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. 2020 Dec 11;11:584849. doi: 10.3389/fphar.2020.584849

Chemical Composition of Pterospermum heterophyllum Root and its Anti-Arthritis Effect on Adjuvant-Induced Arthritis in Rats via Modulation of Inflammatory Responses

Li Yang 1, Ronghua Liu 1, Aiguo Fan 1, Jingjing Zhao 2, Yong Zhang 1, Junwei He 1,*
PMCID: PMC7759541  PMID: 33362544

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease without effective and beneficial drugs. Many traditional folk medicines have been proven to be effective in treating RA. Among these, the root of Pterospermum heterophyllum Hance has been widely used as a traditional remedy against RA in China, but there is no scientific basis yet. The aim of this study was to investigate for the first time the chemical compositions and therapeutic effect of P. heterophyllum on adjuvant-induced arthritis (AIA) model in rats. 73 compounds were identified from P. heterophyllum based on ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-qTOF-MS/MS), and flavonoids may be partly responsible for the major anti-arthritic effect. In parallel, the P. heterophyllum extract at 160, 320, and 640 mg/kg/day were orally administered to rats for 22 days after post-administration adjuvant. The results showed that P. heterophyllum remarkably ameliorated histological lesions of the knee joint, increased body weight growth, decreased arthritis score, reduced thymus and spleen indices in model rats. Moreover, P. heterophyllum treatment persuasively downregulated the levels of rheumatoid factor (RF), C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-17, cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) and matrix metalloproteinase-2 (MMP-2), and observably upregulated IL-4 and IL-10 levels in model rats. These findings suggest that P. heterophyllum has a prominent anti-RA effect on AIA rats by modulating the inflammatory responses, and supports the traditional folk use of this plant.

Keywords: Pterospermum heterophyllum root, rheumatoid arthritis, chemical composition, flavonoid, inflammatory response

Introduction

Rheumatoid arthritis (RA) is a chronic, systematic and autoimmune inflammatory disease that results in progressive synovitis, joint swelling and damage, synovial hyperplasia, and bone and cartilage erosion (Yousefi et al., 2014; Saleem et al., 2020; Zhu et al., 2020). Although the etiology of RA is intricate and vague, inflammatory factors, including pro-inflammatory cytokines, anti-inflammatory cytokines and inflammatory mediators, are responsible for bone and cartilage erosions, and play a crucial role in this disease (Wang et al., 2017a; Rui et al., 2019; Saleem et al., 2020). Additionally, the serum levels of these inflammatory mediators were determined by enzyme-linked immunosorbent assay (ELISA) kits (Lin et al., 2013; Wang et al., 2017a). Currently, immunosuppressants, biological agents and disease-modifying anti-rheumatic drugs (DMARDs), and steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used for the treatment of RA, but most of them display long-term adverse effects, toxicity and comorbidities (Dai et al., 2020; Li et al., 2019a, Li et al., 2019b; Rui et al., 2019). As a result of this, exploring effective and safe anti-RA drug candidates from natural products, especially traditional folk medicines, could be a momentous breakthrough.

Traditional Chinese medicines (TCMs) are decisive complementary and alternative medicines, which have been verified to be effective treating RA for centuries with more safety and little side-effects in China and other Southeast Asian countries (Bao et al., 2018; Li et al., 2019a, Li et al., 2019b; Lin et al., 2013; Jing et al., 2019; Yang et al., 2020). Pterospermum heterophyllum Hance is native only to China and widely distributed in Fujian, Guangdong, Guangxi and Hainan provinces, belonging to the Sterculiaceae family (Editorial Committee of Traditional Chinese Medicine 1999; Yang et al., 2016, 2019a). The root of P. heterophyllum is a vital TCM and has been used for centuries as an empiric treatment for RA and other inflammation-related diseases (Editorial Committee of Traditional Chinese Medicine 1999; Yang et al., 2016; Yang et al., 2019a). Despite good clinical practice and good clinical effects, the phytochemical profiling and anti-RA efficacy of P. heterophyllum are still unknown, leading to numerous obstacles in the clinical application and reasonable development of this plant.

Therefore, in this study, the AIA rat model was adopted to evaluate the therapeutic efficacy and underlying mechanisms of P. heterophyllum. Following this step, ultra-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-qTOF-MS/MS) analysis was performed to explore the phytochemicals present in this plant. Our findings will provide adequate scientific evidence for the development and clinical application of P. heterophyllum.

Materials and Methods

Chemicals and Reagents

Pentobarbital sodium (Shanghai Rongbai Biological Technology Co., Ltd., Shanghai, China), Complete Freund’s adjuvant (CFA) and Histopaque 1,083 (Sigma Co., USA), MTX (Shanghai Xingyi Pharmaceutical Co., China), TNF-α, IL-1β, IL-4, IL-6, IL-10, IL-17, COX-2, 5-LOX and MMP-2 ELISA kits (Chuzhou Shinuoda Biological Technology Co., China) were used in this experiment.

Plant Material and Extracts Preparation

Plant materials of P. heterophyllum roots were collected from the town of Pulu, Lipu Country, Guilin City, Guangxi, China (GPS location: 110.51682262,911,989, 24.576018798,987,043), in October 2017, and was authenticated by professor Ronghua Liu. A voucher specimen (No. PH20171024) for P. heterophyllum root was deposited in the author’s laboratory.

The dried and powdered roots of P. heterophyllum (1.0 kg) were extracted with 95% EtOH (5 L × 3) and subsequently with 50% EtOH (5 L × 3) by maceration at room temperature for 7 days. The ethanol crude extract of P. heterophyllum roots was filtrated and evaporated to obtain a black residue (PH, 160 g), with a yield of 16.0%.

According to the TCM clinical practice (9–30 g/day) (Editorial Committee of Traditional Chinese Medicine 1999), the dosage of P. heterophyllum roots for rat was 0.8–2.7 g/kg/day (body weight). Thus, the dosages of PH for rat were 1.0 g/kg (equivalent to 160 mg/kg crude extract, low-dose), 2.0 g/kg (320 mg/kg, medium-dose) and 4.0 g/kg (640 mg/kg, high-dose) in this experiment. All these extracts were dissolved in 0.3% sodium carboxymethyl cellulose (CMC-Na) for oral administration.

Ultra-Performance Liquid Chromatography-Quadrupole Time-of-Flight Tandem Mass Spectrometry Analysis for Chemical Profiling

The identification of phytochemicals in the ethanol crude extract of PH was carried out using UPLC-qTOF-MS/MS in a Shimadzu UHPLC System (Kyoto, Japan) coupled with an AB SCIEX Triple TOF™ 5600 + system (Foster City, CA, USA) (Yang et al., 2019b). The chromatographic separation was conducted in an ACQUITY UPLC®BEH C18 (100 × 2.1 mm, 1.7 μm) maintained at 35°C. 0.1% aqueous formic acid (v/v, A) and acetonitrile (B) were used as mobile phases. The gradient elution with the flow rate of 0.3 ml/min was performed as follows: 0–8 min 5–8% B; 8–12 min 8–8% B; 12–17 min 8–12% B; 17–28 min 12–35% B; 28–35 min 55–55% B; 35–45 min 55–95% B; 45–47 min 95–95% B; 47–47.1 min 95–5% B; 47.1–50.0 min 5–5% B. The sample inject volume was 3 μL.

