INTRODUCTION:

Polianthes tuberosa Linn [Family: Amaryllidaceae] distributed in hotter parts ,mainly Mexico or the andes of South America Besides its popularity as an ornamental garden plants with beautiful blossoms. Throughout India wild as well as cultivated especially in Karnataka as an ornamental in gardens in Dehradun. The tuberose is a night blooming plant thought to be a native of Mexico along with other species of polianthes. The Azteas called it bone flower. It grown as an ornamental plant for its fragrant flowers. The bulb of young plant is 5-10 cm in diameter, narrowed into the neck and clothed with old leaf sheaths. An erect herb, elongated spikes up to 45cm long with stout tuberous stock. Leaves are long, bright green, clustered at the base of the plant and smaller clasping leaves along the stem. The flowers are clustered, scented waxy white funnel shaped^1-6. Preliminary phyto chemical screening was performed as per the standard procedure. The vacuum dried extracts were screened for anti-inflammatory and anti microbial activites.

MATERIALS AND METHODS:

The details regarding the description and identification of plant were already given. The plant polianthes tuberosa linn was collected from Madurai, during the month of may- june. That was identified by Dr. Stephen, MSc. Ph.D, Department of Botany, The American College , Madurai -2. The leaves were cut off and dried in shade for 45 days. Then the shade dried leaves was made in to a coarse granules and was used for different investigation.

Extraction:

The leaves of polianthes tuberosa linn were dried in the shade. Then the shade dried leaves were powdered to get a coarse powder. About 350 gms of dried course granules of polianthes tuberosa leaves was soaked into 4 liter of petroleum ether for 2 days. Then it was extracted first with petroleum ether at 40 - 60 ^OC by continuous hot percolation method using R.B flask apparatus. The extraction was continued for 72 hours. The petroleum ether extract was filtered and concentrated to a dry mass by distillation (16 gm).A dark blackish green residue was obtained. The marc left, after petroleum ether extraction was taken and then subsequently extracted with ethanol for 72 hours. The ethanolic extract was taken and then filtered and concentrated to the dry mass .A dark brown residue was obtained (20 gms)

Physico- Chemical standards:

Physico- chemical parameters of the powdered drug such as ash value, extractive value, loss on drying were performed according to the method^7-12.Extracts were prepared by various solvents by standard methods and percentage of dry extract was calculated in terms of air-dried leaf powder. (Table 1, 2, 3)

Table 1: Ash values

S. no	Type of ash	Results
1.	Total ash	12.18 % w/w
2.	Acid insoluble ash	0.76 % w/w
3.	Water soluble ash	2.13 % w/w

Table 2: Extractive value, Percentage yield and colour of extracts

Solvent used	Percentage yield	Colour of extract
Petroleum ether	1.7	Green
Ethanol	9.8	Blackish green

Table 3. Loss on drying

Loss on drying

7.41%

Fluorescence characteristics:

When physical and chemical parameters are inadequate as it often happens with the powdered drugs, the plant material may be identified from their adulterants on basis of fluorescence study ^13,14. (Table 4)

Behavior of leaf powder with different chemical reagents:

Behaviour of leaf of polianthes tuberosa with different chemical reagents was performed to detect the occurrence of phytoconstituents along with colour changes under ordinary daylight by standard method¹⁵ (Table 5)

Determination of Saponin:

According to the results obtained from positive foaming test and high foaming index¹⁶ of leafs of polianthes tuberosa study was carried out for the estimation of total saponin content ^17,18 (Table 6).

Preliminary phytochemical investigation:

The qualitative chemical test of various extracts of polianthes tuberosa was carried out using standard procedure^19-22. Glycosides, Phytosterols, Saponins, Flavonoids and Alkaloids are present in petroleum ether and methanol extracts.

Thin Layer Chromatography: ^23-25

About 30gms of silica gel – B was weighed out and it was shaken with 100ml of water to form a homogenous suspension. The suspension was poured into a thin layer chromatography applicator which was adjusted to 0.25mm thickness. 20 to 40 Carrier plates (20.5cm) were laid down for air drying. The plates were kept in the hot air oven at 110°C for one hour to activate the silica gel – G. The plates were stirred in a dry atmosphere and used whenever required. By using the capillary tube the extracts are spotted on the T.L.C plates 2cm above the bottom and in the chromatogram in various solvent systems for different compounds. The spots are developed in solvent system were identified by means of different spraying reagents.

Screening of Anti-Inflammatory activity:

Anti-inflammatory Activity of polianthes tuberosa was studied in adult albino rats of either sex weighing between 120-150gms by Carrageenan Induced hind paw oedema method^26-28. Animal were divided into four groups containing 6 animals per each.

Table values shows that ethanolic extract of polianthes tuberosa exhibited significant anti - inflammatory activity than petroleum ether.

