Phytochemical and pharmacological studies on Mikania micrantha H . B . K . ( Asteraceae )

Asteraceae is one of the largest families of Angiospermae, and contains the Mikania genus. Several species of this family contain polyacetylenic and thiophenic compounds which are used as taxonomic markers. Also, their phototoxic activity acts as plant defense mechanism. In this paper we report the absence of these compounds in Mikania micrantha.We also describe the composition of the volatile oil from the seeds and inflorescence in the species. Linalool and α-pinene were the main components of this M. micrantha’s essential oil. This study tested the ability of extracts of Mikania micrantha to inhibit the mouse ear inflammation in response to topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA). The antibacterial activity of extracts was also evaluated against Bacillus subtilis and Escherichia coli. Ethyl acetate extracts of this plant exhibited significant antibacterial and anti-inflammatory properties. Therefore, it could be used as a medicinal plant.


INTRODUCTION
Mikania micrantha H.B.K. (Asteraceae) is original from Central and South America.This genus is found in the tropics of America and Asia, and is widely known as guaco.It comprises about 300 identified species, but only 20 of them have been studied.It is used to treat fever, rheumatism, influenza and respiratory diseases.(Cabral et al., 2001;Muceneeki et al., 2009).
Terpenes are the major constituents isolated from plants of this genus and its essential oil compounds have large applications in pharmaceutic and cosmetic industries (Silva- Santos et al., 2004).
We analyzed the composition of the volatile oil from seeds and inflorescences of Mikania micrantha.We seeked also for the presence of phototoxic compounds, important taxonomic markers, and natural defence mechanisms against insects in species belonging to the Asteraceae family.
In this study, the pharmacological potential of M. micrantha was evaluated (to treat erisipela, snake beat and gastrointestinal disorders) because it is an important natural resource in the traditional medicine (Martínez et al., 2001).
Mikania micrantha has not been tested in the tetradecanoylphorbol acetate (TPA) mouse ear model of inflammation.This model may block the inflammatory response to topical TPA (Gabor, 2003).Because this plant is being used as a natural product applied to the skin, it is important to test M. micrantha in this model.
Antibacterial activity was tested by microplate method for determining the susceptibility of E. coli and B. subtilis to extracts of M. micrantha and ampicillin.
The broad activity spectrum of the extracts explains the widespread use of this plant for wound healing and other applications.

MATERIALS AND METHODS
Plant material.The plant was collected in Cuetzalan del Progreso, Puebla, Mexico, on March 13, 2006.Vouchers were deposited in the National Herbarium, Instituto de Biología, Universidad Nacional Autónoma de México (MEXU).
Preparation of extracts.The plant was divided in stem with leaves, and inflorescences with seeds, which were successively extracted at room temperature with hexane, ethyl acetate and methanol.Solvents were eliminated at reduced pressure, and the dry extracts were used for the different tests.
Essential oil.The essential oil was obtained by steam distillation of inflorescence and seeds, and was analyzed in a CG-LCR-IQUI chromatograph with an AT Aquawax column.
Pharmacological activities.The anti-inflammatory and antibacterial potential were measured using the three (hexane, ethyl acetate, methanol) extracts.The anti-inflammatory potential was tested with the mouse ear edema test, and the antibacterial potential was measured against Bacillus subtilis (ATCC-6051) and Escherichia coli (ATCC-6051).
The anti-inflammatory activity of the 3 extracts was carried out by the mouse ear edema test induced with TPA (12-Otetradecanoyl-phorbol-13-acetate) (De Young et al., 1989).For each determination, three male CDI mice (25-30 g) were used; 10 mL of an ethanolic solution (0.25 mg/ml) of TPA (2.5 mg/ear) were applied to the surface of the right ear; the left ear was used as the blank; 10 min.after the application of TPA, 20 mL of the extracts (1 mg dissolved in ethanol) were applied topically After the fourth hour, the animals were sacrificed and a section (7 mm) of the ear was weighted.The increase in weight of the right ear with respect to the left one indicates the swelling produced by TPA.Percentage inhibition was calculated by comparison with the control (left ear).Indometacine (0.046, 0.085, 0.15 mg / ear) was used as drug reference.
The following experiments were carried out: Edema A: edema induced by TPA alone, Edema B: edema induced by TPA plus sample, Inhibitory ratio (%) = [(Edema A -Edema B) / Edema A] x 100 Each value represents the mean of individual determinations from 3 mice.The experiment was repeated 3 times.
The bactericidal potential was determined by the microwell dilution method (Ozturk et al., 2006).The inoculum of bacteria was prepared from 18h broth cultures, and suspensions were adjusted to 0.5 McFarland standard turbidity.The Minimal Inhibitory Concentration (MIC) of Mikania micrantha extracts (hexane, ethyl acetate and methanol) against bacterial strains [Escherichia coli (ATCC-6051) and Bacillus subtilis (ATCC-6633)] was determined.Ninety-six well plates were prepared by dispensing 95 µl of nutrient broth and 5 µl of the inoculum into each well.A 100 µl extract from Mikania micrantha initially prepared at concentration of 8 µg/µl, was added into the first well.Then, 100 µl from their serial dilutions was transferred into twelve consecutive wells.Wells containing 195 µl of nutrient broth without compound and 5 µl of the inoculum were used as a test for growth.The negative control contained: 195 µl of nutrient broth, the solution solvents and 5 µl of the inoculum.This ensured that the extraction solvents had no inhibitory effects on the bacterial strains.
The final volume in each well was 200 µl.Ampicille anhydrous (Sigma) at the concentration range of 0.8 -0.001 µg/ µl was prepared in nutrient broth and used as standard drug for positive control.Contents of each well were mixed on a plate shaker at 300 rpm for 20s and then incubated at 37 °C for 20h.After the incubation period, microbial growth was determined by reduction of TTC (colorless compound) to formazan (red compound).With this purpose, 40 µl of 2, 3, 5-triphenyl tetrazolium chloride (TTC) (Sigma) (0.2 mg/ml) were added to each microplate well.The covered microplates were incubated at 37 °C and 100% relative humidity for 4h Pérez-Amador MC et al., FYTON 79 (2010) FYTON ISSN 0031 9457, (2010) 79: 77-80 and 12h.The extracts analyzed in this study were screened three times against each organism.The MIC of each extract was taken as the lowest concentration that showed no growth, and it was confirmed by plating 5µl samples from clear wells on nutrient agar medium.