Experimental Animals

Sprague-Dawley rats (weighing 160–180 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in cages at a room temperature of 21–25°C with 12 light/dark reverse cycles.

Experimental Design

After adaptive feeding, the rats were randomly assigned to six groups (n = 8): normal control (Control), AIA model (AIA), AIA model + MTX (AIA + MTX, 0.35 mg/kg), AIA model + PH low-dose (AIA + PH-L, 160 mg/kg), AIA model + PH medium-dose (AIA + PH-M, 320 mg/kg), and AIA model + PH high-dose (AIA + PH-H, 640 mg/kg). In accordance with the previous method, the AIA rat model was induced by a single intradermal injection of 100 μL CFA into the rat’s left hind footpad (day 1) (Yang et al., 2016; Pan et al., 2017). After the establishment of the AIA model, all PH crude extracts were administered orally once a day from day 7 to day 28. MTX was used as a positive drug and administered intragastrically (i.g.) twice a week. Meanwhile, the rats in the normal control group and the AIA model group were treated with an equal volume of 0.3% CMC-Na. The experimental protocol of PH effect on CFA-induced RA in rats was shown in Figure 1.

FIGURE 1.

FIGURE 1

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The experimental schedule of P. heterophyllum in the AIA rats.

Evaluation of Rheumatoid Arthritis

The body weight and arthritis score of rats were measured every 4 days. The arthritis scores of rat paws were evaluated using a 5-point scale (Yang et al., 2016; Jing et al., 2019): 0 = no erythema or swelling; 1 = erythema or toe joints swelling; 2 = toes and joints swelling; 3 = toes swelling and ankle joints swelling; 4 = the entire paw swelling and ankle joints swelling. The maximum arthritis score of each rat was set at 16 (4 points ×4 paws).

On day 29th day after immunization, all rats were killed after anesthesia (1% pentobarbital sodium, 40 mg/kg), and the immune organs including thymus and spleen were harvested and weighed. The index of thymus or spleen (mg/g) = thymus or spleen wet weight/body weight (Lin et al., 2013; Jing et al., 2019).

Biochemical and Hematological Analysis

Blood was collected from the carotid artery of rats after being anesthetized. The peripheral blood mononuclear cells (PBMC) isolation process was performed according to the previous method (Lin et al., 2013). The serum levels of RF, CRP, TNF-α, IL-1β, IL-4, IL-6, IL-10, and IL-17, and the PBMC levels of COX-2, 5-LOX and MMP-2, were quantified by commercially available ELISA kits based on the manufacturer’s instructions (Chuzhou Shinuoda Biological Technology Co., China).

Histopathological Examination

The ankle joints of the rats were removed and fixed in 4% (w/v) paraformaldehyde, decalcified in 10% ethylene-diamine-tetraacetic acid (EDTA) at 4°C for 30 days. Tissues were embedded in paraffin and 4 µm joint sections were obtained. Subsequently, the sections were deparaffinized, dehydrated and stained with hematoxylin and eosin (HE). These sections were examined with a DS-F12 microscope (magnification, ×100, Nikon Corporation, Japan) for histopathological analysis.

Statistical Analysis

Graphpad Prism6 was used for statistical analysis, and the data were presented as the means ± standard deviation (SD). One-way analysis of variance (ANOVA) and Tukey’s test were used for comparison differences groups. Differences with p < 0.05 indicated statistical significance.

Ethics Statement

All the experiments were carried out in adherence with the guidelines of the Institutional Animal Care and Use Committee of China and were approved by the Animal Care and Research Committee of Jiangxi University of Traditional Chinese Medicine. All surgical procedures were performed under sodium pentobarbital anesthesia to minimize suffering.

Results

Phytochemicals Identification of P. heterophyllum Using Ultra-Performance Liquid Chromatography-Quadrupole Time-of-Flight Tandem Mass Spectrometry

The chemical constituents corresponding to the chromatographic peaks were determined by MS/MS analysis using negative- and positive-ion modes based on literature and databases (Yang et al., 2019b) (Figure 2).

FIGURE 2.

FIGURE 2

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The base peak chromatogram of P. heterophyllum by UPLC-Q-TOF-MS/MS in negative and positive-ion modes.

As a result, a total of 73 compounds, including 34 flavonoids, eight fatty acids, seven triterpenoids, six steroids, six alkaloids, five phenylpropanoids, and seven others were identified from P. heterophyllum based on UPLC-qTOF-MS/MS (Table 1). Therefore, this study has greatly enriched the chemical constituents and diversity. Among them, 15 flavonoids, including procyanidin B2 (peak 9) (Wang et al., 2017b), dihydromyricetin (peak 10) (Chu et al., 2018), (-)-epicatechin (peak 11) (Osman et al., 2019), puerarin (peak 12) (Wang et al., 2016), rutin (peak 24) (Sun et al., 2017), naringin (peak 28) (Ahmad et al., 2014), hesperidin (peak 32) (Li et al., 2019a), myricetin (peak 35) (Yuan et al., 2015), eriodictyol (peak 40) (Lei et al., 2020), quercetin (peak 41) (Saccol et al., 2019), naringenin (peak 43) (Zhu et al., 2015), kaempferol (peak 44) (Pan et al., 2018), diosmetin (peak 46) (Chen et al., 2019), nobiletin (peak 51) (Li et al., 2019a), and tangeretin (peak 53) (Li et al., 2019c), have been reported to have arthritis inhibitory effect in rats. Additionally, cinnamaldehyde (peak 8, phenylpropanoid) (Mateen et al., 2019), ursolic acid (peak 65, triterpenoid) (Kim et al., 2018), linoleic acid (peak 67, fatty acid) (Wang et al., 2011) and emodin (peak 54, other) (Zhu et al., 2013) were also exhibited anti-arthritis activities in vivo. Consequently, flavonoids may be responsible for the major active constituents in the roots of P. heterophyllum against RA.

TABLE 1.

Chemical constituents of P. heterophyllum identified by UPLC-qTOF-MS/MS in negative- and positive-ion modes.

