Anti-Arthritic Activities of Supercritical Carbon Dioxide Extract Derived from Radiation Mutant Perilla Frutescens Var. Crispa in Collagen Antibody-Induced Arthritis

We investigated the anti-arthritic effects of the radiation mutant Perilla frutescens var. crispa leaf extract (SFE-M) and wild type leaf extract (SFE-W), both prepared by supercritical carbon dioxide (SC-CO2) extraction, on collagen antibody-induced arthritis (CAIA) in Balb/c mice. Animals were randomly divided into four groups: control, CAIA, CAIA + SFE-M (100 mg/kg/day), and CAIA + SFE-W (100 mg/kg/day). The mice were subjected to the respective treatments via oral gavage once daily for 4 days. Mice treated with SFE-M developed less severe arthritis than the CAIA mice. They showed significantly improved arthritic score, paw volume, and paw thickness compared to the CAIA mice from days 3 through 7. Furthermore, histopathological analysis of ankle for inflammation showed that SFE-M treatment reduced inflammatory cell infiltration and edema formation. Similarly, the neutrophil-to-lymphocyte ratio (NLR) in the whole blood was 37% lower in mice treated with SFE-M compared with the CAIA mice. However, treatment with SFE-W did not result in any significant difference compared with the CAIA group. In conclusion, SFE-M treatment delays the onset of arthritis and alleviates its clinical manifestations in CAIA mice.


Introduction
Perilla frutescens (L.) Britt. is an annual herbaceous plant that belongs to family Lamiaceae. Its leaves are used as food in Asian cuisines, and its seeds are used to make edible oil in Korea. Traditionally, P. frutescens has also been used to treat a variety of illnesses, including cough, phlegm, back pain, and diabetes [1,2]. In previous studies, extracts derived from P. frutescens var. crispa were obtained using various methods to analyze various pharmacological activities. For example, both the ethanol extract [3] and the supercritical carbon dioxide (SC-CO 2 ) extract showed anti-inflammatory effects. Both water and ethanol extracts exhibited antioxidant effects [4]. The methanol extract exerted a preventive effect against Alzheimer's disease [5]. In this experiment, we used the extract obtained by SC-CO 2 method to acquire maximum anti-inflammatory ingredients from the leaves of P. frutescens [6]. SC-CO 2 extraction is a novel and powerful technique for the extraction of lipophilic components [7,8].

Sample Preparation and Treatment
SFE-M and SFE-W were suspended in corn oil at a concentration of 20 mg/mL and treated with 100 µL per mouse via oral gavage. Mice were fasted at 7 p.m. and were fed at 10 a.m. after oral administration from days 2 to 6.

Assessment of Clinical Signs of Inflammation
Paw volumes were measured using a plethysmometer (Panlab, S.L.U., Digital Water Plethysmometer LE7500, Spain) every day, after LPS injection. The hind leg was soaked in the buffer calibrated with 1 mL standard sinker, and the increased volume was measured. The average volume of both hind legs was used. Paw thickness was measured using a digital caliper (Mitutoyo, Andover, UK) every day, after LPS injection. The average thickness of both hind legs was used. Arthritic score was evaluated blindly using a system based on the number of inflamed joints in the front and hind paws. Inflammation was defined by swelling and redness on a scale from 0 (no redness and swelling) to 3 (severe swelling with joint rigidity or deformity; maximal score for four paws, 12).

Histopathological Assessement
Hind paws were removed after euthanasia and fixed using 4.5% buffered formalin. Hind paws were decalcified in buffered formalin containing 5.5% EDTA. Upon decalcification, paws were embedded in paraffin (wax) to create paraffin blocks, which were sectioned and stained with hematoxylin and eosin for microscopic evaluation by an expert blinded to the treatments received. Each section was screened for synovial tissue infiltrated by neutrophils and every joint was scored as follows: 0, normal; 1, minimal; 2, mild; 3, moderate; and 4, marked.