RESULTS
Phototoxic compounds.We didn't find any phototoxic compounds in Mikania micrantha despite the fact that the Asteraceae family contains phototoxic compounds (polyacetylenes and thiophenes).The hexane extracts did not show the characteristic blue pale spots on TLC and the UV bands were not characteristic (Fig. 1).The phototoxic activity was negative.Essential oil.Ten Components were separated by gas chromatography.The two major compounds were linalool (15.86%) and α-pinene (10.14%) (Table 1).

Pharmacological activities
Anti-inflammatory potential: The hexane and ethyl acetate extracts showed a significant anti-inflammatory activity; the methanol extracts were inactive.The hexane extract from stems and leaves showed 49.69% inhibition of the mouse ear edema; the indometacine control had 71.02%, and the hexane extract had 21.33% (Table 2).Seed and inflorescence extracts had 51.75% inhibition.Inhibition from the ethyl acetate extract from stem and leaves was 88.30%.Seeds and inflorescence extracts showed 54.62% of ear edema inhibition, corresponding to 16.4% (Table 2).Antibacterial activity: The MIC tests revealed that both bacterial strains are sensitive to the three crude extracts from leaves and stems, and seeds and inflorescences of Mikania micrantha between 0.5 -4 µg/µl.
Escherichia coli had a MIC of 1 µg/µl in presence of the ethyl acetate extract from each part of the plant.The methanol extract from seeds and inflorescences was less active, while the Phytochemistry and pharmacology in Mikania hexane extracts, and methanolic extract from leaves and stems have the same inhibitory effect at 2 µg/µl.
Bacillus subtilis was more sensitive to the ethyl acetate extract from each part of the plant, and had a small MIC 0.5 µg/µl.Leaf and stem extracts were twice as active as seed and inflorescence extracts against this bacteria.

DISCUSSION
Apparently this Asterace, collected in March 2006 in Cuetzalan del Progreso, Puebla, Mexico, did not present any phototoxic compounds.
Linalool and α-pinene are the main volatile oil components of seeds and inflorescences in Mikania micrantha.Such composition can explain the antibacterial and antiinflamatory properties of this species.
The activity of leaves plus stem, and seeds and inflorescences, was bigger in the ethyl acetate than in the hexane extract.
Many authors attributed the pharmacological effect of guaco to coumarin (1,2-benzopirone) (Dos Santos et al., 2006).However, our results made clear that this is not the only bioactive component present in the extracts we tested.Ethyl acetate is a solvent capable of extract coumarin and several terpenes and phenolic compounds, which have a wide range of biological activities.These include plant growth-regulators, secondary compounds against insects, and anti-bacterial properties (Huang et al., 2009).Some flavonoids and dicaffeoylquinic acid butyl esters have been recently described as bioactive for M. micrantha (Wei et al., 2004).
We showed by the TPA model of mouse ear inflammation that the secondary metabolites tested, which were contained in the ethyl acetate extract of M. micrantha, have the best biological activity.However, the methanol extracts were inactive.
The highest growth inhibitory concentration against E. coli was in the methanol extract, followed by the hexane and ethyl acetate extracts.For Bacillus subtilis the smallest concentration for both plant parts was in the ethyl acetate extract, followed by the concentration in the methanol and hexane extracts.Therefore, the ethyl acetate extract has the bioactive compounds that can produce a desirable pharmacological effect.
Our results demonstrated that M. micrantha can be used as a medicinal plant.

Table 1 .
Identification of essential oil components by gas chromatography.