No t R (min) Ion mode Molecular weight Measured mass Error (ppm) Formula Fragments Identification Type
1 1.01 [M–H]− 342.11621 341.10944 1.5 C12H22O11 341.1091,179.0589,161.0489,119.0400,113.0295 Sucrose Other
2 1.71 [M + H]+ 267.09675 268.10428 0.9 C10H13N5O4 136.0613,119.0343 Adenosine Alkaloid
3 1.98 [M–H]− 283.09167 282.0853 3.2 C10H13N5O5 150.0451,133.0200,108.0287 Guanosine Alkaloid
4 4.09 [M–H]− 316.07943 315.07218 0.1 C13H16O9 153.0218,109.0346 Protocatechuic acid-hexoside Other
5 4.38 [M–H]− 330.09508 329.0879 0.3 C14H18O9 167.0369,152.0134,133.0353,123.0495,108.0259 Phaseoloidin Phenylpropanoid
6 5.87 [M–H]− 360.10565 359.09827 -0.3 C15H20O10 197.0470, 182.0245,167.0008,153.0603,138.0366,123.0128 Methoxypolygoacetophenoside Other
7 10.36 [M–H]− 306.07395 305.06722 1.8 C15H14O7 305.0599,287.0550,269.0455,243.0298,225.0553,201.0592,164.0125,161.0262,137.0256,130.9705,125.0273,121.0311,109.0347 (-)-Eplgallocatechin Flavonoid
8 11.15 [M + H]+ 132.05751 133.06506 2 C9H8O 105.0696,103.0551 Cinnamaldehyde Phenylpropanoid
9 14.93 [M–H]− 578.14243 577.13328 −3.2 C30H26O12 577.1320,451.1019,425.0859,407.0758,289.0718,245.0823,161.0265,125.0277 Procyanidin B2 Flavonoid
10 15.01 [M–H]− 320.05322 319.04616 0.7 C15H12O8 193.0153,191.0347,161.0227,151.0060,137.0269,125.0268 Dihydromyricetin Flavonoid
11 16.12 [M–H]− 290.07904 289.07286 3.8 C15H14O6 289.0721,245.0833,221.0836,205.0518,203.0733,125.0287,123.0491,109.0340 (-)-Epicatechin Flavonoid
12 17.27 [M–H]− 416.11073 415.10294 −1.3 C21H20O9 415.1047,307.0607,295.0621,277.0515,267.0671,253.0519 Puerarin Flavonoid
13 18.84 [M–H]− 446.1213 445.11343 −1.3 C22H22O10 445.1113,325.0720,310.0493,297.0781,282.0549 3′-methoxypuerarin Flavonoid
14 19.05 [M–H]− 522.21011 521.20164 −2.3 C26H34O11 359.1508，344.1279,313.1090,241.0523 Urolignoside Phenylpropanoid
15 19.67 [M–H]− 548.15299 547.14409 −3 C26H28O13 547.1443,295.0624,277.0529,267.0678 Mirificin Flavonoid
16 20.16 [M–H]− 866.20582 865.19658 −2.3 C45H38O18 865.1956,847.1881,739.1656,713.1496,695.1387,577.1328,575.1178,451.1030,425.0877,407.0768,287.0569,243.0317 B-type procyanidin trimer Flavonoid
17 20.40 [M–H]− 594.13734 593.12836 −2.9 C30H26O13 593.1302,575.1200,557.1046,467.0988,423.0719,405.0596,387.0524,313.0353,305.0671,287.0568,243.0316,195.0295,161.0267,125.0281 Proanthocyanidins dimer Flavonoid
18 20.47 [M–H]− 438.11621 437.1081 −1.9 C20H22O11 437.1076,311.0772,297.0618,269.0698,167.0362,149.0269,125.0277 Loquatoside Flavonoid
19 20.72 [M–H]− 882.20073 881.19123 −2.5 C45H38O19 881.1929,863.1863,755.1616,745.1931,729.1377,711.1371,593.1273,575.1173,467.0996,423.0719,305.0674,287.0577,243.0293,125.0260 Proanthocyanidins trimer Flavonoid
20 21.02 [M–H]− 1,154.692 1,153.2568 −4.4 C60H50O24 1,153.2545,865.1957,739.1650,575.1177,449.0873,287.0573 B-type procyanidin tetramer Flavonoid
21 21.88 [M–H]− 482.14243 481.1338 −2.8 C22H26O12 481.1326,357.1341,311.0779,297.0618,168.0446,167.0368,154.0296,153.0210,108.0243 Heterophylloside B Other
22 22.06 [M + H]+ 304.0583 305.06576 0.6 C15H12O7 287.0529,231.0641,213.0530,153.0178 Dihydroquercetin Flavonoid
23 22.69 [M–H]− 464.09548 463.08696 -2.7 C21H20O12 463.0886,316.0212,301.0367,300.0290,151.0074 Hyperin Flavonoid
24 22.71 [M–H]− 610.15339 609.14485 −2.1 C27H30O16 609.1452,301.0360,300.0288,271.0241,255.0296 Rutin Flavonoid
25 23.20 [M–H]− 576.12678 575.1178 −3 C30H24O12 575.1203,557.1119,539.0974,449.0889,423.0716,407.0776,289.0726,285.015,245.0831 Procyanidin A2 Flavonoid
26 23.33 [M–H]− 864.19016 863.18099 −2.2 C45H36O18 863.1832,711.1340,693.1239,575.1184,539.0976,449.0871,423.0724,285.0419 A-type procyanidin trimer Flavonoid
27 23.85 [M–H]− 594.15847 593.14921 −3.3 C27H30O15 593.1485,285.0411,284.0330,255.0312 Kaempferol-3-O-[2-rhamnose (1–2)]-glucopyranoside Flavonoid
28 24.12 [M + H]+ 580.17921 581.18707 1 C27H32O14 581.2823,417.1038,315.0841,273.0740,219.0270,153.0175,129.0538 Naringin Flavonoid
29 24.4 [M–H]− 448.10056 447.09232 −2.2 C21H20O11 447.0945,301.0359,285.0408,271.0266,257.0490,255.0323,229.0536,151.0046 Kaempferol-7-O-D-glucoside Flavonoid
30 24.85 [M + COOH]- 678.50438 723.50142 −0.1 C36H66N6O6 723.5011,677.4934,419.0733,225.1590 cyclo hexaleucyl (isoleucyl) Alkaloid
31 24.92 [M + H]+ 302.07904 303.08638 0.2 C16H14O6 303.0843,177.0552,153.0172,145.0283,137.0585,117.0329 Hesperetin Flavonoid
32 24.94 [M–H]− 610.18977 609.18109 −2.3 C28H34O15 609.1807,489.1382,343.0826,301.0722,286.0493 Hesperidin Flavonoid
33 24.97 [M–H]- 462.11621 461.10857 −0.8 C22H22O11 461.1071,446.0864,298.0483,283.0272,255.0309 Chrysoeriol-7-O-galactoside Flavonoid
34 25.09 [M + H]+ 274.08412 275.09143 0.1 C15H14O5 169.0490,107.0490 Phloretin Other
35 25.10 [M–H]− 318.03757 317.03072 1.4 C15H10O8 317.0319,289.0733,258.0553,207.0678,192.0442,178.9972,152.0151,151.0075,125.0286,109.0331 Myricetin Flavonoid
