In Vitro and In Vivo Anti-Inflammatory Activity of the Rhizomes of Globba pendula Roxb

Abstract

In this study, we aimed at evaluating in vitro and in vivo anti-inflammatory activity of various extracts of the rhizomes of Globba pendula Roxb. Three extracts (n-hexane, ethyl acetate, and water) were screened for their inhibitory effect on NO production by lipopolysaccharide-stimulated RAW 264.7 macrophages. The ethyl acetate extract of G. pendula rhizomes (EGP) showed a potential effect with an IC₅₀ value of 32.45 µg/mL. For in vivo study, the ethyl acetate extract was further investigated for its anti-inflammatory effect using collagen antibody-induced arthritic mice (CAIA). The level of arthritis in experimental mice significantly reduced (P < .05) after treatment with EGP at a dose of 500 mg/kg body weight (b.w.). This study also revealed that EGP is orally non-toxic. Ethyl p-methoxy cinamate was identified as the main constituent of EGP, which may result in its anti-inflammatory effect.

Introduction

In folk medicine, the rhizomes of Globba pendula Roxb (Zingiberaceae family) have been used to treat many diseases, such as rheumatism and osteoarthritis in Vietnam. ¹ In Malaysia and Indonesia, they have also been used as a decoction to treat flatulence in postpartum women. ² Ten compounds have been reported from G. pendula, including a labdane diterpene, benzofuran, phenolics, and steroids. ³ The essential oil of this plant was found to possess moderate NO inhibitory effect, and significant cytotoxic activity against MCF7 and Hep3B cells. ⁴ We previously reported the isolation of one new polyoxygenated cyclohexene, globbanol A (1), along with two other cyclohexene derivatives, crotepoxide (2) and boesenboxide (3), and a chalcone, 2′-4,4′,6′-trimethoxy-chalcone (4), from the ethyl acetate extract of the rhizomes of G. pendula. ⁵ In this study, various extracts of G. pendula rhizomes were screened for in vitro anti-inflammatory activity on lipopolysaccharide-stimulated RAW 264.7 macrophages. The extract possessing the best activity was then further investigated for in vivo anti-inflammatory activity in an experimental mouse model with collagen antibody-induced arthritis (CAIA), as well as for acute toxicity. The results provide a scientific basis to use the biologically active extract for pharmaceutical purposes.

Results and Discussion

Effect on NO Production

Nitric oxide (NO) is a signaling molecule that plays a key role in the pathogenesis of inflammation. It produces an anti-inflammatory effect under normal physiological conditions. On the other hand, NO is considered as a pro-inflammatory mediator that in abnormal situations induces inflammation due to over production. The overproduction of NO as an inflammatory mediator can lead to tissue destruction such as in inflammatory autoimmune diseases. NO is not only a marker but also a pro-inflammatory mediator of arthritis. It is produced by nitric oxide synthase (NOS). An increased serum concentration of nitrate indicates enhanced NO production in serum and synovial fluid of the inflamed joints in patients with rheumatoid arthritis (RA), osteoarthritis (OA), and ankylosing spondylitis. For this reason, synthetic NOS inhibitors have been evaluated for clinical use. ⁶

N-hexane, ethyl acetate, and water extracts of the rhizomes of G. pendula were screened for their inhibitory activities on LPS-induced NO production using RAW 264.7 cells by the Griess reaction. L-NMMA (NG-methyl-L-arginine acetate) was used as a positive control (IC₅₀ value 8.34 µg/mL). The ethyl acetate extract of EGP displayed significant NO production inhibitory activity with an IC₅₀ value of 32.45 µg/mL, while the n-hexane, and water extracts were inactive (IC₅₀ > 100 µg/mL). In addition, the ethyl acetate extract had a minor impact on RAW 264.7 cell viability; more than 87% of cells were alive after treatment with 100 μg/mL of the extract for 24 h.

