Phytochemical and Antimicrobial Studies of Medicinal Plant Costus Speciosus ( Koen . )

The present paper deals with the phytochemical and antimicrobial screening of therapeutic importance from Costus speciosus (Koen.), an important medicinal plant. The study involves the preliminary screening and qualitative HPTLC separation of secondary metabolites from the rhizome of Costus speciosus (Koen.). The in vitro antibacterial activity was performed against a few pathogens viz. E. coli, Staphylococcus aureus, Klebsiella pnuemoniae and Pseudomonas aeruginosa. The generated data has provided the basis for its wide use as the therapeutic both in traditional and folk medicine.


Introduction
Plants have an almost limitless ability to synthesize aromatic substances, mainly secondary metabolites of which 12000 have been isolated, a number estimated to be less then 10% of the total 1.These substances serve as molecules of plant defense against predation by microorganisms, insects and herbivores and at the same time also exhibit medicinal properties for treating several ailments 1 .The steroidal sapogenin, diosgenin has been reported from the rhizome of costus speciosus (Koen.).Natural products of higher plants may give a new source of antimicrobial agents with possibly novel mechanism of action [2][3][4][5] .In recent years, multiple drug resistance has developed due to indiscriminate use of existing antimicrobial drugs in treatment of infectious diseases 6 .In addition to this, antibiotics are sometimes associated with adverse effects on the hosts like hypersensitivity.Therefore, there is a need to develop alternative antimicrobial drug for the treatment of infectious disease from other sources, such as plants 7 .Natural products of higher plants may be a new source of antimicrobial agents possibly with novel mechanism of action 5 .The storage organs of higher plants show great biological activities.Chemical substances that produce definite physiological actions on human bodies accumulate in storage organs of the plants.The most important of these bioactive compounds are alkaloids, flavanoids and phenolic compounds 8 .
In the present study was concentrated on the preliminary screening, qualitative screening of metabolites and antibacterial activity from the rhizomes of Costus speciosus.

Preliminary screening of secondary metabolites
The shade dried plant material was powdered using mixer grinder and subjected to soxhlet extraction with petroleum ether, chloroform, 95% ethanol and distilled water for 18 h in the order of increasing polarity of solvents.The condensed extracts were used for preliminary screening of phytochemicals such as alkaloids (Wagner, Mayers and Dragendorff's tests), flavonoids (Shinoda and NaOH tests), cardiac glycosides (Keller-Kiliani, conc.H 2 S0 4 tests), saponins (foam and haemolysis tests), sterols (Liberman-Burchard and Salkowski tests) and tannins (gelatin test) were carried out [9][10] .

Chromatographic conditions
Chromatography was performed on silica gel 60 F 254 HPTLC plates (10 x 20 cm; 0.25 mm layer thickness).Samples and standard compounds were applied to the layer as 8 mm wide bands positioned 10 mm from the bottom of the plate, using an automated TLC applicator Linomat IV (Camag, Muttenx, Switzerland) with nitrogen flow providing delivery from the syringe at a speed of 10 s/µL.

Anthra-glycosides, arbutin, bitter principles and flavanoids
Powdered drug (1 g) was extracted by heating on a water bath for 15 minutes with 5 mL methanol.2 µL and 10 µL of the filtrate were applied to the chromatogram.The bitter principles spots were separated using solvent system: 5% acetic acid and anisaldehyde H 2 SO 4 as spray reagent 11 .

Alkaloids
The powdered rhizome were wet with a half diluted NH 4 OH and lixiviated with EtOAc for 24 h at RT.The organic phase is separated from the acidified filtrate and basified with NH 4 OH (pH 11-12).It was extracted with chloroform (3X), condensed by evaporation and used for chromatography.The alkaloid spots were separated using the solvent mixture chloroform and methanol (15:1).The colour and R f values of the separated alkaloids were recorded both under ultraviolet (UV 254 nm) and visible light after spraying with Dragendorff s reagent 11 .

Saponins
Two grams of powdered rhizome were extracted with 10 mL 70% EtOH by refluxing for 10 min.The filtrate was condensed, enriched with saturated n-BuOH, and thoroughly mixed.The butanol was retained, condensed and used for chromatography.The saponins were separated using chloroform, glacial acetic acid, methanol and water (64:34:12:8) solvent mixture.The colour and R f values of these spots were recorded by exposing chromatogram to the iodine vapours 11 .

Cardiac-glycosides
Powdered drug (1 g) was mixed with 5 mL of 50% methanol and 10 mL of 10% lead(II) acetate solution and then heated for 10 minutes on water bath.The cooled filtrate was extracted with 2 separate 10 mL quantities of dichloromethane (DCM).The combined DCM extracts were completely evaporated.The residue was dissolved in DCM: methanol (1:1). 2 µL and 10 µL of this solution was applied to the chromatogram.The cardiac-glycosides spots were separated using solvent mixture.Solvent system of ethyl acetate: methanol: water (10:1.4:1)and methanolic H 2 SO 4 as spray reagent 12 were used.

