Antifungal Activity of Mimosa pudica, Isolation and NMR Characterization of Bioactive Components

In this study, the whole plant of Mimosa pudica was extracted using absolute ethanol. The crude ethanolic extract and its isolated triterpenoid glycoside were tested for antifungal activity towards Aspergillus flavus and Trichophyton rubrum using Well Diffusion Method. At concentrations of 25 – 100 mg/mL, the extract possesses antifungal activity (from being partially active to very active) against Aspergillus flavus and Trichophyton rubrum. The TLC profile of the crude extract indicated many compounds with RF values of 0.41, 0.43, 0.56, 0.68, 0.89, and 0.90. The isolation and purification were established using extensive thin layer and column chromatographic processes by employing various solvents with varied polarity. The results of thin layer and column chromatography gave rise to the isolation of a triterpenoid glycoside with RF value of 0.41. The spectral analyses involving H-NMR, C-NMR, C-DEPT, COSY, HMBC, HMQC and infrared were useful in proposing the structure of the compound. Original Research Article Chukwu et al.; AJOCS, 2(4): 1-5, 2017; Article no.AJOCS.34744 2


INTRODUCTION
The harmful side effects, high cost of other forms of treatment and their non availability to the poor populations who live in the remote areas are some of the reasons for the demand for herbal medicine.The increasing prevalence of multidrug-resistant strains of bacteria and the recent emergence of strains with reduced susceptibility to antibiotics raise the spectre of untreatable bacterial and viral infections.Thus, adding to the urgency to search for new infection fighting strategies (Sieradzki et al. [1]).Contrary to the synthetic drugs, antimicrobial substances of plant origin are not associated with many side effects and have an enormous therapeutic potential to heal many infectious disease (Iwu et al. [2]).
Mimosa pudica Linn is a creeping annual or perennial herb often grown for its curiosity value, as the compound leaves fold inward and drop when touched reopens within minutes.It belongs to the Fabaceae family.The generic name Mimosa is derived from the Greek mimos,meaning "mimic", alluding to the fact that the leaves move in response to something moving against them.The specific epithet is taken from the Latin word pudica, meaning bashful or shrinking to contact (Barneby [3]).Mimosa is a genus of about 400 species of herbs and shrubs, in the subfamily Mimosoideae of the legume family Fabaceae.The plant is native to Brazil, but is now a pan tropical weed.The species is known by numerous common name including: sensitive plant, humble plant, shameful plant, touch-me-not, chuimui, and antplant.The vernacular names in the three major languages of Nigeria include: agboghomechieukwu (Igbo language), kamawalkinka (Hausa language) and ewe padimo or patomo (Yoruba language).In Portugal, Africa, Rio de Janeiro, Sao Paulo City and other southern capitals, the name is nao-me-toque (touch-me-not).
In Brazil, the name is dormideira (roughly "sleeper").In Spanish, the name varies as mori-vivi or morivivi(roughly "I died, I lived") in the Dominican Republic, Puerto Rico and other Spanishspeaking Caribbean islands (Union County College Biology Department [4]).In Costa Rica, it is dormilona.In the Philippines, it is called "makahiya" which means "quite shy".It is putrimalu (shy princess) in Indonesia; pokoksemalu (shy plant) in Malaysia; nidikumba (sleeping plant) in Sri Lanka; mateloi (false death) in Tonga (Churchward [5]).
In south Asia many unrelated names are also common.In Hindi, it is known as chhui-mui (that which dies upon touch).In Bengali, the shrub is called lojjaboti (that bashful girl).In Malayalam, it is called thottavaadi (wilts by touch).In Marathi, it is called lazalu (shy).In Tamil, it is called thottasiningi (acts when touched) and in Kannada, it is known as muttidare muni (angered by touch).In Burmese (Myanmar) it is called htika-yoan (crumbles when touched).In Liberia, it is known as the picker weed (Union county College Biology Department, [4]).
This plant has a history of use for treatment of various ailments and the most commonly used plant part for this purpose is the root; but flowers, back and fruit can also be utilized.Several research works have been carried out to study about the phytochemical components of Mimosa pudica and also about the antimicrobial activity of the plant (Gandhiraja et al. [6]).Phytochemical studies on M. pudica have revealed the presence of alkaloids, fatty acids, non-protein amino acid (mimosine), flavonoids, C-glycosides, sterols, terpenoids, and tannins (Genest et al. [7]).Reported major pharmacological activities are:antiviral properties, aphrodisiac properties, antimicrobial properties, anti-venom activities, anti-hepatotoxic and antioxidant effects, diuretic effect, hyperglycemic effect, wound healing effect etc. (Amalraj and Ignacimuthu [8]).Some of the isolated secondary metabolites are bufadienolide, D-pinitol, norepinephrine, Pcoumaric acid, mimopudine, mimosine, potassium-5-O-β-glucopyranosy gentisate, etc.Two well-known movements are observed in M. pudica: one is the very rapid movement of the leaves when it is stimulated by touch, heat and other external factors, and the second is the very slow, periodical movement of the leaves called nyctinastic movement which is controlled by a biological clock (Ueda et al. [9]).

Sample Collection and Preparation
Fresh and wholesome parts of Mimosa pudica were collected during the month of October 2015, from Ndi-Ojigwe compound in Okoko Item, Bende Local Government Area of Abia State, Nigeria.The plant was identified and authenticated by Mr. I. Ndukwe in Plant Taxonomy Section of the Forestry Department of Michael Okpara University of Agriculture, Umudike, Nigeria.The fresh plant materials were dried under shade to prevent interference of uvradiation from the sun.Dried plant materials were powdered using electric blender.Then the powdered material was preserved in an air-tight container; ready for extraction.