Effect on Arthritis Score

RA is a chronic systemic autoimmune disease that primarily affects the lining of the synovial joints and is associated with progressive disability, premature death, and socioeconomic burdens. ⁷ Collagen antibody-induced arthritis (CAIA) is a simple mouse model of rheumatoid arthritis that can be used to address questions of pathogenic mechanisms and to screen candidate therapeutic agents. ⁸ Based on the promising in vitro anti-inflammatory results, we decided to search for in vivo activity of EGP using the CAIA model. The joints of the negative control mice revealed serious inflammatory symptoms such as red and swollen digits and paws. The arthritis score in this group gradually increased over time and reached a peak at day 10 before a decrease from day 10 to day 14. In the group treated with EGP at a dose of 500 mg/kg b.w., the arthritis score witnessed a significant decline from day 8 to day 14. However, a dose of 250 mg/kg b.w. of EGP caused just a slight decrease in the arthritis score of the experimental mice (Figure 1).

Figure 1.

Effect of EGP on arthritis score compared with controls (P <.05).

Histopathological Results

The histopathology of the joints in the negative control group revealed strong edema, inflammatory leukocytes, macrophages, and synovial hyperplasia. Inflammatory cell infiltration and edema in the joints were reduced by administration of 500 mg/kg b.w. of EGP (Figure 2).

Figure 2.

Histopathology of joint sections of experimental mice at 200 X magnification. (A) Normal mice; (B) negative control mice show remarkably increased fibrin and infiltration of leukocytes; (C) mice treated with EGP at a dose of 500 mg/kg b.w.

Effect on Cytokine Production

As an autoimmune disease, the pathology of RA is related to the presence of cytokines in the joints. Cytokines are involved in the development of the immune system and induce autoimmunity. ⁹ Cytokines play an important role in developing new therapeutic targets in pharmacology. ¹⁰ There are three groups of cytokines playing roles in the development of RA, pro-inflammatory (IL-1, IL-6, and TNF-α), anti-inflammatory (IL-4 and IL-10), and supplemental cytokines. ¹¹ TNF-α is present in the joints and blood of RA patients. TNF-α is produced mainly by macrophages and induces the synthesis and secretion of matrix-degrading proteases, prostanoids, IL-6, IL-8, and granulocyte-macrophage colony stimulating factor by synovial fibroblasts.^12,13 IL-6 is a multifunctional cytokine that not only promotes inflammation and articular joint destruction, but also induces systemic symptoms, including inflammatory anemia, coagulation, and thrombopoietin activation. ¹⁴ IL-10, a cytokine with anti-inflammatory properties, has a central role in infection by limiting the immune response to pathogens. ¹⁵ In inflammation, PGE2 is of particular interest because it is involved in all processes leading to the classic signs of inflammation: redness, swelling, and pain. Redness and edema result from increased blood flow into the inflamed tissue through PGE2 mediated augmentation of arterial dilatation and increased microvascular permeability. Pain results from the action of PGE2 on peripheral sensory neurons and on central sites within the spinal cord and the brain. ¹⁶ Therefore, PGE2 and other cytokines, such as IL-6 and TNF-α, and IL-10, were measured in the serum of mice treated with EGP. The results (Figure 3) showed that the IL-6 level in the serum of mice oral administrated with EGP at a dose of 500 mg/kg b.w. declined significantly compared with the control, while TNF-α and PGE2 levels just decreased slightly compared with the control, but this difference was not statistically significant. In addition, the anti-inflammatory cytokine IL-10 level tended to decrease slightly when treating mice with EGP at a dose of 500 mg/kg b.w. The decrease in IL-6 level in serum is probably the main factor causing the anti-arthritic effect of EGP in experimental mice.

Figure 3.

The effects of EGP at in vivo level (A) IL-6 level; (B) TNF-α level; (C) PGE-2 level; (D) IL-10 level. Student's t-test was used for statistical comparison between treatment and control groups. *P < .05; **P < .01.

Figure 4.

Chemical structure of ethyl p-methoxy cinnamate.

