NF-κ B Inhibitory Activities of the Methanol Extracts and some Constituents therein of some Vietnamese Medicinal Plants

Eighty-seven methanol extracts of medicinal plants, most of them are currently used in Vietnamese oriental medicine, were screened for NF-κB inhibitory activity. Seven methanol extracts showed strong to moderate inhibitory activity. These include the extracts of Crinum latifolium (leaves), Evodia rutaecarpa (fruits), Polygonum cuspidatum (rhizoma), Perilla ocymoides (leaves), Rubia cordifolia (leaves), Scutellaria barbata (leaves) and Sparganium stenophyllum (leaves). The NF-κB inhibitory activities of several metabolites isolated from some of these plants were also reported.


Introduction
Nuclear factor-kappa B (NF-κB) is an inducible and ubiquitous transcriptional factor required for the gene expression of many inflammatory mediators [1].Inappropriate and prolonged activation of NF-κB has been linked to several diseases associated with inflammatory events, including septic shock, acute respiratory distress syndrome, ischemia, and reperfusion injury [1].Because of the pathophysiological importance of an enhanced production of inflammatory mediators through NF-kB activation, selective inhibitors of NF-κB activation may have broad application as novel therapeutics, for example, antiinflammatory agents.A number of other scientific evidences have also linked the role of NF-κB activation to several oncogenic processes.Thus, NF-κB inhibitors may also have a potential for the treatment of some types of malignancy.In deed, a bulk of evidences has validated NF-κB as a target for antiinflammatory and anticancer agents.
In the past decade, extensive efforts have been devoted to discover selective NF-κB inhibitors.Several reports have documented a few NF-κB inhibitors, naturally occuring [2][3][4][5][6] or synthetic [7].However, to date, no NF-κB inhibitor has been approved for use in clinics.Thus, the development of NF-κB inhibitors continues to be an appealing need.Medicinal plants have been and continue to be a powerful source of novel structures with diverse biological activities.Vietnamese flora in particular is still relatively underinvestigated.From these perspectives, we have chosen Vietnamese medicinal plants as a source for screening of NF-κB inhibitory activity in our continuous program to discover novel and potent compounds as NF-κB inhibitors.As a part of this program, we have screened eighty-seven kinds of herbal drugs used in Vietnamese oriental medicine for NF-κB inhibitory activity.This paper describes and discusses the results obtained from this screening and some preliminary results from phytochemical studies carried out on some active plants found in the preceding screening.

Results and discussion
The results of the screening of eighty-seven Vietnamese herbal medicine and medicinal plants for NF-κB inhibitory activity are presented in table 1.This NF-κB inhibition assay used RAW264.7 cells and was carried out as detailed in the Experimental part.To choose a non-cytotoxic concentration of each sample for the NF-κB assay, an in vitro cytotoxicity assay was also performed and the ED 50 values (the concentration of the sample that inhibits the growth of cells by 50%) were included in this table.Based on the results of the cytotoxicity assay we have chosen a noncytotoxic concentration for each plant or herbal medicine sample.These concentrations were used in the NF-κB inhibition assay.Seven plant samples were found to exhibit strong to moderate inhibitory activity.These samples include Crinum latifolium (folium), Evodia rutaecarpa (herba), Perilla ocymoides (folium), Polygonum cuspidatum (radix), Rubia cordifolia (folium), Scutellaria barbata (folium) and Sparganium stenophyllum (folium).The list of the active medicinal plants in the NF-κB inhibition screening is extracted from table 1 and presented in table 2.
Chemically, the plant Evodia rutaecarpa has been reported to contain evodiamine, an alcaloid which exhibits antiproliferative, antimetastatic, and apoptotic activities.In a recent study Takada and colleagues have demonstrated that evodiamine inhibits both constitutive and induced NF-κB activation and NF-κB-regulated gene expression [8].Thus, the NF-κB inhibitory activity of the Evodia rutaecarpa sample might well be attributable to that of evodiamine.
Regarding Polygonum cuspidatum, it was postulated that the NF-κB inhibitory activity might be resulted from resveratrol and emodin.Emodin is one of the main chemical constituents of Polygonum cuspidatum.Resveratrol has also been widely reported to be present in this plant.In our separate study on the Vietnamese medicinal plants we have also isolated both resveratrol and emodine from Polygonum cuspidatum growing in Vietnam [9].Especially, the content of resveratrol therein was determined to be 0.28%, relatively high [10].In our NF-κB inhibition assay system, resveratrol and emodin showed IC 50 values of 5.2 and 5.9 μM, respectively.From Perilla ocymoides we have isolated two compounds which structurally were determined to be (2E,4E)-5-(1,3-benzodioxol-5-yl)-1-(piperidin-1-yl)penta-2,4-dien-1-one (piperine) and 4-hydroxycinnamic acid [11].Piperine is a well-known alkaloid that has been found naturally in plants belonging to the Piperaceae family, such as Piper nigrum L, commonly known as black pepper, and Piper longum L, commonly known as long pepper.However, this was the first report on the presence of this compound in Perilla ocymoides.Piperine has been reported to be an NF-κB inhibitor [12].Reexamination of the NF-κB inhibitory activity of piperine revealed that this compound had an IC 50 value of 2.7 μM in our assay.Compound p-hydroxycinnamic acid also exhibited moderate inhibitory effects on the activation of NF-κB with an IC 50 value of 14.5 μM.
Aiming at the investigation of the chemical constituents responsible for the NF-κB inhibitory activities of the remaining three plants including Rubia cordifolia, Scutellaria barbata, and Sparganium stenophyllum we have fractionated the corresponding methanol extracts against dicloromethane (DCM), ethyl acetate (EA), and n-butanol to give the DCM, EA, and BuOH fractions, respectively.The remaining water layers were concentrated to give the AQ fractions.Subsequent assays located the NF-κB inhibitory activity of Rubia cordifolia in the DCM soluble part, meanwhile the NF-κB inhibition of the other two methanol extracts of Scutellaria barbata, and Sparganium stenophyllum was mainly caused by the AQ fractions.Efforts have been undertaken to isolate the active metabolite(s) from the DCM fraction of Rubia cordifolia and the results will be reported in due course.

