Pharmacological activities of extracts from different parts of Cirsium japonicum var spinossimum

Purpose: To evaluate the antioxidant, anti-inflammatory, inhibitory and cell proliferation activities of Cirsium japonicum (C. japonicum) var. spinossimum. Methods: Phytochemicals (polyphenols, flavonoids, and silymarin) in leaves, roots, and seeds of C. japonicum var. spinossimum were quantified using high-performance liquid chromatography (HPLC). Antioxidant activity was investigated on 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. Anti-inflammatory activity was investigated on nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 cells. Immunostimulant and hair loss prevention was confirmed through 3'-{1-((phenylamino)-carbonyl)-3, 4-tetrazolium}bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate (XTT)-based cell proliferation assays using Jurkat cells and human follicle dermal papilla cells (HFDPC). Results: Extract from seeds exhibited the highest total polyphenol content (610.08 ± 32.40 mg GAE/g), as well as the highest total flavonoid content, while the total polyphenol content of leaves and root extracts were similar. The RC50 values for seed extract against ABTS and DPPH radicals were 8.20 ± 0.02 µg/mL and 40.13 ± 2.47 µg/mL, respectively. In particular, all six Silymarin derivatives were found to be highest in the seed extract. The C. japonicum var. spinossimum seed extract (CSE) showed significant (p < 0.05) nitric oxide (NO) inhibitory activity in RAW264.7 cells induced by LPS at all concentrations (12.5, 25, 50, 100 and 200 µg/mL) and exhibited significant (p < 0.05) cell proliferation activity in Jurkat and hair follicle dermal papilla cells (HFDPC) at 25 µg/mL. Conclusion: Extract of C. japonicum var. spinossimum seeds contain polyphenols and flavonoids and show significant NO inhibitory and cell proliferation activity in Jurkat and HFDPC. Therefore, Cirsium japonicum is a promising source of anti-inflammatory and anticancer agents.

The Cirsium genus of plants contains about 78 species of flavonoids with excellent pharmacological activities, including apigenin, luteolin, myricetin, kaempferol, pectolinarin, 5,7dihydroxy-6,4'-dimethoxyflavone, and hispidulin-7-O-neoheperioside [2,3].Apigenin has been shown to have anticancer and neuroprotective effects, as well as anti-inflammatory, antispasmodic, and antimicrobial effects [4][5][6][7][8].Cirsium plants also have liver-protective effects by inhibiting lipid peroxidation, increasing the activity of glutathione reductase, and promoting alcohol detoxification.They can also decrease serum lipid levels in cases of hyperlipidemia and delay liver damage by reducing the concentration of total cholesterol and neutral lipids in the liver [9].Silymarin found in Silybum marianum has been reported to have hepatoprotective effects against alcohol-induced lipid peroxidation, alcohol-induced liver fibrosis, and other liver diseases due to its flavolignan content [10].Cirsimaritin has been detected in Cirsium pendulum, and linarin has been found in the roots of Cirsium xanthocanthum [11].
In recent times, there has been a growing interest in health-related issues such as disease prevention and treatment, which has led to the development of various drugs and functional foods.
Various pharmacological activity experiments have been conducted to demonstrate the functionality of C. japonicum var.spinossimum as a basic material for the development of functional products.In this study, phytochemicals such as polyphenol and silymarin content were quantified, the extract with the most significant activity was selected, and its pharmacological activity was investigated on inflammatory T cell (Jurkat cell), and human follicle dermal papilla cells (HFDPC) proliferation.
Therefore, this study was aimed at investigating the antioxidant, anti-inflammatory, and immunostimulant activities of the leaves, roots, and seeds of C. japonicum var.spinossimum.

Materials and extraction
Cirsium japonicum var.spinossimum was cultivated in Osu-myeon, Imsil-gun, Jeollabuk-do, Korea.It was taxonomically identified by a plant biotechnologist, Dr. Jong Bo Kim (College of Biotechnology, Konkuk University Glocal Campus, Chungju, Republic of Korea).A voucher specimen was deposited at the College of Health Science, Dankook University, Korea.The leaves and roots were dried at 55 ℃ and stored at ≤ 4 ℃, while the seeds were frozen.
Each sample was prepared by grinding 100 g of the material into powder and extracted three times with 70 % ethanol (1 L) for 24 h.The extracts were filtered using a 0.45 μm filter and then freeze-dried (Eyela FDU-2100, Tokyo Rikakikai Co., Tokyo, Japan) into powder form, which was stored at -20 ℃ for use.The extraction yields were 12.92 % for leaves, 15.68 % for roots, and 16.77 % for seeds.

