Rheumatoid arthritis is an immune-related inflammation disorder that affects over 60 million adults in the world wide. Its predominant symptoms include pain, stiffness, and swelling of peripheral joints. Rheumatoid arthritis may rapidly progress into a multisystem inflammation with irreversible joint destruction and increase the risk of mortality. However, the current treatment medications, such as nonsteroidal anti-inflammatory drugs, glucocorticosteroid, and immune-depressant are limited for their efficacy and frequently toxicity. Therefore, more and more patients look for medical botany options in coping with this debilitating disease. The recent investigation indicated that an estimated 60–90% of patients with rheumatoid arthritis were very likely to use botanicals. This growing interest in alternative medical practices clearly indicates the need for more safety and effective botanicals [
The genus Urtica (Urticaceae Family) is about 30 species and widely distributes in north and south temperate regions, also in mountainous areas of tropics [
Dexamethasone, indometacin, aspirin, morphine, and CMC were purchased from Shanghai National Pharmaceutical Co., Ltd. (Shanghai, China). Salicylic acid, phaselic acid, chlorogenic acid, and rutin for pharmacological test were purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd. (Shanghai, China). Acetonitrile was HPLC grade from Shanghai Xingke Biochemistry Co., Ltd (Shanghai, China). Carrageenin (type IV) and Freund's complete adjuvant agent (FCA) were obtained from Sigma-Aldrich Corporation (Shanghai, China).
Kunming mice (18–22 g) and SD rats (180–220 g) were obtained from Shanghai Laboratory Animal Center of Chinese Academy of Sciences. They were acclimatized for 5 days before use and provided food and water
The dried aerial part of
Adjuvant-induced rats arthritis was induced according to the method in the literature [
A blinded independent observer with no knowledge of the treatment protocol performed the arthritis index evaluation for each rat every other day from the 8th day after FCA injection. Each noninjected paw was graded separately from 0 to 4, depending on the severity. The assessment was made as follows: 0, no response; 1, slight edema of the digital joints; 2, edema of the digital joints and footpad; 3, gross edema of the entire footpad below the joint; 4, edema of the entire foot. In the case of the more severe responses, swellings of the tail and ears also were generally observed, but no additional score was ascribed for these signs. The arthritis index for each rat was the sum of the scores of the three measured joints [
Autoclaved cotton pellets weighing 5.0 ± 0.1 mg each were implanted subcutaneously through small incision made along the flank region of the male mice anesthetized with ether. The mice were orally administered the sample, dexamethasone, or CMC solution once daily for seven consecutive days from the day of cotton pellet insertion. On the 7th day, the mice were sacrificed and the cotton pellets covered by the granulomatous tissue were excised and dried in hot air oven at 60°C till a constant weight was achieved. Granuloma weight was obtained by subtracting the weight of cotton pellet on 0 d (before the start of experiment) from the weight of the cotton pellet on seventh day (at the end of experiment) [
Male rats were treated orally with sample, indomethacin, and CMC solution 60 min prior to an injection of 0.1 mL of 1% carrageenin sterile (w/v) in saline into the plantar tissue of the right hind paw. The contralateral hind paws were injected with 0.1 mL of saline as control. Paw pad thickness was measured at 0, 2, 4 and 6 h after carrageenin injection, and paw swelling was obtained by subtracting the right pad thickness from the left pad thickness [
Mice were kept singly in a clear plastic observational cage (35 cm × 25 cm × 15 cm) and were pretreated with sample, aspirin or CMC solution by intragastric administration 60 min prior to intraperitoneal injection of 0.6% acetic acid in a volume of 0.1 mL per mouse. After the injection of acetic acid, the number of writhes exhibited for 15 min were counted. The percent inhibition was calculated as follows [
Female mice were placed on an YLS-6A intelligent hot-plate apparatus (Shandong Academy of Medical Sciences, China). The temperature of metal surface was maintained at 55.0 ± 0.5°C. Latency to a discomfort reaction (licking hind paw) was taken as pain threshold in mice and a cut-off time of 60 seconds was maintained to prevent scald. The fifty valid mice were selected (the pain threshold was determined in 5 to 30 seconds) and divided into five groups randomly. The deferent groups of mice were orally administered the sample, CMC solution, or intraperitoneally injected morphine. The pain threshold was determined before and 30, 60, 90, and 120 min after administration, respectively [
Data were expressed as mean ± standard deviation (SD). Differences between groups were evaluated by Kruskal-Wallis test for the arthritic scores, or one-way analysis of variance (ANOVA) followed by Dunnett’s test for the others indices with the aid of SPSS11.5 software package. Statistical significance is expressed as *
TFUA was fractionated by silica gel (200–300 mesh, Qingdao Haiyang chemical Co., Ltd.) column chromatography and gradually eluted with dichloromethane-methanol. The fractions obtained were further purified by repeat silica gel and Sephadex LH-20 (Pharmacia) column chromatography to yield pure compounds.
