production of ROS and MPO activity suggests that the protective effect of PIN may be beneficial in controlling inflammation in experimental AA (Macickova et al*.*, 2010). PIN was also the most effective in reducing HPV on day 28, when administered in the dose of 30 mg/kg b.w. per os (Fig. 1). According to these findings we chose PIN as a suitable candidate for combination therapy with methotrexate (MTX). In the combination AA-PIN+MTX, arthritic animals were treated twice a week with MTX in the oral dose of 0.4 mg/kg b.w. and daily with PIN in the oral dose of 50 mg/kg b.w. Monotherapy was performed with the same doses. In addition to the routine statistical analysis the combination treatment was compared to individual MTX treatment (#-symbol). In arthritic rats, PIN potentiated the antiarhritic effect of MTX on days 14 and 21, evaluated as decrease of HPV (Table 1). Activity of GGT in spleen homogenate (Table 2), plasma levels of MCP-1 and CRP (Table 3) were not improved by addition of PIN to MTX, due to the prominent effect of MTX alone on these parameters. Arthritic animals showed an increase in OS, evaluated as plasma levels of TBARS. PIN enhanced the antioxidant effect of MTX

> **(%) Day 14 Day 21 Day 28 CO** 8.48 ±0.69 12.98 ±0.97 17.05 ±0.83

**AA-PIN** 26.30 ±2.61 86.02 ±4.56 84.03 ±5.51

Table 1. Hind paw volume (HPV) changes in an experiment with methotrexate (MTX) and pinosylvine (PIN) in monotherapy and in combination therapy PIN+MTX measured in time profile. The data were expressed as arithmetic mean ± S.E.M. Statistical significance was evaluated applying Student's t-test for independent variables: \*P < 0.05, \*\*P < 0.01, and \*\*\*P < 0.001 compared to control healthy animals (CO); +P < 0.05, ++P < 0.01, and +++P < 0.001 compared to untreated arthritic animals (AA); #P < 0.05, ##P < 0.01, and ###P < 0.001

87.24 ±7.50 \*\*\*

13.14 ± 2.22 +++

7.49 ± 1.14 +++ / #

81.62 ±7.20 \*\*\*

16.22 ± 2.10 +++

11.44 ± 1.16 +++

increased acetylcholine-evoked relaxation of aorta (Sotnikova et al., 2009).

HPV (excluding pinosylvin) although some of the compounds and extracts tested (e.g. UV, Ar , CU, Bo-So or Bo-UV-Zg) achieved a biochemical improvement in the body redox state expressed as reduction of plasmatic TBARS and GGT activity assessed in joint and spleen. Moreover, CU was found to be a potent inhibitor of neutrophil functions in experimental arthritis. AA was accompanied by an increased number of neutrophils in blood and by a more pronounced spontaneous as well as PMA stimulated chemiluminescence. Whereas the arthritis-related alterations in neutrophil count and in spontaneous chemiluminescence were not modified by CU, the increased reactivity of neutrophils to PMA was less evident in CUtreated animals. The effects of CU were comparable with those of methotrexate. CU was found to be a potent inhibitor of neutrophil functions (Jancinova et al., 2009). As neutrophils are considered to be cells with the greatest capacity to inflict damage within diseased joints, the observed effects could support the beneficial use of CU in RA treatment. Further the beneficial effect of sesame oil (So) administered alone on markers of OS accompanying AA was demonstrated not only by decrease of plasma TBARS, decrease of GGT activity in the joint and spleen tissues, but also the level of protein carbonyls, TAC in plasma and activity of NAGA in serum and in the kidney were improved, yet not significantly. In HPV the maximal increase was found on day 28 of AA, and at the same time we observed a

Fig. 1. Comparison of the effect of different plant treatments in adjuvant arthritis (AA) on reduction of hind paw volume (HPV) and on GGT (-glutamyltransferase) activity in spleen and joint and level of TBARS (thiobarbituric acid reactive substances) in plasma measured on experimental day 28. Changes in parameters are illustrated in relation to untreated arthritic rats (100% represented by dot-and-dash line). The data were expressed as arithmetic mean ± S.E.M. Statistical significance was evaluated applying Student's t-test for independent variables: +P < 0.05, ++P < 0.01, and +++P < 0.001 compared to untreated arthritic animals. *Boswellia serrata extract* (Bo), *Arctostaphylos uva-ursi extract* (UV), *Zingiber officinale extract* (Zg), combination of three previous extracts (Bo-UV-Zg), sesame oil in combination with *Arctostaphylos uva-ursi* extract (Bo-So), arbutin (Ar), curcumin (CU), Pycnogenol® (PYC), pinosylvin (PIN) and pterostilbene (PTE).

HPV (excluding pinosylvin) although some of the compounds and extracts tested (e.g. UV, Ar , CU, Bo-So or Bo-UV-Zg) achieved a biochemical improvement in the body redox state expressed as reduction of plasmatic TBARS and GGT activity assessed in joint and spleen. Moreover, CU was found to be a potent inhibitor of neutrophil functions in experimental arthritis. AA was accompanied by an increased number of neutrophils in blood and by a more pronounced spontaneous as well as PMA stimulated chemiluminescence. Whereas the arthritis-related alterations in neutrophil count and in spontaneous chemiluminescence were not modified by CU, the increased reactivity of neutrophils to PMA was less evident in CUtreated animals. The effects of CU were comparable with those of methotrexate. CU was found to be a potent inhibitor of neutrophil functions (Jancinova et al., 2009). As neutrophils are considered to be cells with the greatest capacity to inflict damage within diseased joints, the observed effects could support the beneficial use of CU in RA treatment. Further the beneficial effect of sesame oil (So) administered alone on markers of OS accompanying AA was demonstrated not only by decrease of plasma TBARS, decrease of GGT activity in the joint and spleen tissues, but also the level of protein carbonyls, TAC in plasma and activity of NAGA in serum and in the kidney were improved, yet not significantly. In HPV the maximal increase was found on day 28 of AA, and at the same time we observed a

Fig. 1. Comparison of the effect of different plant treatments in adjuvant arthritis (AA) on reduction of hind paw volume (HPV) and on GGT (-glutamyltransferase) activity in spleen and joint and level of TBARS (thiobarbituric acid reactive substances) in plasma measured on experimental day 28. Changes in parameters are illustrated in relation to untreated arthritic rats (100% represented by dot-and-dash line). The data were expressed as

arithmetic mean ± S.E.M. Statistical significance was evaluated applying Student's t-test for independent variables: +P < 0.05, ++P < 0.01, and +++P < 0.001 compared to untreated arthritic animals. *Boswellia serrata extract* (Bo), *Arctostaphylos uva-ursi extract* (UV), *Zingiber officinale extract* (Zg), combination of three previous extracts (Bo-UV-Zg), sesame oil in combination with *Arctostaphylos uva-ursi* extract (Bo-So), arbutin (Ar), curcumin (CU),

Pycnogenol® (PYC), pinosylvin (PIN) and pterostilbene (PTE).

significant decrease of aortic endothelium-dependent relaxation. Administration of So resulted in mild, non-significant decrease of hind paw swelling and in significantly increased acetylcholine-evoked relaxation of aorta (Sotnikova et al., 2009).