36 25.12 [M–H]− 436.13695 435.12907 −1.4 C21H24O10 273.0775,179.0355,167.0366,125.0267,123.0482,119.0527 Phloridzin Other
37 25.62 [M + COOH]- 791.58845 836.58353 −2.4 C42H77N7O7 836.5830,790.5797 cyclo heptaleucyl (isoleucyl) Alkaloid
38 26.26 [M + COOH]- 904.67251 949.66779 −1.9 C48H88N8O8 949.6680,903.6638 cyclo octaleucyl (isoleucyl) Alkaloid
39 26.78 [M + COOH]- 1,017.75658 1,062.7511 −2.5 C54H99N9O9 1,062.7547,1016.7439 cyclo nonaleucyl (isoleucyl) Alkaloid
40 27.01 [M–H]− 288.06339 287.05721 3.8 C15H12O6 151.0078,135.0492,134.0409,107.0191 Eriodictyol Flavonoid
41 27.70 [M–H]− 302.04265 301.03621 2.8 C15H10O7 301.0357,273.0418,245.0464,179.0002,151.0067,121.0331 Quercetin Flavonoid
42 29.19 [M + H]+ 386.13655 387.14419 0.9 C21H22O7 387.1431,357.1300,191.0687,181.0491,163.0741,137.0587 Kushenol W Flavonoid
43 29.36 [M–H]− 272.06847 271.06247 4.7 C15H12O5 271.0618,187.0418,151.0063,119.0541 Naringenin Flavonoid
44 30.08 [M + H]+ 286.04774 287.0549 −0.4 C15H10O6 287.0531, 258.0480,213.0528,153.0141 Kaempferol Flavonoid
45 30.21 [M + H]+ 488.35018 489.35729 −0.3 C30H48O5 453.3349,435.3228,425.3228,407.3288,205.1577 Trihydroxy-urs-12-en-28-oic acid Triterpenoids
46 30.27 [M–H]− 300.06339 299.05697 2.9 C16H12O6 299.0566,284.0340,256.0396,227.0370 Diosmetin Flavonoid
47 30.91 [M–H]− 330.24062 329.23415 2.4 C18H34O5 329.2344,311.2236,293.2138,229.1463,211.1364,183.1420,171.1059,139.1173 Trihydroxy-octadecaenoic acid Fatty acids
48 32.10 [M + H]+ 260.10486 261.1119 −0.9 C15H16O4 243.1019,213.0533,189.0533,187.0393,159.0432,131.0484,103.0539 Meranzin Phenylpropanoid
49 32.79 [M + H]+ 342.11034 343.1179 0.8 C19H18O6 343.1151,328.0910,313.0691,285.0751,181.0113,153.0186 5,7,2′,3′-tetramethoxyflavone Flavonoid
50 33.30 [M + COOH]- 740.43469 785.42943 −3 C39H64O13 739.4244,577.3648 20 (22)-en-5β-furost-3β,15β-diol-3-O-β-d-glucopyranosyl-(1→2)-β-d-galactopyranoside Steroid
51 33.88 [M + H]+ 402.13147 403.13911 0.9 C21H22O8 403.1384,388.1158,373.0912,327.0850,183.0273 Nobiletin Flavonoid
52 34.01 [M + H]+ 344.0896 345.09727 1.1 C18H16O7 345.0955,330.0707, 315.0534,287.0523,281.0426,181.0426 Santin Flavonoid
53 35.24 [M + H]+ 372.1209 373.12858 1.1 C20H20O7 373.1286,358.1045,343.0810,325.0700,312.0994,297.0748 Tangeretin Flavonoid
54 36.43 [M–H]−- 270.05282 269.04621 2.5 C15H10O5 269.0459,241.0511,225.0575,213.0602 Emodin Other
55 36.44 [M + H]+ 202.02661 203.03372 −0.8 C11H6O4 203.0334,175.0511,159.0434,147.0438,131.0459,129.0335,119.0508 Xanthotoxol Phenylpropanoid
56 36.52 [M–H]−- 314.24571 313.2389 1.5 C18H34O4 313.2389,295.2283,277.2172,201.1150,171.1049 Dihydroxy-octadecaenoic acid Fatty acids
57 39.83 [M + H]+ 472.35526 473.36283 0.6 C30H48O4 437.3402,409.3445,391.3338,205.1565,203.1769,189.1616 Maslinic acid Triterpenoids
58 39.84 [M–H]− 518.36074 517.35111 −4.6 C31H50O6 471.3456 (1,3,9)-24-hydroperoxy-1,3-dihydroxy-5-methyl-9,19-cyclolanost-25-en-28-oic acid Triterpenoids
59 40.29 [M–H]− 294.2195 293.21285 2.1 C18H30O3 293.2125,197.1216,185.1200,125.0991 Hydroxy-octadecatrienoic acid Fatty acids
60 41.46 [M + H]+ 352.26136 353.26885 0.6 C21H36O4 353.2658,261.2203,243.2099,173.1313,135.1160,121.1007,107.0854 Pregnane-3,11,17,20-tetrol Steroid
61 41.75 [M–H]− 312.30283 311.2958 0.8 C20H40O2 311.2597,293.2483,275.2358,171.1047 Arachidic acid Fatty acids
62 41.80 [M–H]− 296.23515 295.22824 1.3 C18H32O3 295.2290,277.2197,251.2395,221.1934,169.1618 Hydroxy-octadecadienoic acid Fatty acids
63 42.12 [M–H]− 312.23006 311.22319 1.3 C18H32O4 311.2230,171.1051,155.1469,127.1163,111.0860,109.0698 Dihydroxy-octadecadienoic acid Fatty acids
64 43.55 [M + H]+ 438.34978 439.35744 0.9 C30H46O2 439.3556,393.3476,203.1779,191.1774,189.1617 3-Oxolup-20 (29)-en-28-aL Triterpenoids
65 43.57 [M + H]+ 456.36035 457.36785 0.5 C30H48O3 457.2337,411.3602,297.2544,203.1785,189.1635,121.1007 Ursolic acid Triterpenoids
66 43.93 [M–H]− 340.24023 339.23292 0.9 C23H32O2 339.2330,163.1155 Dimethisterone Steroid
67 44.15 [M–H]− 280.24023 279.23385 3.2 C18H32O2 279.2334,261.2223 Linoleic acid Fatty acids
68 44.62 [M + H]+ 454.3447 455.35225 0.6 C30H46O3 455.3163,437.3401,409.3455,329.2449,283.2401,203.1777,189.1626 Oleanonic acid Triterpenoids
69 44.62 [M + H]+ 436.33413 437.3416 0.4 C30H44O2 437.3410,391.3345,215.1770,203.1785,189.1626,133.1000 Ursa-2,9 (11),12-trien-24-oic acid Triterpenoids
70 45.14 [M + H]+ 428.36543 429.37266 −0.1 C29H48O2 429.3692,411.3598,393.3497,357.3497,175.1106 5α-stigmastan-3,6-dione Steroid
71 45.48 [M + H]+ 278.22458 279.23161 −0.9 C18H30O2 279.0936,201.0436,149.0213,121.0999 Estrane-3,17-diol Steroid
72 46.86 [M–H]− 284.27153 283.26528 3.6 C18H36O2 283.2648,265.2529 Stearic acid Fatty acids
73 48.00 [M–H]− 576.43899 575.43149 −0.4 C35H60O6 575.4606,557.4487,295.2381,241.2207 β-daucosterin Steroid
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Amelioration of Body Weight Loss and Arthritis Score in Adjuvant-Induced Arthritis Rats by P. heterophyllum