Analysis of Neutrophils and Lymphocytes
Neutrophil-to-lymphocyte ratio (NLR) is a useful marker for the evaluation of inflammatory activity in chronic inflammatory disease such as ulcerative colitis [16], prostate cancer [17], and RA [18]. Whole blood samples were collected via cardiac puncture. The blood was placed in Vacutainer TM tubes containing EDTA (BD science, Franklin Lakes, NJ, USA). Anti-coagulated blood was used for hematological testing including neutrophil and lymphocytes in a HEMAVET 950 (Drew Scientific Inc., Miami Lakes, FL, USA).

Statistical Analysis
One-way analysis of variance (ANOVA) was used to determine the overall differences among groups, followed by Fisher's LSD test for individual group comparisons. The results from all comparisons were considered significant at P < 0.05. Data were reported as mean ± SD. All data were analyzed using the SPSS 21.0 program (SPSS Inc., Chicago, IL, USA).

Composition of SFE-M and SFE-W
The leaf extracts from radiation mutant P. frutescens var. crispa and wild type were acquired using the SC-CO 2 method. Figure 1 shows the composition of the two extracts. The isoegomaketone (IK) content in SFE-M was 76.0 ± 0.7 mg/g, approximately 7-fold higher compared with the IK concentration (10.8 ± 0.3 mg/g) in SFE-W.

Statistical Analysis
One-way analysis of variance (ANOVA) was used to determine the overall differences among groups, followed by Fisher's LSD test for individual group comparisons. The results from all comparisons were considered significant at P < 0.05. Data were reported as mean ± SD. All data were analyzed using the SPSS 21.0 program (SPSS Inc., Chicago, IL, USA).

Composition of SFE-M and SFE-W
The leaf extracts from radiation mutant P. frutescens var. crispa and wild type were acquired using the SC-CO2 method. Figure 1 shows the composition of the two extracts. The isoegomaketone (IK) content in SFE-M was 76.0 ± 0.7 mg/g, approximately 7-fold higher compared with the IK concentration (10.8 ± 0.3 mg/g) in SFE-W.

Effects of SFE-W and SFE-M Treatments on the Development of RA in CAIA Model
Initially, whether SFE-M or SFE-W treatment by oral gavage prevented initiation of disease in Balb/c mice with CAIA was investigated. SFE-M-treated mice developed less severe arthritis ( Figure  2). Redness and swelling of joints observed in the CAIA group were significantly attenuated with the SFE-M-treated (100 mg/kg). Histopathological examination also revealed that SFE-M treatment reduced synovial hyperplasia and inflammatory cell infiltration in the joint space ( Figure 2). The mean histopathological arthritic scores of CAIA-group, SFE-M-treated group, and SFE-W-treated group were (2.33 ± 0.82), (0.00 ± 0.00), and (1.00 ± 0.89), respectively (Table 1 and Figure 3).

Effects of SFE-W and SFE-M Treatments on the Development of RA in CAIA Model
Initially, whether SFE-M or SFE-W treatment by oral gavage prevented initiation of disease in Balb/c mice with CAIA was investigated. SFE-M-treated mice developed less severe arthritis ( Figure 2). Redness and swelling of joints observed in the CAIA group were significantly attenuated with the SFE-M-treated (100 mg/kg). Histopathological examination also revealed that SFE-M treatment reduced synovial hyperplasia and inflammatory cell infiltration in the joint space ( Figure 2). The mean histopathological arthritic scores of CAIA-group, SFE-M-treated group, and SFE-W-treated group were (2.33 ± 0.82), (0.00 ± 0.00), and (1.00 ± 0.89), respectively (Table 1 and Figure 3).

Effects of SFE-W and SFE-M Treatments on Paw Volume in CAIA Model
To evaluate whether SFE-W and SFE-M affected the progression of RA in the CAIA model, male Balb/c mice were gavaged with corn oil with or without SFE-M and SFE-W once a day between days 3 and 6. The CAIA group showed a significant increase in paw volume on days 5, 6, and 7 (17.3%, 14.4% and 20.7%, respectively) compared with the control group ( Figure 4). Paw volume was significantly lower in the SFE-M-treated group compared with the control CAIA group on days 5, 6, and 7 (17.4%, 22.8%, and 22.4%, respectively). However, SFE-M treatment did not result in a significant difference in paw volume compared with the CAIA group.