Compound identification was carried out by determination spectra data. The 1H (400 MHz) and 13C (100 MHz) NMR data were obtained with the solvent as reference, employing Bruker Avance 400 spectrometers. Molecular weight was obtained on Finnigan/MAT 4510 mass spectrometer. Optical rotation values were determined with a JASCO Dip-360 digital polarimeter.
Chemical analysis of TFUA was performed with an Agilent 1200 liquid chromatograph equipped with a DAD detector, quaternary pump, on-line degasser, autosampler, and thermostatic column compartment. Phenolic compounds were separated on an Agilent Zorbax Elipse XDB-C18 column (5
TFUA showed the obvious therapeutic effect on the adjuvant-induced arthritis. It could significantly inhibit the paw swell induced by FCA and decreased the arthritis index, indicating it can inhibit the primary inflammation and second inflammation (Table
Effect of phenolic compounds-rich fraction from
Group | Dose (mg/kg) | Paw swell (mm) | Arthritis index | |||||||
12 d | 16 d | 20 d | 12 d | 16 d | 20 d | |||||
R | L | R | L | R | L | |||||
Vehicle | — | 4.21 ± 0.84 | 3.52 ± 1.01 | 3.86 ± 0.72 | 3.63 ± 0.71 | 3.47 ± 0.45 | 3.29 ± 0.73 | 7.6 ± 2.1 | 9.9 ± 2.6 | 10.3 ± 1.9 |
Dexamethasone | 30 | 3.34 ± 0.66* | 2.61 ± 0.76* | 3.09 ± 0.56* | 2.52 ± 0.76** | 2.88 ± 0.27** | 2.07 ± 0.62** | 5.2 ± 2.3* | 4.4 ± 1.8** | 3.5 ± 2.7** |
TFUA | 50 | 3.77 ± 0.75 | 3.07 ± 0.94 | 3.61 ± 0.62 | 2.82 ± 0.97* | 3.02 ± 0.46* | 2.52 ± 0.56* | 7.1 ± 1.9 | 8.7 ± 1.5 | 8.4 ± 2.3 |
150 | 3.51 ± 0.61* | 2.89 ± 0.85* | 3.20 ± 0.67* | 2.77 ± 0.85* | 2.81 ± 0.61* | 2.27 ± 0.68** | 6.5 ± 2.0 | 7.8 ± 1.5* | 7.6 ± 2.2* |
Values are expressed as mean ± SD.
The inhibiting activity of nonspecific inflammation was evaluated by the models of mice cotton pellet granuloma and carrageenin-induced rats paw edema. The results showed that TFUA had the moderate anti-inflammatory activity. It could inhibit the granuloma hyperplasia caused by cotton pellet at the dosages of 50 and 150 mg/kg (Figure
Effect of phenolic compounds-rich fraction from
Group | Dose (mg/kg) | Paw swell (mm) | ||
2 h | 4 h | 6 h | ||
Vehicle | — | 4.72 ± 0.88 | 5.15 ± 1.06 | 4.93 ± 0.92 |
Indometacin | 30 | 3.78 ± 0.93* | 3.22 ± 0.73** | 2.37 ± 0.95** |
TFUA | 50 | 4.42 ± 1.26 | 4.08 ± 0.95* | 3.69 ± 0.71* |
150 | 4.38 ± 0.65 | 3.64 ± 0.78** | 3.20 ± 0.83** |
Values are expressed as mean ± SD.
Effect of phenolic compounds-rich fraction from
The analgesic activity of TFUA was evaluated by the mice models of acetic acid-induced writhing response and hot-plate test. The results of acetic acid-induced writhing response revealed that TFUA had the obviously analgesic activity. It could significantly decrease the number of writhes induced by acetic acid (Table
Effect of phenolic compounds-rich fraction from
Group | Dose (mg/kg) | Number of writhes in 15 min | Inhibition rate (%) |
---|---|---|---|
Vehicle | — | 29.5 ± 8.4 | — |
Aspirin | 150 | 14.3 ± 4.8** | 51.5 |
TFUA | 50 | 20.5 ± 6.4** | 30.5 |
150 | 11.7 ± 3.2** | 63.4 |
Values are expressed as mean ± SD.