The body weight and arthritis score of the rats in this experiment were evaluated at 4-day intervals from day 0 to day 28. As shown in Figure 3A, the body weight of the normal control rats increased steadily throughout the process, whereas the body weight slowly increased in AIA model rats. Importantly, P. heterophyllum treatment in three doses (160, 320, and 640 mg/kg) ameliorated the body weight loss of the model rats to some extent.

FIGURE 3.

FIGURE 3

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Effect of P. heterophyllum on the body weight (A) and arthritis score (B) in the AIA rats. Data are shown as mean ± SD (n = 8). Differences were analyzed using ANOVA by Tukey’s test. & p < 0.05 and && p < 0.01 compared with the model group.

As presented in Figure 3B, the rats in the model group had markedly higher arthritis scores compared to the normal control group (arthritis scores = 0, p < 0.01). After drug treatment, the positive drug methotrexate (MTX) showed prominently decreased arthritis scores compared to the model group from day 8 (p < 0.01). Similar to the MTX treatment, after administration of PH-M (320 mg/kg) and PH-H (640 mg/kg), the arthritis scores values decreased significantly from day 24 (p < 0.01 or p < 0.05). These results indicate that P. heterophyllum possesses a potent anti-RA effect in AIA model rats.

Improving the Histopathology Lesions in Adjuvant-Induced Arthritis Rats by P. heterophyllum

Histopathological examination is the most informative and intuitive technique for exploring the manifestations of RA disease. Compared to the normal control rats, histopathological changes of the ankle joint in AIA model rats were characterized by massive inflammatory cell infiltration into synovial tissue, pannus formation, synovial hyperplasia, and bone and cartilage erosions (Figure 4). These abnormal histopathological changes were prominently alleviated in AIA model rats after treatment with MTX and P. heterophyllum, especially P. heterophyllum at a dosage of 640 mg/kg.

FIGURE 4.

FIGURE 4

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Effect of P. heterophyllum on histopathological changes of ankle joints in the AIA rats (×100, HE staining).

Decrease in Thymus and Spleen Indices in Adjuvant-Induced Arthritis Rats by P. heterophyllum

The results summarized in Figure 5 indicate that the weights of the thymus (Figure 5A) and spleen (Figure 5B) increased remarkably in the rats of the AIA model group in contrast to the rats of the normal control group (p < 0.01). After treatment with three crude extracts of P. heterophyllum and MTX, the weight of the thymus and spleen persuasively decreased (p < 0.01 or p < 0.05) compared to the model group. The results showed that rats in the AIA model group had hyperimmune response after intradermal injection of CFA, while P. heterophyllum could suppress abnormal immune function.

FIGURE 5.

FIGURE 5

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Effect of P. heterophyllum on the indexes of thymus (A) and spleen (B) of AIA rats. Data are shown as mean ± SD (n = 8). Differences were analyzed using ANOVA by Tukey’s test. ## p < 0.01 compared with the control group, & p < 0.05 and && p < 0.01 compared with the model group.

Decreasing Serum Levels of Rheumatoid Factor and C-Reactive Protein in Adjuvant-Induced Arthritis Rats by P. heterophyllum

As summarized in Figure 6, the serum levels of RF and CRP in AIA model rats were significantly higher than those of rats in normal control group (p < 0.01). The three crude extracts from treatment with P. heterophyllum and MTX observably downregulated the levels of RF and CRP in serum (p < 0.01).

FIGURE 6.

FIGURE 6

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Effect of P. heterophyllum on the serum levels of RF (A) and CRP (B) in AIA rats. Data are shown as mean ± SD (n = 8). Differences were analyzed using ANOVA by Tukey’s test. ## p < 0.01 compared with the control group, && p < 0.01 compared with the model group.

Decreasing Serum Levels of Pro-inflammatory Cytokines in Adjuvant-Induced Arthritis Rats by P. heterophyllum

The results showed that serum concentrations of TNF-α, IL-1β, IL-6 and IL-17 increased prominently (p < 0.01) in the AIA model group compared to the normal control group. Treatment with P. heterophyllum markedly decreased (p < 0.01) the serum levels of all the above-mentioned anti-inflammatory cytokines (Figure 7).

FIGURE 7.

FIGURE 7

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Effect of P. heterophyllum on the serum levels of TNF-α (A), IL-1β (B), IL-6 (C) and IL-17 (D) in AIA rats. Data are shown as mean ± SD (n = 8). Differences were analyzed using ANOVA by Tukey’s test. ## p < 0.01 compared with the control group, & p < 0.05 and && p < 0.01 compared with the model group.

Increasing Serum Levels of Anti-inflammatory Cytokines in Adjuvant-Induced Arthritis Rats by P. heterophyllum

Compared to rats in the normal control group, the levels of anti-inflammatory cytokines including IL-4 and IL-10 in the serum of AIA model group rats were significantly up-regulated (p < 0.01, Figure 8). Treatment with 640 mg/kg of P. heterophyllum remarkably down-regulated the levels of IL-4 and IL-10 in the serum of AIA model rats.

FIGURE 8.

FIGURE 8

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Effect of P. heterophyllum on the serum levels of IL-4 (A) and IL-10 (B) in AIA rats. Data are shown as mean ± SD (n = 8). Differences were analyzed using ANOVA by Tukey’s test. ## p < 0.01 compared with the control group, & p < 0.05 and && p < 0.01 compared with the model group.

Decreasing Peripheral Blood Mononuclear Cells Levels of Cyclooxygenase-2, 5-Lipoxygenase and Matrix Metalloproteinase-2 in Adjuvant-Induced Arthritis Rats by P. heterophyllum

The levels of inflammatory mediators (COX-2 and 5-LOX) and MMP-2 in the rat PBMC were also evaluated by ELISA kits (Figure 9). The results showed that the levels of COX-2, 5-LOX and MMP-2 in PBMC of model rats were remarkably reduced than those of normal control rats (p < 0.01). After treatment with P. heterophyllum and MTX, the levels of COX-2, 5-LOX and MMP-2 were significantly elevated (p < 0.01 or p < 0.05) compared to those of the model group.

FIGURE 9.

FIGURE 9

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Effect of P. heterophyllum on the PBMC levels of COX-2 (A), 5-LOX (B) and MMP-2 (C) in AIA rats. Data are shown as mean ± SD (n = 8). Differences were analyzed using ANOVA by Tukey’s test. ## p < 0.01 compared with the control group, & p < 0.05 and && p < 0.01 compared with the model group.

Discussion

RA is the most prevalent chronic and long-term autoimmune inflammatory disease (Wang et al., 2017a; Li et al., 2019a; Saleem et al., 2020; Zhu et al., 2020). Although there are many anti-RA drugs in clinic, such as immunosuppressants, biological agents, DMARDs, steroidal drugs, and NSAIDs, most of them are associated with long-term adverse effects and costs (Li et al., 2019b; Dai et al., 2020; Zhu et al., 2020). In addition, the CFA-induced arthritis (AIA) model and the collagen-induced arthritis model are two typical preclinical experimental animal models of RA, and the former is a classic, easy-to-measure, short duration, reliable and reproducible test animal model, which is extensively used for the preclinical evaluation of anti-RA drugs since its pathological and morphological characteristics were similar to those of human RA (Yang et al., 2016; Pan et al., 2017; Voon et al., 2017; Zhang et al., 2019; Saleem et al., 2020).

The roots of P. heterophyllum have been widely used to treat RA as a vital TCM for centuries (Editorial Committee of Traditional Chinese Medicine 1999; Yang et al., 2016), but their anti-RA effect and chemical profiling have not been reported so far. Previous phytochemical studies have found that only 42 secondary metabolites, including six phenylpropanoids, eight triterpenoids, four flavonoids, 14 phenols and 10 others, were reported. In parallel, only 5-hydroxy-2-methoxy-1,4-naphtoquinone and taraxer-14-ene-1α,3β-diol exhibited antitumor effects in vitro (Yang et al., 2019a). In this work, we reported for the first time the anti-RA effect and chemical profiling of P. heterophyllum, thereby identifying 34 flavonoids and 39 others, while 15 flavonoids, including procyanidin B2, dihydromyricetin (-)-epicatechin, puerarin, rutin, naringin, hesperidin, myricetin, eriodictyol, quercetin, naringenin, kaempferol, diosmetin, nobiletin, and tangeretin, have been reported to have anti-RA effects in rats. Consequently, flavonoids may be responsible for the major active constituents in the roots of P. heterophyllum against RA as traditional folk medicine in China for centuries; however, further studies are needed to isolate and identify the bio-constituents directly related to anti-RA activity and its probable mechanism in vivo and in vitro of this plant.