Acute Toxicity

After being treated with EGP for 3 days at doses of 6, 8, and 10 g/kg b.w., all groups of experimental mice were still alive, and moved, consumed food and water normally, and had a good reflection of light and sound. Thus, EGP did not cause acute toxicity at the highest dose of 10 g/kg b.w. The body weight of mice after treatment with EGP for one, four, and seven days in the control and studied groups were not significantly different (P > .05). The findings showed that EGP did not affect the normal development of the mice. According to the classification of oral toxins by the World Health Organization (WHO, 1993), Organization for Economic Cooperation and Development ¹⁷ , and Globally Harmonized System of Classification and Labelling of Chemicals, samples that were either not toxic at the maximum dose or had LD₅₀ values > 5000 mg/kg b.w. are considered non-toxic. Therefore, EGP is considered to be orally non-toxic.

Chemical Profile of EGP

EGP displayed potential anti-inflammatory properties, which may result from several biologically active constituents. Our previous phytochemical study reported the isolation of globbanol A (1), crotepoxide (2), boesenboxide (3), and 2′-4,4′,6′-trimethoxy-chalcone (4) from EGP. In the present study, we have continuously isolated ethyl p-methoxy cinnamate as the main component of EGP, with an isolation efficiency of about 4.5% (0.9 g/20 g). The structure of the compound (Figure 4) was elucidated by using NMR spectra and comparison with a reference. ¹⁸ Interestingly, ethyl p-methoxy cinnamate is known to possess significant anti-inflammatory and analgesic properties. ¹⁹ This suggested that p-methoxy cinnamate may be the major element contributing to the anti-inflammatory activity of EGP.

Conclusions

In summary, our findings showed that the ethyl acetate extract of Globba pendula rhizomes (EGP) containing ethyl p-methoxy cinnamate as its main constituent, displayed anti-inflammatory activity. The in vitro test presented a significant NO production inhibitory ability of EGP with an IC₅₀ value of 32.45 µg/mL. EGP, at a dose of 500 mg/kg b.w., also decreased significantly the arthritis score and IL-6 level in the CAIA model. This may be due to the presence of the main component, ethyl p-methoxy cinnamate. Additionally, EGP was also proved to be non-toxic. These promising results not only suggested the potential of EGP for preparing functional foods and pharmaceutical medicines to treat inflammation, rheumatism, and joint disorders, but also provided the scientific basis for the use of this plant in folk medicine.

Experimental

Plant Materials

The fresh rhizomes of Globba pendula Roxb. were collected in An Giang province, Vietnam in February 2019 and were identified by Dr Nguyen Van Du, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology (VAST). The voucher specimens (NGM-02.2019) were deposited in the Pharmaceutical Chemistry Laboratory, Institute of Natural Product Chemistry, VAST.

General Experimental Procedures:

The ¹H-NMR (500MHz) and ¹³C-NMR (125MHz) spectra were recorded on a Bruker AM500 FT-NMR spectrometer. Column chromatography (CC) was performed on silica gel (0.040-0.063 mm). Thin layer chromatography (TLC) was conducted on pre-coated silica gel 60 F254. Compounds were visualized by UV light at 254 and 365 nm, and by spraying with a solution of 10% H₂SO₄ in ethanol, followed by heating.

Lipopolysaccharides (LPS) from Escherichia coli were purchased from Sigma Chemical Co. (St. Louis, MO, USA), Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) from Life Technologies, Inc., (Gaithersburg, MD, USA), and sodium nitrite, sulfanilamide, N-1-napthylethylenediamine dihydrochloride, and dimethyl sulfoxide (DMSO) from Sigma Chemical Co. (St. Louis, MO, USA). Other chemicals were purchased from Sigma, GIBCO, Invitrogen, and Promega. The murine macrophage cell line (RAW 264.7) was obtained from Prof. Domenico Delfino, Perugia University, Italy. TNF-alpha, IL-10 and IL-6, PGE2 ELISA kits were obtained from Biovision (Chester Springs, PA, USA). The mix of anti-collagen antibodies was provided by Modi Quest Research (Nijmegen, The Netherlands). Male albino BALB/c mice at a range of 8 to 10 weeks old were obtained from the Institute of Biotechnology, Vietnam Academy of Science and Technology (Hanoi, Vietnam).

Extraction and Isolation:

The fresh rhizomes of Globba pendula were washed, sliced, and dried in an oven at 50 °C. The dried rhizomes (1.5 kg) were powdered and extracted with methanol at 50 °C (3 times x 2 h per time) using a heated ultrasonic machine to give the total dry extract, which was then suspended in water and successively partitioned with n-hexane and ethyl acetate to give three corresponding extracts (n-hexane, ethyl acetate, water).