Effect of phenolic compounds-rich fraction from
Group | Dose (mg/kg) | Anterior pain threshold (s) | Posterior pain threshold (s) | |||
---|---|---|---|---|---|---|
30 | 60 | 90 | 120 | |||
Vehicle | — | 18.0 ± 3.5 | 20.1 ± 5.7 | 21.5 ± 5.6 | 21.2 ± 6.7 | 19.9 ± 8.1 |
Morphine | 30 | 18.5 ± 3.5 | 30.4** ± 3.8 | 35.0** ± 6.0 | 28.6** ± 4.1 | 27.4** ± 4.6 |
TFUA | 50 | 19.4 ± 3.8 | 21.6 ± 5.5 | 23.2 ± 6.1 | 21.7 ± 7.2 | 21.3 ± 8.8 |
150 | 19.0 ± 3.7 | 21.2 ± 5.4 | 22.7 ± 5.9 | 21.4 ± 7.0 | 22.0 ± 8.5 |
Values are expressed as mean ± SD.
TFUA (83 g) was fractionated by silica gel column chromatography and gradually eluted with dichloromethane-methanol (95 : 5–60 : 40) to give 76 fractions of 500 mL each. These fractions were purified by repeat silica gel and sephadex LH-20 column chromatography and resulted in the isolation of 18 compounds (Figure
The chemical structures of 18 compounds isolated from TFUA. 1: bis (5-formylfurfuryl) ether; 2: scopoletin; 3: (–) olivil; 4: (–) secoisolariciresinol; 5: (–) matairesinol; 6: ferulic acid; 7:
Colorless needle (chloroform); HR EI-MS
Colorless needle (MeOH). 1H and 13C-NMR spectral data were consistent with those reported in the literature [
Colorless powder (MeOH);
Colorless powder (MeOH).
Colorless powder (MeOH).
Colorless styloid (MeOH); EI-MS
Colorless needle (acetone); 1H and 13C-NMR spectral data were consistent with those reported in the literature [
Yellowish needle (MeOH). 1H and 13C-NMR spectral data were consistent with those reported in the literature [
Colorless needle (MeOH); 1H and 13C-NMR spectral data were consistent with those reported in the literature [
Colorless needle (MeOH); 1H and 13C-NMR spectral data were consistent with those reported in the literature [
Yellow needle (MeOH); 1H and 13C-NMR spectral data were consistent with those reported in the literature [
Yellow needle (MeOH); 1H and 13C-NMR spectral data were consistent with those reported in the literature [
Yellow needle (MeOH); 1H and 13C-NMR spectral data were consistent with those reported in the literature [
Yellow granule (MeOH); 1H and 13C-NMR (CDCl3, 100 MHz) spectral data were consistent with those reported in the literature [
Yellow powder (MeOH); 1H and 13C-NMR spectral data agreed with those reported in the literature [
Yellow needle (MeOH); 1H-NMR (CD3OD, 400 MHz)
Yellow needle (MeOH);
Colorless needle (MeOH); 1H and 13C-NMR spectral data agreed with those reported in the literature [
The chemical compositions of TFUA were further investigated by HPLC analysis. The results showed that phenolic acids and flavonoids were the main constituents. The relatively rich phenolics (contents above 5 percent) included rutin, chlorogenic acid, phaselic acid, and salicylic acid (Figure
The content determination results of phenolic compounds-rich fraction from
Phenolic acids | Flavonoids | The others | |||
---|---|---|---|---|---|
Chlorogenic acid | 88.3 | Rutin | 124.1 | Secoisolariciresinol | 37.5 |
Phaselic acid | 62.4 | Quercetin | 30.9 | Scopoletin | 21.6 |
Salicylic acid | 52.9 | Luteolin | 4.4 | ||
Caffeic acid | 17.6 | ||||
Protocatechuic aldehyde | 10.3 |
HPLC profile of phenolic compounds-rich fraction from
In order to screen the antirheumatoid arthritis constituents in TFUA, the pharmacological activities of the main phenolic acids, flavanoids, and their combination (the ratio was based on their contents in TFUA) were evaluated by FCA-induced rats arthritis model. The phenolic acids exhibited the statistically significant inhibition of edema in the injected paw (primary response) at dose levels of 50 mg/kg orally, when compared with the vehicle control group. Rutin showed moderate activity of inhibition of edema in the uninjected paw (second response). It is noted that neither phenolic acids nor flavanoids exhibited the significant suppressive effect on both primary swelling and second pathological changes of the experimental rats. However, their combination showed the obvious inhibition activity of paw edema in both the injected paw and the uninjected paw (Table
The antirheumatoid arthritis activities of the main phenolic compounds and their combination.