In the animal model of AIA, histopathological lesions were aggravated due to massive inflammatory cell infiltration into synovial tissue, synovial hyperplasia, pannus formation, and bone and cartilage erosion (Lin et al., 2013; Voon et al., 2017; Rui et al., 2019; Yang et al., 2020). In the present research, P. heterophyllum exhibited the possible anti-RA effect, which prominently alleviates the above-mentioned abnormal histopathological changes in AIA model rats, accompanied by the reduction of inflammatory cytokines. Moreover, there is a straightforward relationship between weight loss/slow gain in rats and the massive inflammatory cell infiltration into synovial tissue (Lin et al., 2013; Pan et al., 2017; Jing et al., 2019). In this study, with P. heterophyllum treatment, body weight rose continuously in AIA model rats compared to rats in the model group. In addition, the arthritis score is a vital index to measure the anti-RA effect of drugs (Lin et al., 2013; Pan et al., 2017; Saleem et al., 2020) and is employed here to evaluate the possible therapeutic effect of P. heterophyllum, which was significantly decreased from day 24 compared to the model group. Finally, the spleen and thymus are two important immune organs, and their hyperfunction is closely related to the stimulation of the immune system in the AIA model rat (Lin et al., 2013; Zuo et al., 2018; Xiong et al., 2019), and the simultaneous decrease of the thymus and spleen indices by P. heterophyllum indicate the conceivable immunosuppressive effect.

In RA, serum RF and CRP are considered to be two important biomarkers of systemic inflammation in RA, indicating an active inflammatory response and are used to assess arthritic activity in rats with RA (Arjumand et al., 2019). This study shows that the expression of RF and CRP in serum of AIA model rat is remarkably increased, and the significant deduction after treatment with P. heterophyllum also suggests the feasible immunosuppressive activity.

A large number of studies have demonstrated that inflammation is a primary mechanism and a crucial role in rats with RA (Jing et al., 2019; Li et al., 2019c; Lin et al., 2013; Pan et al., 2017). Moreover, infiltration of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6 and IL-17, inflammatory mediators augment like COX-2 and 5-LOX, reduction of anti-inflammatory factors such as IL-4 and IL-10, which have been positively related to RA, causes synovial inflammation and cartilage damage (Jing et al., 2019; Li et al., 2019c; Lin et al., 2013; Pan et al., 2017). In RA, TNF-α, IL-1β, IL-6 and IL-17 play a decisive and synergistic role in synovial inflammation and cartilage damage (Jing et al., 2019; Yu et al., 2019; Saleem et al., 2020). In addition, the overproduction of TNF-α elevates the levels of IL-1β and IL-6, and generates matrix degrading enzymes (Jing et al., 2019). Likewise, IL-1β promotes osteoclast activation and MMP generation, just like increasing the expression of MMP-1, which ultimately leads to bone injury (Jing et al., 2019). On the other hand, IL-6 incites immunological reaction, MMP overproduction, and osteoclast differentiation and formation (Jing et al., 2019). IL-17 also plays a pivotal role in RA, which promotes the overproduction of pro-inflammatory cytokines and MMPs, as well as the activation of the osteoclasts and angiogenesis (Jing et al., 2019). Based on the above, therapeutic substances that particularly impede the production of TNF-α, IL-1β, IL-6 and IL-17 distinguish a crucial target for RA treatment (Jing et al., 2019; Rui et al., 2019; Yu et al., 2019). IL-4 and IL-10 by contrast, are two pivotal anti-inflammatory cytokines, which also play an important role in regulating the levels of endogenous pro-inflammatory cytokines during RA (Jing et al., 2019; Saleem et al., 2020). Our results indicate that treatment of P. heterophyllum obviously reduces the levels of TNF-α, IL-1β, IL-6 and IL-17, and increases the expression of IL-4 and IL-10, implying that the anti-RA effect of P. heterophyllum is achieved to a certain extent via the inhibition of pro-inflammatory cytokines and the elevation of anti-inflammatory cytokines in AIA model rats.

COX-2 is an overexpression of inflammatory tissues such as rheumatoid disease, and is a pivotal enzyme involved in the production of pro-inflammatory cytokines and cartilage destruction (Lin et al., 2013, Lin et al., 2014; Jing et al., 2019; Rui et al., 2019). Moreover, 5-LOX is the decisive enzyme involved in the synthesis of leukotriene, which is directly responsible for RA diseases (Lin et al., 2013, Lin et al., 2014). In addition, MMPs belong to the family of proteolytic enzymes, which play a crucial role during RA and are primarily responsible for bone and cartilage erosion (Jing et al., 2019; Yu et al., 2019). Our results demonstrate that PBMC levels of COX-2, 5-LOX and MMP-2 are highly expressed in AIA model rats, while a significant decrease is observed in PH-treated rats.

Conclusion

In summary, the chemical profiling and anti-RA effects of P. heterophyllum on AIA in rats were studied for the first time. The results demonstrate that flavonoids may be partly responsible for the major anti-RA effect of P. heterophyllum, which can ameliorate joint damage and suppress the hyperimmune response via downregulation of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-17), inflammatory mediators (COX-2 and 5-LOX) and MMP-2, and upregulation of anti-inflammatory cytokines (IL-4 and IL-10). Our findings suggest that P. heterophyllum possesses the therapeutic effect of RA and supports the claim that it is a vital folk medicine in TCM for treating RA and inflammation-related diseases for centuries.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics Statement

The animal study was reviewed and approved by JZLLSC2018-0701.

Author Contributions

JH designed the project and wrote the manuscript. LY, JH, RL, AF, JZ and YZ performed the experiments and analyzed the data. JH and LY discussed the data.

Funding

This work was financially supported by the National Natural Science Foundation of China (NSFC) (No. 81760705), the Natural Science Foundation of Jiangxi Province (Nos. 20192BBHL80008 and 20192BAB215059), and the Jiangxi University of Traditional Chinese Medicine (no. JXSYLXK-ZHYA0031).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

We would like to thank Modern Manuscript Editing Services (http://www.mmanuscripteditserv.com) for providing linguistic assistance during the preparation of this manuscript.

Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2020.584849/full#supplementary-material.