Extraction and Isolation of Plant Material
Two (2) kg of the milled sample was percolated in 98% ethanol for 48 hours.Thereafter, it was filtered through Whatmann Filter Paper (No.42).
The filtrate was concentrated using the Digital Heidolph Rotary-evaporator (4000 series); collecting 48.9 g of crude extract.The crude extract was partitioned between CHCl 3 and water and a CHCl 3 -soluble fraction (16.3g) was obtained.The CHCl 3 fraction (10.0 g) was then partitioned between petroleum ether (at 60and aqueous methanol.The CHCl 3 fraction (5.0 g) was then subjected to column chromatography over silica gel (200 mesh) and eluted gradually with 100 mL of petroleum ether, then petroleum ether:CHCl

Antifungal Activity of Extracts from
Mimosa pudica

Preparation of extract stock solution
A stock of extract was prepared by dissolving 0.2 g of the plant extract in 2.0 mL of dimethyl sulphoxide (DMSO) to get a concentration of 100 mg/mL of the stock solution.This stock solution was diluted with sterile distilled water to give concentration of 50 mg/mL and 25 mg/mL.

Fungal media (Potato dextrose agar)
Potato slices (200 g) were boiled with distilled water.The potato infusion was used as water source of media preparation.Dextrose (20 g) was mixed with the potato infusion.Agar (20 g) was added as a solidifying agent.These constituents were mixed and autoclaved.The solidified plates were bored with 6-mm diameter cork borer.The plates with wells were used for the antifungal studies.

Preparation of discs
Filter paper discs (Whatmann No. 1) were sterilized in hot air oven inside glass Petri dishes at 160°C for 2 hours.Each disc was impregnated with 20 µl of the plant extract solution at three concentrations: 25 mg/mL, 50 mg/mL and 100 mg/mL.The discs were placed in an incubator at 40°C and left for 2 hours to dry.They were used immediately and the remaining discs were stored at 4°C.

Disc diffusion test
This was performed following the Kirby-Bauer method.Plates of Mueller Hinton Agar (MHA, Hardy Diagnostics, USA) were prepared according to manufacturer's instructions.The plates were dried in an incubator at 40°C for 30 min.using a sterile swab stick, standardized cells suspension containing an inoculums size of 5x10 8 CFU/mLwas aseptically spread on the agar surface.The discs of the extracts and antibiotics were placed on the inoculated plates of each test organisms.The plates were incubated at 35-37°C for 16-18 hours.The diameter of any clear zone of inhibition obtained around the discs was measured manually using a transparent ruler.

Antifungal Activity of the Plant Extract
The crude ethanolic extract and the isolated compound labeled MP-19 were tested against the fungal pathogens Aspergillus flavus and Trycophyton rubrum at concentrations of 25 mg/mL, 50 mg/mL and 100 mg/mL.The experiment was replicated three times for each extract and antibiotic.

Mimosa pudica
The results of the antifungal assay of the crude ethanolic extract of M. pudica (MP) that the extract exhibited (from being partially active to very active) antifungal activity against the tested microorganisms at all three different concentrations of 25 mg/mL, 50 mg/mL and 100 mg/mL.At 100 mg/mL the extract was very active against Aspergillus flavus.The zone of inhibition was recorded and presented in Table 1.
Table 2 shows the effect of the isolated compound labelled MP-19 on the microorganisms.The results of the experiment indicated that Aspergillus flavus and Trichophyton rubrum showed resistance against the isolated compound MP-19 from the crude ethanol extract of M. pudica.These results show that the isolated compound may not be responsible for the antifungal activity recorded in Table 1.
Compound MP-19 was eluted with chloroform and petroleum ether at the ratio of 50:50.Thin layer chromatography carried on MP-19 showed one spot (R F 0.41).

Analysis of
1 H-NMR revealed the presence of olefinic protons at ᵹH5.20, cluster of peaks between ᵹH0.8 to ᵹH2.35 clearly indicated the triterpenoid structure, and two methoxy protons signals at ᵹH4.058 and ᵹH4.239.Then the presence of two anomeric protons at ᵹH5.45 and ᵹH5.25 confirmed the presence of two sugars.The spectrum also showed the presence of aromatic protons atᵹH5.043-ᵹH5.315.

CONCLUSION
In the present study, a triterpenoid glycoside was isolated from Mimosa pudica ethanol crude extracts.At concentrations of 25 -100 mg/mL, the extract possesses antifungal activity (from being partially active to very active) against Aspergillus flavus and Trichophyton rubrum, indicating that the plant contains potential antimicrobial components for the therapy of infections.Further investigation should be done to purify and isolate more bioactive components responsible for most of its documented pharmacological activities in order to promote the use of the plant in drug discovery.

Fig. 1 .
Fig. 1.MP-19 13 C-NMR spectrum revealed the presence of six aromatic carbons at ᵹC 124.00, ᵹ126.08,ᵹ126.72,ᵹC 126.87, ᵹC 127.04 and ᵹC 127.22.Methylene carbons chemical shift were seen at ᵹC 21.55 -ᵹC 22.60, methane carbon peaks were observed at ᵹC 25.38 -ᵹC 33.17, while the signal at ᵹC 172.93 indicates the presence of carbonyl carbon, which is an integral part of the triterpenoid.Based on the chromatographic data, IR, NMR, COSY, DEPT, HMBC and HMQC, the structure of MP-19 was proposed as shown in above Fig. 1 with molecular formula C 35 H 46 O 16 .