Abstract: The methanol extracts of thirteen medicinal plants from Okinawa, Japan were examined for Matrix Metalloproteinase-13 (MMP-13) inhibitory activity. Among the thirteen selected species, Curcuma longa, Ocimum basilicum and Curcuma aromatica showed high inhibitory effect with IC50 values of 27.8, 81.7 and 85.8 μg mL-1, respectively. The chemical compositions of these three plant extracts were determined by LC-MS. Curcumin was the predominant constituent of C. longa and C. aromatica (58.6 and 28.7 mg g-1 extract, respectively), whilst O. basilicum mainly contained rosmarinic acid with amount of 47.3 mg g-1 extract. Both of curcumin and rosmarinic acid exhibited excellent MMP-13 inhibitory activity (IC50: 3.6 and 2.9 μM, respectively). The results indicate that curcumin and rosmarinic acid might be potent MMP-13 natural inhibitors.
INTRODUCTION
Medicinal plants have been traditionally used for pharmaceutical and dietary therapy in long history. A number of herbs and many relevant prescriptions have been screened and used for treating and preventing various tumors and inflammations as folk practices. Nowadays medicinal plants are still widely practiced particularly in the country side and remote mountainous regions and even in the urban areas of many Asian countries.
Matrix Metalloproteinases (MMPs) comprise a family of secreted and membrane-bound endopeptidases which hydrolyze extracellular matrix proteins. Based on their preferred substrates and on structural features, MMPs can be divided into collagenases, gelatinases, stromelysins and membrane-type matrix metalloproteinases (Jiang and Bong, 1992; Hooper, 1994). Collagenases were important proteolytic tools for extracellular matrix remodeling during organ development and tissue regeneration. Chronic activation of collagenases results in an excessive degradation of extracellular matrix components and was believed to contribute to many pathological conditions such as tumor progression, osteoarthritis, rheumatoid arthritis and many inflammatory diseases (Clark and Parker, 2003; Ala-Aho and Kähäri, 2005; Blavier et al., 2006; Deryugina and Quigley, 2006). As one of important enzyme of MMPs family, collagenase 3 (MMP-13) was reported to be involved in the development and metastasis of breast and lung carcinomas. Additionally, this enzyme plays an important role in degenerative bone diseases such as rheumatoid arthritis and osteoarthritis (Tardif et al., 2004; Burrage and Brincherhoff, 2007). Preclinical studies have provided compelling evidence that inhibition of MMPs would be therapeutic for inflammatory, malignant, arthritis and degenerative diseases (Murphy and Docherty, 1992; Skiles et al., 2001). In recent years, several highly selective synthetic MMP-13 inhibitors have been tested for their effects against growth and invasion of malignant tumors and for therapeutic of osteoarthritis and rheumatoid arthritis (Ala-Aho et al., 2005; Burrage and Brincherhoff, 2007).
Okinawa locates in subtropical region of Japan, which has a rich plant diversity. It was estimated that about 300 plant species have been traditionally used for disease treatment and pharmaceutical purpose (Hatushima and Nakajima, 1976).
In this study, thirteen medicinal plant species which are well popular in Okinawa were investigated for their efficacies against MMP-13 inhibitory activity. Identification and quantification of potent bioactive compounds from these plants were also performed.
MATERIALS AND METHODS
Plant material: Thirteen folk herbs associated with anticancer, anti-arthritis and anti-inflammatory activities as described by Yosikawa (1983) and Tawata et al. (1985) were selected in this study. Pentrarhizidium orientale Hayata, Alpinia zerumbet (Pers.) B.L., Zingiber officinale Rosc., Curcuma longa L., Curcuma aromatica Salisd., Catharanthus roseus G. Don, Orthosiphon aristatus (BL.) Miq., Elfvingia applanata karst, Houttuynia cordata Thunb., Plantago asiatica L., Ocimum basilicum L. were purchased at a Naha herbalist, Okinawa, Japan, except Smilax sebeana Miq. and Ficus microcarpa L. f., which were collected from the campus of University of the Ryukyus during November of 2005. The authenticity of the plant species was confirmed by Professor Tastuyama Gochi of Faculty of Agriculture, University of the Ryukyus. The voucher specimens have been deposited in Faculty of Agriculture, University of the Ryukyus (Deposit No. R0502-R0515). The scientific names and medicinally used parts are shown in Table 1.