Effects of SFE-W and SFE-M Treatments on Paw Volume in CAIA Model
To evaluate whether SFE-W and SFE-M affected the progression of RA in the CAIA model, male Balb/c mice were gavaged with corn oil with or without SFE-M and SFE-W once a day between days 3 and 6. The CAIA group showed a significant increase in paw volume on days 5, 6, and 7 (17.3%, 14.4% and 20.7%, respectively) compared with the control group (Figure 4). Paw volume was significantly lower in the SFE-M-treated group compared with the control CAIA group on days 5, 6, and 7 (17.4%, 22.8%, and 22.4%, respectively). However, SFE-M treatment did not result in a significant difference in paw volume compared with the CAIA group.

Effects of SFE-W and SFE-M Treatments on Paw Thickness in CAIA Model
To evaluate whether SFE-W and SFE-M had an effect on RA progression in the CAIA model, the paw thickness was measured by digital caliper. The CAIA group showed significant increase in paw thickness on days 6 and 7 (12.5% and 7.8%, respectively) compared to the control group ( Figure 5). Paw thickness was significantly lower in the SFE-M-treated group compared with the control CAIA group on days 5, 6 and 7 (4.7%, 15.3%, and 15.9%, respectively). However, SFE-W-treatment did not result in a significant difference in paw thickness compared with the CAIA group.

Effects of SFE-W and SFE-M Treatments on Paw Thickness in CAIA Model
To evaluate whether SFE-W and SFE-M had an effect on RA progression in the CAIA model, the paw thickness was measured by digital caliper. The CAIA group showed significant increase in paw thickness on days 6 and 7 (12.5% and 7.8%, respectively) compared to the control group ( Figure 5). Paw thickness was significantly lower in the SFE-M-treated group compared with the control CAIA group on days 5, 6 and 7 (4.7%, 15.3%, and 15.9%, respectively). However, SFE-W-treatment did not result in a significant difference in paw thickness compared with the CAIA group.

Effects of SFE-W and SFE-M Treatments on Paw Volume in CAIA Model
To evaluate whether SFE-W and SFE-M affected the progression of RA in the CAIA model, male Balb/c mice were gavaged with corn oil with or without SFE-M and SFE-W once a day between days 3 and 6. The CAIA group showed a significant increase in paw volume on days 5, 6, and 7 (17.3%, 14.4% and 20.7%, respectively) compared with the control group (Figure 4). Paw volume was significantly lower in the SFE-M-treated group compared with the control CAIA group on days 5, 6, and 7 (17.4%, 22.8%, and 22.4%, respectively). However, SFE-M treatment did not result in a significant difference in paw volume compared with the CAIA group.

Effects of SFE-W and SFE-M Treatments on Paw Thickness in CAIA Model
To evaluate whether SFE-W and SFE-M had an effect on RA progression in the CAIA model, the paw thickness was measured by digital caliper. The CAIA group showed significant increase in paw thickness on days 6 and 7 (12.5% and 7.8%, respectively) compared to the control group ( Figure 5). Paw thickness was significantly lower in the SFE-M-treated group compared with the control CAIA group on days 5, 6 and 7 (4.7%, 15.3%, and 15.9%, respectively). However, SFE-W-treatment did not result in a significant difference in paw thickness compared with the CAIA group.

Effects of SFE-W and SFE-M Treatments on Arthritic Score in CAIA Model
Arthritic score was determined blindly by three persons to further evaluate whether SFE-M and SFE-W treatments suppressed RA progression in the CAIA model. The CAIA group showed a significant increase in arthritic score from days 4 through 7 compared with the control group ( Figure 6). The CAIA group showed arthritic symptoms in all joints from day 4 through day 7. The arthritic symptoms were significantly attenuated in the SFE-M-treated group on days 4 through 7. However, SFE-W-treatment did not result in a significant difference in arthritic score compared with the CAIA group.