Measurement of total polyphenol content
Total polyphenol content was measured using the Folin-Denis method [12].Each extract was dissolved in distilled water (DW) at a concentration of 1 mg/mL, and then 1 mL of Folin-Ciocalteau reagent and 1 mL of 10 % sodium trioxocarbonate IV (Na2CO3) (Sigma Co., Ltd., St. Louis, MO, USA) solution were added sequentially.The mixture was left at room temperature (RT) for 1 h and absorbance was measured at 700 nm.
The total polyphenol content of the extract was determined by preparing a standard calibration curve using gallic acid (Sigma Co.) at concentrations ranging from 0 to 100 μg/mL and analyzing it using the same method as the extract.

Measurement of total flavonoid content
Total flavonoid content was measured using the method of Moreno et al [13].A sample solution of 1 mg/mL was prepared by adding 0.1 mL of 10 % aluminum nitrate (Sigma Co.), 0.1 mL of 1 M potassium acetate (Sigma Co.), and 4.3 mL of ethanol in sequence, and then allowed to stand at RT for 40 mins.Absorbance was measured at 415 nm.Total flavonoid content was determined from standard calibration curve obtained from the range of 0 -100 μg/mL using quercetin (Sigma Co.) as the standard reference.

2,2′-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) radical scavenging activity
2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activity was measured using the method of Re et al [14].A solution (7.4 mM) of ABTS (Sigma Co.) and 2.6 mM potassium persulfate (Sigma Co.) were mixed and left at RT for 24 h to form radicals.The ABTS solution was then diluted with methanol to an absorbance of 0.70 ± 0.00 (mean ± SE) at 732 nm just prior to the experiment.Thereafter, 50 μL of the extract was added to 950 μL of prepared ABTS solution, and the mixture was left for 1 min before measuring the absorbance at 732 nm.The sample was analyzed by determining the RC50 value, which is the concentration required to reduce the activity of the negative control by 50 %.

2,2-diphenyl-1-picrylhydrazyl radical scavenging activity
The DPPH radical scavenging activity was measured using Blois' method [15].The samples were dissolved in methanol (MeOH) to a final concentration of 15, 30, 60, 125, 250, and 500 μg/mL and quantified.Then, 100 μL of each sample was added to a 96-well plate along with 100 μL of 0.3 mM DPPH to make a total volume of 200 μL.After incubating at RT for 30 min, the absorbance was measured at 540 nm using an ELISA reader (Molecular Devices, USA).The DPPH radical scavenging activity was expressed as the percentage difference in absorbance between the sample and control groups.Samples were compared and analyzed by calculating the RC50 value, which is the concentration that reduces the activity of negative control group by half.

Analysis of silymarin content
High liquid performance chromatography (HPLC) analysis was performed to determine the content of 6 silymarin standards (Sigma Co.), including silybin A & B, isosilybin A & B, silychristin, and silydian in extracts from different parts of C. japonicum var.spinossimum.Samples and silymarin standards were prepared by dissolving in MeOH to a concentration of 1 mg/mL and filtered through a 0.2 μm filter prior to analysis.An HPLC system (Shimadzu Corporation, Kyoto, Japan) consisting of a CBM-20A communications bus module and SPD-M20A diode array detector was used.A C18 column (250 × 4.6 mm, 5 µm) was selected for analysis, and the column temperature was maintained at RT. Solvent A was 0.01 % acetic acid and solvent B was MeOH, and the solvent gradient was programmed as follows: 0 -3 min, 47 % B; 3 -13 min, 50 % B; 13 -25 min, 60 % B; 25

Nitric oxide (NO) content
Concentration of nitric oxide (NO) was measured by using the Griess Reagent System to determine nitrite concentration in the culture medium.RAW264.7 cells were seeded at a density of 1.0 × 10 5 cells/well in a 96-well plate and cultured for 18 h.The cells were then pretreated with CSE at concentrations of 12.5, 25, 50, 100 and 200 µg/mL for 1 h, followed by treatment with LPS at a concentration of 100 ng/mL and further incubation for 24 h.An equal volume of Griess reagent was added to the culture medium, and after a 10-min incubation at RT, the absorbance was measured at 540 nm [17].The concentration of NO in the culture medium was determined using a standard curve of sodium nitrite (Sigma Co.) at various concentrations.