Preparation of the methanol extract: The samples were ground to fine powder and passed through a sieve (24 mesh), then dried to constant weight in desiccator at 40°C. Six grams of each sample were extracted with 35 mL of 100% methanol for 12 h at room temperature with shaking. The plant materials were extracted twice in the same conditions. The methanol extracts obtained from each sample were collected, filtered, dried under vacuum and then re-dissolved in methanol and stored under refrigeration for further analysis. The quantity of plant extracts is shown in Table 1.
Solvents and reagents: N-Hydroxy-1-(4-methoxyphenyl) sulfonyl-4-(biphenylcarbonyl) piperazine-2-carboxamide (CBC) curcumin and rosmarinic acid were purchased from Sigma Chemicals. All solvents were at analytical grade and purchased from Wako Pure Chemical Industries, Japan.
MMP-13 inhibitory assay: The MMP-13 inhibitory ability of the 13 medicinal plants, curcumin and rosmarinic acid was determined by a MMP-13 inhibitor assay kit (Chondrex, Inc., Redmond, WA, USA, distributed by IWAI Chemicals Company, Japan, Catalog No. 3003). The human CBC was used as a positive control. A designate reaction was performed in the 96-well microtiter plate according to the manufacturer`s protocol. The assay procedure was separated into two stages. First, diluted recombinant human MMP-13 (rh-MMP-13, 10 μg mL-1) with dilution buffer B was activated by adding 5 μL of activator 1 (APMA) at 35°C for 60 min. Second, appropriate amounts of test samples that diluted by solution B and reaction buffer to the wells were added to adjust the final volume to 160 μL. The reaction was initiated by adding 100 μL substrate solution to each well. The collagenase reaction was stopped by adding 10 μL of the stop solution to each well after incubating at room temperature (25°C) for approximately 30 min. The reaction fluorescence intensity was determined at λem = 450 nm and λex = 360 nm with LS- PLATE manager 2001 (Wako, Osaka, Japan). The MMP-13 activity was determined by comparing with a standard response curve using buffer instead of inhibitor in similar conditions. The inhibitory activity was calculated from 100 subtracted by the percentage of enzyme activity. All treatments were carried out in 3 replications.
LC/MS spectrometer: LC/MS spectra were obtained using a Sciex API 2000 LC-MS/MS System (Model Sciex API 2000, Applied Biosystems, Langen, Germany) coupled to a Agilent 1100 LC Binary pump equipped with a Agilent 1100 Thermo Auto-sampler, Agilent 1100 Column Oven and Agilent 1100 Diode Array Detector in combination with a SYNERGI 4 u MAX-RP 80 A C18 reverse phase column (150x4.6 mm, Phenomenex Company, USA.). Five microliter samples were injected for analysis. A gradient elution was performed with solvent A (water: acetic acid, 100: 0.5, v/v) and B (methanol: acetonitrile, 3: 1, v/v) as follows: 0-10 min, 30% B; 10-15 min, 30-40% B; 15-20 min, 40-50% B; 20-25 min, 50-100% B; 25-30 min, 100% B; 30-31 min, 100-30% B; 31-38 min, 30% B and flow rate was 500 μL min-1. Mass spectra were obtained in ion spray voltage of 5000 V (negative mode) and a temperature of 450°C using a Turbolon spray ion source. Spectra were recorded between m/z 40-800 with scan duration of 2 sec scan and an interscan time of 0.1 sec. Spectra were processed using Biosystems/MDS SCIEX instruments Analyst Software (version: Analyst 1.4). UV detector spectral was recorded between 190-400 nm with a 2 nm step width.
| Table 1: | The name, family, medicinally used parts and yield of methanol extracts of selected 13 medicinal plants in Okinawa |
Statistical analysis: The statistical analyses were performed by one-way ANOVA and the Student`s t-test. The results were expressed as the mean±SE (n = 3) to show variations in the various experimental. Differences are considered significant when p<0.05.
RESULTS
MMP-13 inhibitory activity: The methanol extracts from thirteen selected medicinal plants, standard compounds (curcumin and rosmarinic acid) and positive control (CBC) were assayed for MMP-13 inhibitory activity by a MMP-13 inhibitor assay kit. The results are shown in Table 2. Except for Z. officinale, C. roseus and F. microcarpa, the other plants showed certain inhibitory effect against MMP-13, however their IC50, values varied among plant species. The best inhibitory result was obtained from the extract of C. longa (IC50, 27.8 μg mL-1), followed by those of O. basilicum and C. aromatica (IC50, 81.7 and 85.8 μg mL-1, respectively). However, no plant extracts could have greater MMP-13 inhibitory ability than the positive control CBC (a selective synthetic MMP-13 inhibitor, IC50, 0.15 μM). In general, the MMP-13 inhibitory activities of these plants were proportional to applied dose.