Effects of SFE-W and SFE-M Treatments on Arthritic Score in CAIA Model
Arthritic score was determined blindly by three persons to further evaluate whether SFE-M and SFE-W treatments suppressed RA progression in the CAIA model. The CAIA group showed a significant increase in arthritic score from days 4 through 7 compared with the control group ( Figure  6). The CAIA group showed arthritic symptoms in all joints from day 4 through day 7. The arthritic symptoms were significantly attenuated in the SFE-M-treated group on days 4 through 7. However, SFE-W-treatment did not result in a significant difference in arthritic score compared with the CAIA group. Figure 6. Effects of SFE-M and SFE-W treatments on arthritic score in the CAIA mice. Treatment concentrations of SFE-M and SFE-W were 100 mg/kg. Arthritic score was evaluated blindly using a system based on the number of inflamed joints in the front and the hind paws. Data are presented as mean  SD (n = 6). SFE-M-treated mice show significantly lower severity than CAIA mice (p  0.05).

Effects of SFE-W and SFE-M Treatments on Blood Cell Population in CAIA Model
NLR is a measure of the absolute neutrophil count relative to lymphocyte numbers in the whole blood. To further determine whether SFE-M and SFE-W treatments affect blood cell population in the CAIA model, NLR was measured in the whole blood sample. The CAIA group showed a significant increase in NLR on day 7 compared with the control group (Figure 7). The NLR level was lower in the SFE-M-treated group compared with the CAIA group by 37%. However, SFE-Wtreatment did not result in a significant difference in NLR levels compared with the CAIA group.

Effects of SFE-W and SFE-M Treatments on Blood Cell Population in CAIA Model
NLR is a measure of the absolute neutrophil count relative to lymphocyte numbers in the whole blood. To further determine whether SFE-M and SFE-W treatments affect blood cell population in the CAIA model, NLR was measured in the whole blood sample. The CAIA group showed a significant increase in NLR on day 7 compared with the control group (Figure 7). The NLR level was lower in the SFE-M-treated group compared with the CAIA group by 37%. However, SFE-W-treatment did not result in a significant difference in NLR levels compared with the CAIA group.

Effects of SFE-W and SFE-M Treatments on Arthritic Score in CAIA Model
Arthritic score was determined blindly by three persons to further evaluate whether SFE-M and SFE-W treatments suppressed RA progression in the CAIA model. The CAIA group showed a significant increase in arthritic score from days 4 through 7 compared with the control group ( Figure  6). The CAIA group showed arthritic symptoms in all joints from day 4 through day 7. The arthritic symptoms were significantly attenuated in the SFE-M-treated group on days 4 through 7. However, SFE-W-treatment did not result in a significant difference in arthritic score compared with the CAIA group. Figure 6. Effects of SFE-M and SFE-W treatments on arthritic score in the CAIA mice. Treatment concentrations of SFE-M and SFE-W were 100 mg/kg. Arthritic score was evaluated blindly using a system based on the number of inflamed joints in the front and the hind paws. Data are presented as mean  SD (n = 6). SFE-M-treated mice show significantly lower severity than CAIA mice (p  0.05).

Effects of SFE-W and SFE-M Treatments on Blood Cell Population in CAIA Model
NLR is a measure of the absolute neutrophil count relative to lymphocyte numbers in the whole blood. To further determine whether SFE-M and SFE-W treatments affect blood cell population in the CAIA model, NLR was measured in the whole blood sample. The CAIA group showed a significant increase in NLR on day 7 compared with the control group (Figure 7). The NLR level was lower in the SFE-M-treated group compared with the CAIA group by 37%. However, SFE-Wtreatment did not result in a significant difference in NLR levels compared with the CAIA group.