Cell proliferation
Cell proliferation of Jurkat cells and HFDPC in response to the test samples was measured using the XTT kit (XTT II, Boehringer Mannheim, Mannheim, Germany) [18].Cells were seeded at a density of 1 × 10 4 cells/well in a 48-well culture plate and treated with CSE at concentrations of 0, 12.5, 25, 50, 100, and 200 µg/mL, divided into 3 treatment groups.After 24 and 48 h, 50 μL of XTT reagent (sodium 3'-(1-(phenylaminocarbonyl)-3, 4-tetrazolium)-bis (4-methoxy-6-nitro) benzenesulfonic acid hydrate N-methyl dibenzopyrazine methyl sulfate; mixed at a ratio of 50:1) was added, and after 4 h, the absorbance was measured at 490 nm using an ELISA plate reader.The experiment was independently performed in triplicate and the average was taken.

Statistical analysis
Data were analyzed using Statistical Package for Social Sciences (SPSS software, ver.25, IBM Co., Armonk, NY, USA).Results are presented as mean ± standard deviation (SD).Analysis of variance (ANOVA) was conducted to test statistical significance.The statistical significance of the mean values was analyzed using Duncan's multiple range test at a significance level of p < 0.05.

Silymarin content
The chromatogram of the silymarin standard substance showed six major peaks (Figure 1).The C. japonicum var.spinossimum seed extract showed a higher content of all silymarin derivatives than the leaf and root extracts (Table 2).Particularly, the content of Silydianin and Silybin B, among silymarin derivatives in the seed extract, were 240.48 and 220.48 μg/mg, respectively.Therefore, the C. japonicum var.spinossimum seed extract (CSE), which contains excellent antioxidant and pharmacological active components, was used for anti-inflammatory activity, Jurkat cell proliferation, and HFDPC proliferation tests.

Anti-inflammatory activity of CSE
Before confirming the anti-inflammatory effect of CSE on RAW264.7 cells, cell viability was checked (Figure 2 A) and the results showed that the cell survival rate was over 90 % at all concentrations of CSE (12.5, 25, 50, 100, and 200 µg/mL), indicating no toxicity.Furthermore, levels of NO in CSE were examined (Figure 2 B), and the results revealed that the dose-dependent increase (p < 0.05) in NO-induced by LPS treatment was significantly reduced (p < 0.05) at a concentration of 12.5 µg/mL of CSE.
2) The total flavonoid content was analyzed as quercetin equivalence (QE) mg/g of samples. 3)RC50: Amount required for 50 % reduction of scavenging activity.abcd P < 0.05 vs. control