Chemical composition and biological activity: As C. longa, O. basilicum and C. aromatica showed greater MMP-13 inhibitory properties than other plant species, they were therefore analyzed by LC-MS to determine potential chemicals involved in their biological activities. By comparing the retention times, MS and UV spectra with those of standards and from available literatures (Bais et al., 2002; Jayaprakasha et al., 2005), curcumin was identified in C. longa and C. aromatica, while, rosmarinic acid was detected in O. basilicum (Fig. 1). The contents of curcumin and rosmarinic acid were also quantified by LC-MS (Table 3). As shown in Table 3, curcumin was the major compound found in C. longa (58.6 mg g-1 extract) and C. aromatica (28.7 mg g-1 extract). On the other hand, rosmarinic acid was the main constituent in O. basilicum (47.3 mg g-1 extract).
| Table 2: | MMP-13 inhibitory activity of methanol extracts from 13 medicinal plants in Okinawa |
| NI: No inhibitory activity; The values represent means±SE (n = 3) | |
| Table 3: | Contents of curcumin in C. longa, C. aromatica and rosmarinic acid in O. basilicum |
| The values represent means±SE (n = 3); ND: Not detected | |
The MMP-13 inhibitory activity of the 2 compounds was examined and the results are shown in Table 2. Both curcumin and rosmarinic acid exhibited high inhibitory effect (IC50, 3.6 and 2.9 μM, respectively). Obviously, rosmarinic acid exerted higher inhibitory activity than that of curcumin while two compounds exhibited dose-dependent activity. However, the inhibitory strength against the MMP-13 of the two natural compounds was lesser than that of the positive control CBC.
DISCUSSION
Rhizomes of C. longa and C. aromatica have long been used as indigenous medicines for the treatment of a variety of inflammatory conditions and other diseases. It has been previously determined that, the main components present in the rhizome of C. longa were three pyrazole analogues of curcuminoids (curcumin, monodemethoxycurcumin and bisdemethoxycurcumin) (Ramsewak et al., 2000; Jayaprakasha et al., 2005). Hong et al. (2006) also reported the content of curcumin in C. longa and C. aromatica (3.6 and 0.3%, respectively). Curcumin was the active constituent of C. longa and C. aromatica and it has been used in clinical Chinese medicine as aromatic stomachic and choleretic (Ching et al., 2001). Curcumin and related species have a wide array, of pharmacological and biological activities. Ramsewak et al. (2000) and Selvam et al. (2005) found that C. longa extract and curcumin exhibited significant COX-1 and COX-2 inhibitory activity in vitro. Cytotoxicity, antioxidant, anti-inflammatory and anti-cancer activities of curcumin have been assessed in various assays (Jayaprakasha et al., 2005; Maheshwari et al., 2006; Johnson and Mukhtar, 2007). Compounds of C. longa other than curcumin, might be also responsible for its MMP-13 inhibitory activity. However, in this study, the two substances monodemethoxycurcumin and bisdemethoxycurcumin were not quantified or examined for their biological activities as they could neither been purchased nor successfully isolated in our laboratory.
| Fig. 1: |
The LC-MS chromotograph of curcumin in methanol extracts of (A) C. longa, (B) C. aromatica and (C) Rosmarinic acid in methanol extracts of O. basilicum |
On the other hand, O. basilicum is also an important medicinal plant and culinary herb and is marketed worldwide (Loughrin and Kasperbauer et al., 2001). The extract of O. basilicum leaves showed inhibitory activity against HIV-1 reverse transcriptase (Yamasaki et al., 1998). Rosmarinic acid was one of the most abundant caffeic acid esters present in O. basilicum (Chamila et al., 2003) and it has antioxidant, anti-HIV and anti-inflammatory or cyclooxygenase and lipoxygenases inhibitory activities (Kelm et al., 2000; Petersen and Simnonds, 2003). These evidences indicated that curcumin and rosmarinic acid have been shown to possess wide range of pharmacological activities.
CONCLUSION
Among the 13 Okinawa medicinal plants, the plant extracts of C. longa, O. basilicum and C. aromatica showed high inhibitory effect against MMP-13. Curcumin and rosmarinic acid showed promising MMP-13 inhibitory activity and may be responsible for the strong MMP-13 inhibitory activities of the three plants. Our results provide some scientific evidences for the use of several medicinal plants from Okinawa for treating tumours, inflammatory diseases and arthritis.