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References

  1. Abd-Allah S., Shahzad M., Shabbir A., Yousaf M. Z. (2019). Thymoquinone attenuates rheumatoid arthritis by downregulating TLR2, TLR4, TNF-α, IL-1, and NFκB expression levels. Biomed. Pharmacother. 111, 958–963. 10.1016/j.biopha.2019.01.006 [DOI] [PubMed] [Google Scholar]
  2. Ahmad S. F., Zoheir K. M. A., Abdel-Hamied H. E., Ashour A. E., Bakheet S. A., Attia S. M., et al. (2014). Amelioration of autoimmune arthritis by naringin through modulation of T regulatory cells and Th1/Th2 cytokines. Cell. Immunol. 287, 112–120. 10.1016/j.cellimm.2014.01.001 [DOI] [PubMed] [Google Scholar]
  3. Bao Y., Li H., Li Q.-Y., Li Y., Li F., Zhang C.-F., et al. (2018). Therapeutic effects of Smilax glabra and Bolbostemma paniculatum on rheumatoid arthritis using a rat paw edema model. Biomed. Pharmacother. 108, 309–315. 10.1016/j.biopha.2018.09.004 [DOI] [PubMed] [Google Scholar]
  4. Chen Y., Wang Y., Liu M., Zhou B., Yang G. (2019). Diosmetin exhibits anti‐proliferative and anti‐inflammatory effects on TNF‐α‐stimulated human rheumatoid arthritis fibroblast‐like synoviocytes through regulating the Akt and NF‐κB signaling pathways. Phytother Res. 34, 1310–1319. 10.1002/ptr.6596 [DOI] [PubMed] [Google Scholar]
  5. Chu J., Wang X., Bi H., Li L., Ren M., Wang J. (2018). Dihydromyricetin relieves rheumatoid arthritis symptoms and suppresses expression of pro-inflammatory cytokines via the activation of Nrf2 pathway in rheumatoid arthritis model. Int. Immunopharm. 59, 174–180. 10.1016/j.intimp.2018.04.001 [DOI] [PubMed] [Google Scholar]
  6. Dai X., Yang D., Bao J., Zhang Q., Ding J., au M., et al. (2020). Er Miao San, a traditional Chinese herbal formula, attenuates complete Freund's adjuvant-induced arthritis in rats by regulating Th17/Treg cells. Pharmaceut. Biol. 58, 157–164. 10.1080/13880209.2020.1720745 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Editorial Committee of Traditional Chinese Medicine (1999). Chinese materia medica. Shanghai, CN: Zhonghua Bencao, 3, 751–752. [Google Scholar]
  8. Jing R., Ban Y., Xu W., Nian H., Guo Y., Geng Y., et al. (2019). Therapeutic effects of the total lignans from Vitex negundo seeds on collagen-induced arthritis in rats. Phytomedicine 58, 152825 10.1016/j.phymed.2019.152825 [DOI] [PubMed] [Google Scholar]
  9. Kim E. Y., Sudini K., Singh A. K., Haque M., Leaman D., Khuder S., et al. (2018). Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1. Feaeb. J. 32, 6174–6185. 10.1096/fj.201800425r [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lei Z., Ouyang L., Gong Y., Wang Z., Yu B. (2020). Effect of eriodictyol on collagen-induced arthritis in rats by Akt/HIF-1 alpha pathway. Drug Des. Dev. Ther. 14, 1633–1639. 10.2147/dddt.s239662 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Li H. J., Zhang C. T., Du H., Xu T., Li Q., Wang P., et al. (2019a). Chemical composition of Bawei Longzuan granule and its anti-arthritic activity on collagen-induced arthritis in rats by inhibiting inflammatory responses. Chem. Biodivers. 16, e1900294 10.1002/cbdv.201900294 [DOI] [PubMed] [Google Scholar]
  12. Li T. P., Zhang A. H., Miao J. H., Sun H., Yan G. L., et al. (2019b). Applications and potential mechanisms of herbal medicines for rheumatoid arthritis treatment: a systematic review. RSC Adv. 9, 26381 10.1039/c9ra04737a [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Li X., Xie P. G., Hou Y., Chen S. D., He P. H., Xiao Z. F., et al. (2019c). Tangeretin inhibits oxidative stress and inflammation via upregulating Nrf-2 signaling pathway in collagen-induced arthritic rats. Pharmacology 104, 187–195. 10.1159/000501163 [DOI] [PubMed] [Google Scholar]
  14. Lin B., Zhang H., Zhao X. X., Rahman K., Wang Y., Ma X. Q., et al. (2013). Inhibitory effects of the root extract of Litsea cubeba (lour.) pers. on adjuvant arthritis in rats. J. Ethnopharmacol. 147, 327–334. 10.1016/j.jep.2013.03.011 [DOI] [PubMed] [Google Scholar]
  15. Lin H. C., Lin T. H., Wu M. Y., Chiu Y. C., Tang C. H., Hour M. J., et al. (2014). 5-lipoxygenase inhibitors attenuate TNF-α-induced inflammation in human synovial fibroblasts. PloS One 9, e107890 10.1371/journal.pone.0107890 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Liu J., Zeng L., Wei R., Zhong G., Zhu Y., Xu T., et al. (2019). Lagopsis supina exerts its diuretic effect via inhibition of aquaporin-1, 2 and 3 expression in a rat model of traumatic blood stasis. J. Ethnopharmacol. 231, 446–452. 10.1016/j.jep.2018.10.034 [DOI] [PubMed] [Google Scholar]
  17. Mateen S., Shahzad S., Ahmad S., Naeem S. S., Khalid S., Akhtar K., et al. (2019). Cinnamaldehyde and eugenol attenuates collagen induced arthritis via reduction of free radicals and pro-inflammatory cytokines. Phytomedicine 53, 70–78. 10.1016/j.phymed.2018.09.004 [DOI] [PubMed] [Google Scholar]
  18. Osman W. N. W., Tantowi N. A. C. A., Lau S. F., Mohamed S. (2019). Epicatechin and scopoletin rich Morinda citrifolia (Noni) leaf extract supplementation, mitigated osteoarthritis via anti-inflammatory, anti-oxidative, and anti-protease pathways. J. Food Biochem. 43, e12755. [DOI] [PubMed] [Google Scholar]
  19. Pan D. M., Li N., Liu Y. Y., Xu Q., Liu Q. P., You Y. T., et al. (2018). Kaempferol inhibits the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes by blocking activation of the MAPK pathway. Int. Immunopharm. 555, 174–182. 10.1016/j.intimp.2017.12.011 [DOI] [PubMed] [Google Scholar]
  20. Pan T., Cheng T. F., Jia Y. R., Li P., Li F. (2017). Anti-rheumatoid arthritis effects of traditional Chinese herb couple in adjuvant-induced arthritis in rats. J. Ethnopharmacol. 205, 1–7. 10.1016/j.jep.2017.04.020 [DOI] [PubMed] [Google Scholar]
  21. Rui J., Ban Y. F., Xu W. H., Nian H., Guo Y. L., Geng Y. Y., et al. (2019). Therapeutic effects of the total lignans from Vitex negundo seeds on collagen-induced arthritis in rats. Phytomedicine 58, 152825 10.1016/j.phymed.2019.152825 [DOI] [PubMed] [Google Scholar]
  22. Saccol R. D. P., Silveira K. L., Adefegha S. A., Manoni A. G., sa Silveira L. L., Coelho A. P. V., et al. (2019). Effect of quercetin on E-NTPDase/E-ADA activities and cytokine secretion of complete Freund adjuvant-induced arthritic rats. Cell Biochem. Funct. 37, 474–485. 10.1002/cbf.3413 [DOI] [PubMed] [Google Scholar]