DISCUSSION
Recently, due to increase in national income and westernization of lifestyle patterns, dietary habits have been changing, and with increase in interest in health due to high mortality rates from chronic degenerative diseases such as cancer, cerebrovascular disease, and diabetes, there is a growing demand for functional foods with pharmacological functions [19].Research on food in recent years focused on natural ingredients extracted from natural foods.As a result, studying the pharmacological activity of components proven to be effective in folk remedies and traditional Korean medicine, and applying them to the development of functional foods becomes imperative [19].
This study aimed to investigate C. japonicum var.spinossimum as a functional food.Ethanol was used to extract the leaves, roots, and seeds of C. japonicum var.spinossimum, and the antioxidant effects of each extract were quantified.The results showed that seed extract contained the highest total polyphenol and flavonoid content.In addition, it was found that the highest ABTS and DPPH activity was observed in the seed extract.Compounds of polyphenol and flavonoid families are known to have excellent antioxidant effects [20].As a result, the excellent antioxidant activity from C. japonicum var.spinossimum seed extracts may be due to the large number of flavonoids and polyphenols present.Silymarin is the main component of Cardus marianus extract, which is extracted from the fruits of Cardus marianus belonging to the Asteraceae family.It is a complex of flavonolignans, consisting of six substances: silychristin, silydianin, silybin A, silybin B, isosilybin A, and isosilybin B. Cardus marianus extract is known to restore liver function and cell protection effects, and is used as an adjuvant therapy for liver diseases in Europe and Asia [21][22][23].When the silymarin content of C. japonicum var.spinossimum leaves, roots, and seeds were measured, the highest content was found in the seed extract.Therefore, C. japonicum var.spinossimum seed extract (CSE) has the highest potential as a functional material.As a result, the cell proliferation activity of T cells and HFDPC was measured using CSE.
When foreign substances enter the body, T cells induce cellular immune responses and B cells produce antibodies to trigger humoral immune responses [24].To investigate the effect of CSE on T cells, the proliferation of T cells was measured using Jurkat cells.T cells were treated with CSE at 12.5, 25, 50, 100, and 200 μg/mL with a control group that received no treatment and then cultured for 24 and 48 h to determine cell proliferation rate.At 24 and 48 h, there was a significant increase in proliferation rate at 25 μg/mL, indicating that CSE induces T cell proliferation.Hair loss refers to the abnormal shedding of hair at a rate faster than normal hair growth cycle due to genetics, stress, diet, hair and scalp products, and more.Hair serves a protective function against external stimuli, UV radiation, and temperature changes, and is also used as a decorative means of expressing one's individuality and self-confidence in modern society [25].Recently, the number of patients suffering from hair loss is estimated to be in the tens of millions, and various studies are underway to develop treatment methods in line with these trends.However, treatment options and cure have not been identified.Therefore, to investigate the potential of CSE as a material for improving hair loss, the cell proliferation ability of HFDPC was measured in a concentration-and time-dependent manner and a significant cell proliferation effect of CSE was observed at 25 μg/mL.Based on these results, the seed extract of C. japonicum var.spinossimum was considered to be a strong antioxidant and antiinflammatory agent, and it also has immuneenhancing and HFDPC proliferating effects.

CONCLUSION
The seed extract of C. japonicum var.spinossimum possesses a high concentration of polyphenols and flavonoids and also shows the best ABTS and DPPH radical scavenging activity with large amounts of silymarin compounds.Furthermore, it significantly inhibits NO production in LPS-stimulated RAW 264.7 cells and promotes HFDPC proliferation.Therefore, C. japonicum var.spinossimum seed extract may be a valuable candidate for developing immuneenhancing agents and hair loss treatments.

Figure 1 :
Figure 1: Comparative chromatogram of silymarin derivatives in leaf, root, and seed of C. japonicum var.spinossimum.

Figure 2 :
Figure 2: Effect of CSE on the RAW264.7 cell viability and NO inhibition.(A) Cell viability on CSE was determined using an MTT assay in the presence of LPS (100 ng/mL).RAW264.7 cells were treated with CSE at indicated concentrations (12.5, 25, 50, 100, and 200 µg/mL) with LPS (100 ng/mL).(B) NO in the culture supernatant was evaluated using Griess reagent.abcde P < 0.05 vs. control

Figure 3 :
Figure 3: Effect of CSE on the cell proliferation in Jurkat cell line.Cells were incubated with various concentrations of CSE.After (A) 24 h and (B) 48 h of incubation, proliferation was measured by XTT assay.abcde P＜0.05 vs. control

Figure 4 :
Figure 4: Effect of CSE on the cell proliferation in HFDPC.Cells were incubated with various concentrations of CSE.After (A) 24 h and (B) 48 h of incubation, proliferation was measured by XTT assay.abcde P < 0.05 vs. control -35 min, 60 % B; 35 -35.01 min, 47 % B; 35.01 -45 min, 47 % B. The total analysis time for all samples was 45 min.The flow rate was 1.0 mL/min, and the sample injection volume was 10 μL.Quantitative analysis was performed at 288 nm absorbance.

Table 1 :
Total phenolic, flavonoid contents and antioxidant activities of leaf, root, and seed of C. japonicum var.spinossimum

Table 2 :
Silymarin derivatives contents from leaf, root, and seed of C. japonicum var.spinossimum