Group I (control) was given a 1% Sodium Carboxy methyl Cellulose solution. Group II of animals received 10mg/kg of Diclofenac sodium which was considered as standard. Group III and Group IV were treated with 200 mg/kg of petroleum ether and ethanol extracts respectively dissolved in Sodium Carboxy methyl Cellulose solution. Doses were given orally with the help of an oral catheter. 0.1 ml of 1% solution of carragenan was administered to the rats into the plantar surface of the right hind limb to induce paw oedema. Paw volume was measured plethysmographically after 1h, 2h and 3h, 4h of carrageenan injection and paw swelling in groups of drug treated were compared with control. Percentage inhibition of oedema was calculated by using the following formula

%inhibition = x 100

Where,

Vt- means increase in paw volume in rats treated with test compounds

Vc-means increase in paw volume in control group of rats.

Table 4: Fluorescence characteristics of leaf extract of polianthes tuberosa

Powder + Reagent	Color observed in Ordinary light	Color observed under Ultra violet light Short (254 nm)	Color observed under Ultra violet light Long (365 nm)
Powder	Brown	Green	Green
Powder+ 1N NaOH in methanol	Greenish black	Green	Green
Powder+ 1 N NaOH in water	Brownish green	Green	Black
Powder++ 1 N HCl	Brownish yellow	Green	Black
Powder+50% HNO₃	-	Light Green	Black
Powder+50% H₂SO₄	Slight brown	Green	Black
Powder+Methanolic NaOH.dried+ nitrocellulose in aceticacid	Yellowish green	Dark Green	Black
Powder+ 1N NaOH + nitrocellulose in aceticacid	Dark brown	Light Green	Greenish Black

Table 5: Behavior of leaf extract of polianthes tuberosa

Reagent	Color / ppt	Constitituent
Powder	Green	-
Powder + con. H2So4	No brown colour	Carbohydrate absent
Powder + aqueous Fecl3	No Bluish black colour	Tannin absent
Powder + Iodine solution	No black	Starch absent
Powder + Aqs. Hgcl₂	Blue colour is produced	Alkaloids present
Powder + picric acid	Yellow colour is formed	Alkaloids present
Powder + Mg Hcl	Mangoe colour is produced	Flavonoids present
Powder + aqueous AgNo₃	Ppt is not formed	Protein absent
Powder + ammonia solution	Pink colour	Cardiac glycoside present
Powder + Aqs. KOH	Pink colour	Cardiac glycoside present
Powder + Aqs. Na nitrite	Red colour	Phytosterols present
Powder + Water ( shaking )	Foam is produced	Saponins present

Table 6: Results of Quantitative estimation of leaf extracts of polianthes tuberosa

S. No	Estimation		Results
1.	Foaming index		More than 1000
2.	Total saponin content	Method I Method II	9.5% w/w 10.4 % w/w

Table 7: Phytochemical constituents of Thin Layer Chromatography

S. No	Active constituents	Mobile phase	Spraying reagent	Inference
1.	Glycosides	Ethyl acetate :Pyridine:Water 5:1:4	Chloroform	Orange
2.	Saponins	Butanol:Water 1:1	Concentrated HCl	Dark brown
4.	Phytosterols	Hexane : Ethylacetate 1:1	Stannic chloride reagent	Orange brown round
5.	Flavonoids	Butanol : Aceticacid : Water : Ether 9:6:1:3	Phenol sulphuric acid	Greenish brown
6.	Alkaloids	Methanol:Ammonium hydroxide 5:5	Dragendroff’s reagent	Orange brown

Table 8 : Anti inflammatory activity of polianthes tuberose carrageenan induced paw edema method

S. no	Dose	Increase In Paw Volume in ml (MEAN ± SEM)	% Inhibition of Paw Volume
1	Normalsaline 10ml/kg	4.52 ± 0.310	-
2	Diclofenac sodium 10ml/kg	1.09 ± 0.011	75.8 %
3	Petroleum ether extract 200 mg/kg	2.12 ± 0.018	53.1 %
4	Ethanol extract 200 mg/kg	1.48 ± 0.120	67.2 %

Values are expressed as mean ± SEM. Values are significantly different from control (P < 0.01)

Table 9 Antimicrobial activity of ethanol extract of polianthes tuberosa .

Organism Diameter of zone of inhibition (mm)

Ethanol extract

Bacteria Gram(+) A B C Standard

(Amikacin) (10µg/ml)

Bacillus subtilis 9.5±0.5 11.3±1.3 15.6±1.2 20.5±0.5

Staphylococcus aureus 11.4±0.8 13.6±1.2 16.7±0.6 * 21.7±1.6

Micrococcus luteus 8.5±1.2 10.2±0.6 13.5±1.2 19.3±0.7

Bacteria Gram(─)

Escherichia coli NI 9.7±1.5 12.5±1.3 18.6±1.3

Psuedomonos aeruginosa 8.5±1.2 10.3±0.8 13.4±0.5 20.2±0.2

Salmonella typhi NI 7.2±1.4 8.5±0.5 14.5±0.6

Fungi Standard

(Griseofulvin) (20µg/ml)

Aspergillus niger 7.3±0.7 10.1±1.2 12.5±0.3 19.2±0.7

Candilla albicans 12.8±1.1 14.6±1.7 20.1±0.7 24.1±1.2

All the values indicate mean ±SD (n=3) the concentrations were A : 100µg/disc, B :250µg/disc, C :500µg/disc. NI- No Inhibition Zone.

statistical value *p<0.05 when compared to standard.