Essential oils, coumarins, phenols carboxlic acids and valepotriates
Powdered drug (1 g) was extracted by heating under reflux for 15 minutes with DCM.The filterate was evaporated to dryness and the residue was dissolved in 1 mL toluene, 2 µL and 10 µL of the filtrate were applied to the chromatogram.Best solvent system used was toluene: ethyl acetate (9.3:0.7) and 5% vanillin in H 2 SO 4 as spray reagent 11 .

Steroids
Air dried plant powder was extracted with pet ether (40-60 o C).Hot methanol was added to the green coloured extract which was obtained.This was then evaporated to reduce the volume.The steroids spots were separated using solvent mixture.DCM:diethyl ether: methanol: water (7.7:1.5:0.8:0.12) and methanolic H 2 SO 4 as spray reagent 11 .

Flavanoids
One gram powdered rhizome were extracted with 10 mL methanol on water bath (60 °C / 5 min).The filtrate was condensed by evaporation, a mixture of water and EtOAc (10:1 mL) was added and mixed thoroughly.The EtOAc phase thus retained was used for chromatography.The flavanoids spots were separated using chloroform and methanol (19:1) solvent mixture anisaldeyde-sulphuric acid reagent.The colour and R f values of these spots were recorded under ultraviolet (UV 254 nm) light 11 .

Sterols
Two grams of powdered rhizome were extracted with l0 mL methanol in water bath (80 °C /15 min).The condensed filtrate was used for chromatography.The sterols were separated using chloroform, glacial acetic acid, methanol and water (64:34:12:8) solvent mixture.The colour and R f values of these spots were recorded under visible light after spraying the plates with anaisaldeyde-sulphuric acid reagent on heating at 100 0 C for 6 min 11 .

Antimicrobial screening
Extraction of plant material was prepared by cold percolation method.The air-dried and powered plant material 5 g of each was soaked in 50 mL methanol and kept for 48 h with intermittent shaking.The plant extract were filtered through Whatman no. 1 filter paper.The filtrated were dried until a constant dry weight of each extract was obtained.The remaining part of the plant residue was dried and soaked in 50 mL distill water for 48 h sand extract was collected as described earlier.Each extract was dissolved in 1 mL dimethyl sulfoxide (DMSO) and 20 µL of each sample was taken for experiment.

Antimicrobial assay
The methanolic and water extract of plant were screened against four bacteria.The test organisms E. coli, Staphylococcus aureus, Klebsiella pnuemoniae, Pseudomonas aeruginosa were obtained from Department of Microbiology, Institute of Science, Mumbai.Microorganisms were maintained at 4 ºC on nutrient agar slants.The modified agar disc diffusion method 13 was used to screen the antimicrobial activity.Sterile filter paper discs of 6 mm diameter impregnated with 20 µL extract solution equivalent to 4 mg of the dried extract and after evaporation placed on the surface of the inoculated agar plate and the compound was allowed to diffuse for 5 minutes and plates were kept for incubation at 37 °C for 24 h.At the end of incubation, inhibition zones around the disc were measured with transparent ruler in mm (Table 1).The study was performed in triplicate.Amikacin was used as control against all pathogens

Results and Discussion
The curative properties of medicinal plants are perhaps due to the presence of various secondary metabolites such as alkaloids, flavanoids, glycosides, phenols, saponins, sterols etc.The successive extracts of rhizome have revealed the presence of alkaloids, flavanoids, cardiac glycosides, saponins, sterols and tannins (Table 2).Thus the preliminary screening tests may be useful in the detection of the bioactive principles and subsequently may lead to the drug discovery and development.Further, these tests facilitate their quantitative estimation and qualitative separation of pharmacologically active chemical compounds 1 .

Bitter principle
The HPTLC chromatogram can be observed best at any wavelength (254 nm, 366 nm).There is no necessary need for derivatisation since compounds are seen best separated before derivatisation.After derivatisation good band separation was seen under visible light and at 366 nm.The major compounds separated was seen at R f =0.03, 0.07, 0.26, 0.34, 0.48, 0.53, 0.63, 0.79, 0.89 and 0.95 (Track 1 at 366 nm, Figure 1).Since the compound is highly polar in nature it can be seen just below the solvent front.Due its nature of polarity the chromatogram can further be used for preparative HPTLC.The sample concentration 10 µ L is found to be more than suitable as compared to 5 µ L.