Group | Dose (mg/kg) | Paw swell (mm) | Arthritis index | |||||||
12 d | 16 d | 20 d | 12 d | 16 d | 20 d | |||||
R | L | R | L | R | L | |||||
Vehicle | — | 4.45 ± 0.93 | 3.80 ± 0.61 | 4.16 ± 0.89 | 4.03 ± 0.77 | 3.89 ± 0.54 | 3.57 ± 0.61 | 8.5 ± 2.3 | 10.8 ± 1.7 | 11.1 ± 2.4 |
Dexamethasone | 30 | 3.33 ± 0.65* | 3.09 ± 0.87* | 2.92 ± 0.72** | 2.84 ± 0.47** | 2.23 ± 0.42** | 2.24 ± 0.58** | 6.2 ± 1.7* | 4.9 ± 1.8** | 3.7 ± 2.7** |
Salicylic acid | 50 | 3.57 ± 0.89* | 3.67 ± 0.79 | 3.34 ± 0.56* | 3.51 ± 0.83 | 2.95 ± 0.61* | 3.25 ± 0.43 | 7.7 ± 2.8 | 10.1 ± 2.5 | 9.8 ± 1.7 |
Phaselic acid | 50 | 3.88 ± 0.89 | 3.52 ± 0.85 | 3.09 ± 0.67** | 3.62 ± 0.53 | 2.71 ± 0.49** | 3.28 ± 0.73 | 6.9 ± 2.6 | 9.8 ± 1.7 | 10.4 ± 2.1 |
Chlorogenic acid | 50 | 4.21 ± 0.66 | 3.55 ± 0.44 | 3.73 ± 0.75 | 3.66 ± 0.59 | 3.56 ± 0.85 | 3.39 ± 0.76 | 8.1 ± 2.5 | 10.3 ± 1.7 | 10.8 ± 2.3 |
Rutin | 50 | 4.36 ± 0.80 | 3.40 ± 0.67 | 3.92 ± 0.54 | 3.29 ± 0.47* | 3.78 ± 0.92 | 2.83 ± 0.87* | 7.6 ± 2.0 | 9.3 ± 1.3 | 8.7 ± 1.9* |
Combinationa | 50 | 3.48 ± 0.72* | 3.21 ± 0.58* | 3.40 ± 0.71* | 3.04 ± 0.55** | 2.84 ± 0.65** | 2.61 ± 0.77** | 7.2 ± 1.8 | 9.0 ± 1.9* | 8.2 ± 1.5* |
Values are expressed as mean ± SD.
It is well known that salicylic acid and its derivative aspirin had the anti-inflammatory and analgesic activity and often used to cure rheumatoid arthritis. However, these constituents only inhibit primary response, do not affect the second response and immune function, which is similar with the result of the present study. Although immunosuppressive agent and glucocorticosteroid inhibit secondary process, they have obvious side effect and toxicity. So the exploit of the antirheumatoid arthritis agent which could inhibit both the primary and secondary process, and possess good effect and lower toxicity from medical plant, had drawn much attention.
In order to elucidate the constituents in TFUA, the systemic isolation of TFUA was carried out. As a result, eighteen compounds were isolated, of which the phenolic acids and flavanoids were the main constituents (Figure
In order to investigate the active constituents of TFUA, the antirheumatoid arthritis actions of main phenolic constituents were further evaluated. The results suggested that the main phenolic acids (chlorogenic acid, phaselic acid, and salicylic acid) in TFUA inhibited the primary response; the main flavanoids (rutin) inhibited the second response. The antirheumatoid arthritis of TFUA may be the combined action of multiple phenolic constituents.
Some phenolics were reported to possess the therapeutic effect on rheumatoid arthritis. Salicylic acid and its derivates had the potent anti-inflammatory activity and analgesic activity and usually used for rheumatoid arthritis patients to relieve the joint inflammation and pain. Recent research reported that scopoletin had good analgesic activity [
In conclusion, the phenolic compounds-rich fraction from
The authors report no conflict of interests. The authors alone are responsible for the content and writing of the paper.
This work was financially supported by the Research Foundation of Shanghai Health Bureau (no. 2004Y002A) and National Project in Significant Creation of New Drugs during the Eleventh Five-Year Plan Period (2009ZX09502-0130). Shuyan Zhou, Fangdi Qi, Kangmu Ma, and Yiwen Zhu took part in the isolation work and pharmacological evaluation.