Discussion
In the present study, the anti-arthritic effects of the extract from radiation mutant P. frutescens var. crispa prepared via supercritical carbon dioxide extraction (SFE-M) on the development of arthritis in CAIA model was investigated. The efficacy of SFE-M was compared with that of SFE-W. Treatment with SFE-M alleviated immune cell infiltration into joint synovium, paw edema, arthritic score, and NLR levels. Treatment with SFE-W had no significant effect on the development of arthritis. In a previous study, SFE-M showed higher anti-inflammatory activities than SFE-W in LPS-stimulated RAW264.7 cells [15]. Higher anti-inflammatory activities of SFE-M appeared to be due to the higher IK content (almost 7-fold higher level) compared with SFE-W [15]. Similar to the previous study, SFE-M was more effective in delaying the onset of arthritis in CAIA model, compared with SFE-W.
Radiation-induced mutations have been extensively studied and utilized in mutation breeding, following the discovery of X ray-induced mutations in Drosophila [19] and barley [20]. Later, it was found that ionizing radiation induced DNA damage, which is a major factor underlying mutations [21]. Radiation-induced mutation breeding focused on crop improvement [22], enhancing resistance to abiotic and biotic stresses [23], and development of new flower varieties [24]. However, increased functional metabolites in radiation-induced plant mutants has never been reported. This study is significant, in that it provides the evidence for the possibility of therapeutic use of radiation-induced plant mutants by increasing functional phytochemical content. One of the biggest challenges in the investigation of functional foods or phytomedicines using natural resources is that natural resources usually contain very small amounts of functional components. In this study, the possible selection of new resources containing higher levels of functional constituents derived from radiation-induced plant mutants was confirmed. The radiation-induced mutant P. frutescens var. crispa used in this study was acquired using gamma rays. It contained about 7-fold higher IK than the wild type species. IK is biosynthesized from egomaketone (EK), and this reaction is inhibited by gene I in P. frutescens [25]. Therefore, it seems that gene I was affected by gamma radiation, and consequently exhibited lower activity, compared with wild type. The correlation between gene variation and changing IK content is currently being studied. The amount of IK in 100 mg SFE-M is about 6.38 mg. However, treatment with SFE-M (100 mg/kg) showed more effective anti-arthritic action compared with pure IK treatment (10 mg/kg), in CAIA animal model [26]. This may be attributed to other ingredients contained in the extracts, in addition to IK.
Rheumatoid arthritis (RA) is a systemic autoimmune disease in which chronic joint inflammation leads to cartilage destruction and bone erosion [9]. Generally, the use of standard drugs in RA triggers numerous side effects such as infusion hypersensitivity reactions, nausea, dry mouth, somnolence, fatigue, and severe infection [27][28][29]. Currently, there is a growing interest in medicines of botanical origin, which lack severe side effects and have proven efficacy in traditional medicine [11]. These remedies may be effective not only in alleviating the symptoms but also in ameliorating the pathogenesis of the disease [12]. Several anti-arthritic medicinal plants have been tested in animal and human studies: Arnica montana [30], Boswellia spp. [11]; Curcuma spp. [31]; Equisetum arvense [32]; Harpagophytum procumbens [33]; Salix spp.; and Sesamum indicum [34]. Radiation-induced mutant P. frutescens var. crispa used in this study exhibited higher anti-inflammatory activities, compared with wild type. Its extract obtained by supercritical carbon dioxide extraction also displayed adequate potential as an anti-arthritic medicinal plant. To the best of our knowledge, this is the first report that describes radiation-induced plant mutants containing higher anti-arthritic properties, compared with wild type species. However, this experiment was conducted only with male mice; but given that women are more likely to have arthritis than man [35], it will be necessary to use female mice, before applying the treatments to humans. Moreover, it was reported that female mice carrying the Cia40 congenic locus are more affected by collagen-induced arthritis, than are male mice [36].

Conclusions
The extracts of radiation mutant P. frutescens var. crispa (SFE-M) obtained via SC-CO 2 method contained 7-fold higher IK content than the extracts obtained from wild type (SFE-W). SFE-M showed anti-arthritic activity in CAIA model, unlike SFE-W. Therefore, it is thought that radiation mutant P. frutescens var. crispa may be a potential candidate for the treatment of inflammatory diseases, such as RA.