Microorganisms and Media:

The following bacterial strains used were staphylococcus aureus (ATCC 25923), Bacillus Subtilis (ATCC 6633), Micrococcus luteus (ATCC 10240) Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 7853) and Salmonella typhi (ATCC 43579). The fungal species used were Candida albicans (ATCC 10231) , Aspergillus niger(ATCC 16404) , for the present study. The bacterial and fungal cultures were maintained on Muller Hinton Agar Medium and Sabouraud dextrose agar slants respectively, which were stored at 4º C. Eight microorganisms maintained on nutrient agar base were used to assess the antimicrobial activity of the plant extracts. The fungi were maintained on Sabouraud dextrose agar, which is often used with antibiotics for the isolation of pathogenic fungi.

Antimicrobial screening:

The anti bacterial activity of the extract was performed by disc diffusion method on nutrient agar plates^29-31. Three to five similar colonies were selected and transferred to 5ml broth with a loop and the broth cultures were incubated for 24 h at 37°C. The ethanol extract was dissolved in DMSO with a magnetic stirrer. For screening, sterile 6-mm diameter filter paper discs were impregnated with 100-500µg of the ethanol extract of polianthes tuberosa and then placed in Muller Hinton Agar Medium. The inoculum for each organism was prepared from broth cultures. The concentration of cultures was 1×10⁵ colony forming units/ ml. The results were recorded by measuring the zones of growth inhibition surrounding the disc indicate the presence of antimicrobial activity. All the data regarding antimicrobial activity are the average of triplicate analyses were recorded. (Table 2). The antibacterial Amikacin (10µg/ml) and antifungal Griseofulvin (20µg/ml) was used as reference standards as recommended by the National Committee for clinical laboratory standards³². The selected microorganisms included Bacillus subtilis ,Staphylococcus aureus, Micrococcus luteus ,Escherichia coli Pseudomonas aureginosa and Salmonella typhi at concentrations of 100, 250 and 500 µg/disc respectively. The antifungal activity against Aspergillus niger, Candida albicans at the same concentrations.

Statistical analysis:

Data are reported as the mean ± SD of three measurements. Statistical analysis was done by one way ANOVA followed Newmann level’s multiple range test. A.P value < 0.01 was considered as significant³³.

RESULTS AND DISCUSSION:

The result of the experiments are shown in Table no: 1 in this acute inflammation model ethanolic extract of polianthes tuberosa (200 mg /kg) and the standard drugs produced significant inhibition of paw oedema as compared to the control . The pet ether extract was found to be less effective than ethanolic extract. The results were found to be highly significant (P< 0.01) in comparison to control. Three distinct phases are observed during inflammation which are the histamine and serotonin released in the first phase, Kinin and Prostoglandin are released in the second and third phases respectively³⁴. The aqueous extract of polianthes tuberosa of 500 mg/kg body weight caused a significant inhibition only during the 3^rd h ( the phase of prostaglandin release), whereas the inhibiton it caused at other times was insignificant. Carrageenin induced hind paw oedema in the standard experimental model of acute inflammation. Carrageenin in the phlogistic agent of choice for testing anti-inflammatory drugs as it is not known to be antigenic and is devoid of apparent systemic effects. The polianthes tuberosa extract produced significant inhibition of Carrageenin induced paw oedema. The inhibition was however less than that of the standard drug.

The antimicrobial activity was observed (Table 2) that the ethanol extract showed antibacterial activity against all the tested organisms. However no activity was seen against Salmonella typhi at 100 µg/ml concentration. It was observed that ethanol extracts exhibited antifungal activity against Aspergillus niger, Candida albicans in a dose dependent manner. Tested extracts at higher concentration exhibits comparable antimicrobial activity with that of standard drugs. Disc diffusion methods are used extensively to investigate the antibacterial activity of natural substances and plant extracts. These assays are based on the use of discs as reservoirs containing solutions of the substances to be examined. In the case of solutions with a low activity, however, a large concentration or volume is needed. Due to limited capacity of discs, holes or cylinders are preferably used. Most of the bacterial species and the fungal species were inhibited by the plant extract as shown in Table 2. In this study, eight different bacterial and fungal species were used to screen the possible antimicrobial activity of EEPT. It showed a broad spectrum of activity against all the bacterial strains at the tested concentration of 100, 250, 500 µg/ml. Amikacin (10µg/ml) and Griseofulvin (20µg/ml) were used as positive controls for bacteria and fungi, respectively. As reported earlier secondary metabolites like flavonoids, saponins are likely responsible for the observed antibacterial activity of plants^35-37. The presence of said constituents in the methanol extract of polianthes tuberosa as found in phytochemical test may be responsible for the antibacterial activities.

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