  23. Saleem A., Saleem M., Akhtar M. F., Shahzad M., Jahan S. (2020). Moringa rivae leaf extracts attenuate complete Freund’s adjuvant-induced arthritis in wistar rats via modulation of inflammatory and oxidative stress biomarkers. Inflammopharmacology 28, 139–151. 10.1007/s10787-019-00596-3 [DOI] [PubMed] [Google Scholar]
  24. Sun C. L., Wei J., Bi L. Q. (2017). Rutin attenuates oxidative stress and proinflammatory cytokine level in adjuvant induced rheumatoid arthritis via inhibition of NF-kappa B. Pharmacology 100, 40–49. 10.1159/000451027 [DOI] [PubMed] [Google Scholar]
  25. Tong Z. W., Cheng L., Song J. Z., Wang M., Yuan J. Z., Li X. Z., et al. (2018). Therapeutic effexts of Caesalpinia minax Hance on complete Frund’s adjuvant (CFA)-induced arthritis and the anti-inflammatory activity of cassane diterpenes as main active components. J. Ethnopharmacol. 226, 90–96. 10.1016/j.jep.2018.08.011 [DOI] [PubMed] [Google Scholar]
  26. Voon F. L., Sulaiman M. R., Akhtar M. N., Idris M. F., Akira A., Perimal E. K., et al. (2017). Cardamonin (2',4'-dihydroxy-6'-methoxychalcone) isolated from Boesenbergia rotunda (L.) Mansf. inhibitits CFA-induced rheumatoid arthritis in rats. Eur. J. Pharmacol. 794, 127–134. 10.1016/j.ejphar.2016.11.009 [DOI] [PubMed] [Google Scholar]
  27. Wang A., Leong D. J., He Z. Y., Xu L., Liu L. D., Kim S. J., et al. (2017b). Procyanidins mitigate osteoarthritis pathogenesis by, at Least in part, suppressing vascular endothelial growth factor signaling. Int. J. Mol. Sci. 17, 2065 10.3390/ijms17122065 [DOI] [PMC free article] [PubMed] [Google Scholar] [Research Misconduct Found]
  28. Wang C., Meriwether D., Lee Y. Y., Reddy S. T. (2011). Oxidation products of arachidonic acid and linoleic acid are increased in high density lipoprotein and low density lipoprotein from patients with active rheumatoid arthritis. Arthritis Rheum-US 63, s299. [Google Scholar]
  29. Wang C. X., Wang W. D., Jin X. P. (2016). Puerarin attenuates inflammation and oxidation in mice with collagen antibody-induced arthritis viaTLR4/NF-kappa B signaling. Mol. Med. Rep. 14, 1365–1370. 10.3892/mmr.2016.5357 [DOI] [PubMed] [Google Scholar]
  30. Wang X., He X., Zhang C. F., Guo C. R., Wang C. Z., Yuan C. S. (2017a). Anti-arthritic effect of berberine on adjuvant-induced rheumatoid arthritis in rats. Biomed. Pharmacother. 89, 887–893. 10.1016/j.biopha.2017.02.099 [DOI] [PubMed] [Google Scholar]
  31. Xiong H., Ding X., Wang H., Jiang H. Q., Wu X. Y., Tu C. Y., et al. (2019). Tibetan medicine Kuan-Jin-Teng exerts anti-artitic effects on collagen-induced arthritis rats vvia inhibition the production of pro-inflammatory cytokines and down-regulation of MAPK signaling pathway. Phytomedicine 57, 271–281. 10.1016/j.phymed.2018.12.023 [DOI] [PubMed] [Google Scholar]
  32. Yang L., He J. W., Wang S. F., Wang L. F., Liu R. H. (2019a). Research progress of chemical constituents, pharmacological activities and quality control of Pterospermum heterophyllum Hance. J. Jiangxi Univi. TCM. 31, 113–116. [Google Scholar]
  33. Yang L., Liu R. H., He J. W. (2019b). Rapid analysis of the chemical compositions in Semiliquidambar cathayensis roots by ultra-high-performance liquid chromatography and quadrupole time-of-flight tandem mass spectrometry. Molecules 24, 4098 10.3390/molecules24224098 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yang L., Wang Y. Q., Liu S. C., He J. W. (2016). Research progress of chemical constituents and pharmacological activities from three commonly used Ban-feng-he medicinal plants. Chin. J. Exp. Tradit. Med. Form. 22, 191–196. [Google Scholar]
  35. Yang P., Chen G., Qin W.-Y., Zhong Y., Yang J., Rong X.-F. (2016). Xitong Wan attenuates inflammation development through inhibiting the activation of nuclear factor-κB in rats with adjuvant-induced arthritis. J. Ethnopharmacol. 193, 266–271. 10.1016/j.jep.2016.08.006 [DOI] [PubMed] [Google Scholar]
  36. Yang P., Qian F. Y., Zhang M. F., Xu A. L., Wang X., Jiang B. P., et al. (2020). Zishen Tongluo formula ameliorates collagen-induced arthritis in mice by modulation of Th17/Treg balance. J. Ethnopharmacol. 250, 112428 10.1016/j.jep.2019.112428 [DOI] [PubMed] [Google Scholar]
  37. Yousefi B., Jadidi-Niaragh F., Azizi G., Hajighasemi F., Mirshafiey A. (2014). The role of leukotrienes in immunopathogenesis of rheumatoid arthritis. Mod. Rheumatol. 24, 225–235. 10.3109/14397595.2013.854056 [DOI] [PubMed] [Google Scholar]
  38. Yu H. H., Zeng R., Lin Y., Li X,, Shumaila T., Yang Z., et al. (2019). Kadsura heteroclita stem suppresses the onset and progression of adjuvant-induced arthritis in rats. Phytomedicine 58, 152876 10.1016/j.phymed.2019.152876 [DOI] [PubMed] [Google Scholar]
  39. Yuan X. L., Liu Y. G., Hua X., Deng X. X., Sun P. J., Yu C. M., et al. (2015). Myricetin ameliorates the symptoms of collagen-induced arthritis in mice by inhibiting cathepsin Kactivity. Immunopharmacol. Immunotoxicol. 37, 513–519. 10.3109/08923973.2015.1096942 [DOI] [PubMed] [Google Scholar]
  40. Zhang C. F., Zhang W. F., Shi R. Y., Tang B. Y., Xie S. C. (2019). Coix lachrymal-jobi extract ameliorates inflammation and oxidative stress in a complete Freund’s adjuvant-induced rheumatoid arthritis model. Pharm. Biol. 57, 792–798. 10.1080/13880209.2019.1687526 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Zhu L. J., Zhang Z. S., Xia N. N., Zhang W. F., Wei Y. L., Huang J. S., et al. (2020). Anti-arthritic activity of ferulic acid in complete Freund’s adjuvant (CFA)-induced arthritis in rats: JAK2 inhibition. Inflammopharmacology 28, 463–473. 10.1007/s10787-019-00642-0 [DOI] [PubMed] [Google Scholar]
  42. Zhu L. P., Wang J., Wei T. T., Gao J., He H., Chang X. Y., et al. (2015). Effects of naringenin on inflammation in complete Freund's adjuvant-induced arthritis by regulating Bax/Bcl-2 balance. Inflammation 38, 245–251. 10.1007/s10753-014-0027-7 [DOI] [PubMed] [Google Scholar]
  43. Zhu X. F., Zeng K., Qiu Y., Yan F. H., Lin C. Z. (2013). Therapeutic effect of emodin on collagen-induced arthritis in mice. Inflammation 36, 1253–1259. 10.1007/s10753-013-9663-6 [DOI] [PubMed] [Google Scholar]
  44. Zuo J., Yin Q., Wang Y. W., Lu L. M., Xiao Z. G., Wang G. D., et al. (2018). Inhibition of NF-Kb pathway in fibroblast-like synoviocytes by α-magostin implicated in protective effects on joints in rats suffering from adjuvant-induced arthritis. Int. Immunopharm. 56, 78–89. 10.1016/j.intimp.2018.01.016 [DOI] [PubMed] [Google Scholar]

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