The EtOAc fraction (20.0 g) was separated by silica gel CC using a gradient of n-hexane-EtOAc (100:0, 75:25, 50:50, 25:75, 0:100, v:v) to obtain 5 fractions (E1→E5). Fraction E4 (6.5 g) was chromatographed on a silica gel column, eluting with n-hexane-acetone (20:1), to give 3 sub-fractions (E4.1-E4.3). Sub-fraction E4.2 (2.8 g) was further subjected to a silica gel column using n-hexane-acetone (15:1) as the mobile phase to produce 4 smaller sub-fractions (E4.2.1- E4.2.4). Sub-fraction E4.2.2 (1.5 g) was then recrystallized from n-hexane-acetone (2:1) to give ethyl p-methoxy cinnamate (0.9 g) as the main constituent of EGP.

Assay for Inhibition of NO Production:

RAW264.7 cells were initially grown in a 96-well plate (2 × 10⁵ cells/well) and incubated in a humidified atmosphere with 5% CO₂ at 37 °C for 24 h. After that, the medium was removed and replaced by DMEM (free FBS) for 3 h. The cells were treated with a sample for 2 h followed by 1 µg/mL of LPS treatment for 24 h. The nitrite accumulated in the culture medium was measured as an indicator of NO production based on the Griess reaction. Briefly, 100 µL of culture medium was incubated with 100 µL of Griess reagent {50 μL of 1% (w/v) sulfanilamide in 5% (v/v) phosphoric acid and 50 μL of 0.1% (w/v) N-1-naphthylethylenediamine dihydrochloride in water} in a 96-well plate, and incubated at room temperature for 10 min. After incubation, the absorbance was determined using an ELISA reader at 540 nm. The DMEM (free FBS) medium was used for the blank-reading in all experiments, and the positive control was N^G-methyl-L-arginine acetate (L-NMMA). The remaining cell solutions in cultured 96-well plates were used to evaluate cell viability by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.

Anti-Inflammatory Evaluation Using Collagen Antibody-Induced Arthritic Mice (CAIA):

BALB/c male mice (24) were injected intraperitoneally (i.p.) with 3 mg of the cocktail of anti-collagen antibodies on day 0, followed by intravenous injection with 25 μg of LPS on day 3 and day 9 to boost arthritic development. Mice having initial symptoms of arthritis on day 4 were divided into 4 groups (6 mice/group). Group 1 received water daily for 10 days served as a negative control; Groups 2 and 3 were orally administered EGP at a dose of 250 mg/kg b.w. and 500 mg/kg b.w. daily for 10 days, respectively; group 4, serving as a positive control, was given dexamethasone by gavage at a dose of 0.5 mg/kg b.w. daily for 10 days. The score of clinical arthritis was examined every 2 days of the experiment. Each paw was scored on a scale of 0 to 2 based on signs of swelling and inflammation. At the end of the experiment, the hind paws of 3 experimental mice per group were collected, fixed immediately in 10% neutral formalin for pathological analyses. After decalcifying, all tissues were embedded in paraffin. Finally, 3 mm thick joint sections were stained by hematoxylin and eosin (H&E) for histopathological analyses. The levels of examined cytokines in the sera were measured by using commercial ELISA kits (Biovision, Chester Springs, PA, USA), following the manufacturer's protocols.

Acute Toxicity Assay

The acute toxicity assay was conducted according to the Vietnam Ministry of Health ²⁰ . Twenty-four healthy albino BALB/c mice were divided into 4 groups (6 mice/group). Group 1 receiving water daily for 10 days served as a negative control while groups 2, 3, and 4 were treated with EGP at doses of 6, 8, and 10 g/kg b.w. Their general movements, behavior, eating, and death were monitored and recorded within 72 h, and then for a further 4 days in the case of previously having no signs of abnormality.

Statistical Analysis

The results are shown as mean  ±  SD. Data were analyzed by GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego CA). Student's t-test was used for statistical comparison between the different groups. In all comparisons, P < .05 was considered statistically significant.