Alkaloids
The HPTLC chromatogram can be observed best only in wavelength 366 nm, before derivatisation.There is no need to derivatise since compounds are not seen after derivatisation.A single band of alkaloid is seen to be separated before derivatisation at 366 nm.The compounds separated was seen at R f =0.02 (Track 1 at 366 nm, Figure 2).However there is still need for improvements and modification in the process of extraction in order to optimize the concentration of separation of bands.The compound is non polar in nature.The sample concentration 10 µL is found to be more than suitable as compared to 5 µL.

Saponins
Saponins was best observed at visible light after derivatisation but not seen under visible light before derivatisation.It is necessary to derivatise since compounds are seen well separated after derivatisation.The major compounds separated was seen at R f =0.30, 0.45, 0.49, 0.53, 0.59, 0.79, 0.91, 0.93 and 1.0 (Track 1 at 366 nm, Figure 3).Since the compound is highly polar in nature it can be seen just below the solvent front.Due its nature of polarity the chromatogram can further be used for preparative HPTLC.The sample concentration 10 µL is found to be more than suitable as compared to 5 µL.

Cardiac glycosides
The HPTLC chromatogram for cardiac glycoside was best observed at 366 nm before derivatisation.There is no necessary need for derivatisation since compounds are seen best separated before derivatisation.After derivatisation good band separation is seen at 366 nm (Track 1 at 366 nm, Figure 4).Since the compound is highly polar in nature it can be seen just below the solvent front.The sample concentration 10 µL is found to be more than suitable as compared to 5 µL.

Essential oils, coumarin, phenols and carboxlic acid
Essential oils, coumarin, phenols and carboxlic acid was found to be present.The chromatogram can be observed at 254 nm and 366 nm before derivatisation and under visible light after derivatisation.No specific need for derivatisation, as they are seen to be best separated before derivatisation.The major compounds separated was seen at R f =0.63, 0.82 and 0.95 (Track 1 at 366 nm, Figure 5).However there is still need for improvements and modification in the process of extraction in order to optimize the concentration of separation of bands.Since the compound is highly polar in nature it can be seen just below the solvent front.Due its nature of polarity the chromatogram can further be used for preparative HPTLC.The sample concentration 10 µL is found to be more than suitable as compared to 5 µL.

Anthraquinones
The HPTLC chromatogram for anthraquinones was best observed at 366 nm before and after derivatisation.There is no necessary need for derivatisation since compounds are seen best separated before derivatisation.The major compounds separated was seen at R f =0.02, 0.05, 0.31, 0.33, 0.44, 0.54, 0.65, 0.66, 0.85 and 0.89 (Track 1 at 366 nm, Figure 6).The sample concentration 10 µL is found to be more than suitable as compared to 5 µL.

Steroids
The HPTLC chromatogram for steroids was best observed at 366 nm before and after derivatisation.There is need for derivatisation since the bands of the compound separated appears prominent after derivatisation.The major compounds separated was seen at R f =0.27, 0.47 and 0.68 (Track 1 at 366 nm, Figure 7).However there is still need for improvements and modification in the process of extraction in order to optimize the concentration of separation of bands.

Flavanoids
The HPTLC chromatogram can be best observed under fluorescence 366 nm before and after derivatisation.The sample concentration (5 µL) is sufficient to generate the compounds.The major compounds separated was seen at R f =0.56, 0.60, 0.74, 0.78, 0.84 and 0.88 (Track 2 at 366 nm).

Sterols
Sterols were found to be best observed at 254 nm and 366 nm before derivatisation and at 366 nm and under visible light after derivatisation with optimum sample application of 5 µL.The major compounds separated was seen at R f =0.02, 0.07, 0.20, 0.38, 0.75, 0.81 and 0.86 (Track 2 at 366 nm, Figure 8).

Antimicrobial studies
The aqueous extracts appear to have antibacterial activity only against Staphylococcus aureus.This is interesting in that the traditional method of treating a bacterial infection was by administering a decoction of the plant or apart there by boiling it in water; our results are in accordance to the traditional system of administration.The methanolic extract did not show inhibitory activity against any bacteria), this may be because the active compound(s) may be present in insufficient quantities in the crude extracts to show activity with the dose levels employed 14 .Lack of activity can thus only be proven by using large doses 15 .Alternatively, if the active principles are present in high enough quantities, there could be other constituents exerting anatagonistic effects or negating the positive effects of the bioactive agents 16 .With no antimicrobial activity, extracts may be active against other bacterial species, which were not tested 17 .
The data generated from these experiments have provided the chemical basis for the wide use of this plant as therapeutic agent for treating various ailments.However, there is need to further carry out advanced hyphenated spectroscopic studies in order to elucidate the structure of these compounds.Furthermore, this data may be handy in probing of biochemistry of this plant in the future.

Table 1 .
Inhibition zones (in mm) in accordance to test microorganisms by agar disc