Plant materials
Most of the plant materials were purchased from an oriental herbarium in Hanoi, Vietnam.Some medicinal plants were provided by Professor Tran Cong Khanh, Department of Botany, Hanoi University of Pharmacy, Vietnam.

Cells and cell culture
Cell lines were obtained from a Cancer Cell Bank at Korea Research Institute of Bioscience and Biotechnology (KRIBB) and cultured in DMEM supplemented with FBS in 10% and L-glutamine (0.2 mM/ mL).

Extraction, fractionation and sample preparation
Dried plant materials (100 gr each) were extracted with MeOH (3 times x 200 mL) under reflux.The combined methanol extracts were concentrated to dryness in vacuo.In some cases, the MeOH extracts were suspended in water and partitioned with dichloromethane (DCM), ethyl acetate (EA) and butanol (BuOH) successively to give DCM, EA, BuOH fractions, respectively.The remains in water suspension were concentrated to give corresponding water fractions (WTR).For assays, the fractions were dissolved in dimethyl sulfoxide (DMSO) or ethanol at the initial concentration of 100, 30 or 10 mg/mL, respectively and serially diluted into 30, 10, 3, and 1 mg/mL concentrations.The insoluble parts were filtered off.The filtrates were stored at 4 °C and used as stock solutions.

NF-κB inhibition test
The assay was carried out using the procedure described in literature [4].Briefly, NF-κB mediated reporter plasmid vector was constructed as previously described with minor modification [15].This reporter vector has 9x binding site for the NF-κB and secretable alkaline phosphatase (SEAP) as a reporter.The RAW264.7 cells stably transfected with the reporter construct were seeded in a 96-well plate at 5 x 104 cells/200 μL/well and incubated for 3 h to let the cells attach.The cultures were treated with compounds tested at a predetermined concentration (non-cytotoxic) and stimulated with 1 μg/mL of LPS.After 6 h incubation, the medium was replaced with 200 μL of fresh DME medium containing 0.5% FBS followed by twice washing with serum-free medium, and then the cultures were incubated for 24 h.One hundred μL of each culture supernatant were transferred a new 96-well plate, heated at 65 °C for 5 min, and then mixed with equal volume of 2 x SEAP assay buffer (2M diethanolamine, 1 mM MgCl 2 , 20 mM Lhomoarginine).The reaction was initiated by the addition of 20 microL of 120 mM pnitrophenyl phosphate dissolved in 1 x SEAP assay buffer and incubated at 37 °C.The absorbance (A) of the reaction mixture was measured at 405 nm with a microplate reader (Molecular Devices Co., Menlo park, CA, USA).The inhibition percentage (IP) was calculated using the following equation: IP = [(A blk − A spl )/A blk ] x 100%, in which A blk = average absorbance of the blank wells; A spl = average absorbance of the sample wells.

In vitro cytotoxic assay
To choose a noncytotoxic concentration for the above NF-κB inhibition test, a cytotoxicity assay was conducted using SRB method [16].The ED 50 values (the concentration of the sample that inhibits the growth of cells by 50%) were calculated and included in Table 1.