Phytochemical screening, investigations & antimicrobial activity on the various extracts obtained from the aerial parts of Alternanthera sessilis (Joyweed) and Crepidium acuminatum (Jeevak)

Identification

Stems rooting at the nodes, cylindrical and faintly hairy, erect branches.

Leaves are opposite and clustered, simple type, lanceolate to spathulate in shape, obtute apex, entire margin and cuneate base.1, 2, 3, 4, 5

Inflorescence

White, Axillary spikes, Sessile, Dense,

Seeds/fruits

Fruit: Compressed, small and flat, enclosing the seed

Seeds: Orbicular, dark brown to black

Habit / Habitat

It is a Perennial herb. It typically grows on disturbed parts of wetlands and often observed in a species-rich association with other plants of variety of habitat such as flood-plain wetland, margins of river, streams, canals, ponds, ditches, and damp forest.

Habit / Habitat

Around the world it is found in Pakistan, Bhutan and Tibet between 1500-3100 mts elevation. In India found in Central and Eastern Himalaya from Uttarakhand to Assam and Sikkim up to an altitude of 3300 mts in alpine grassland, grassy hill sides, damp gullies and stony slopes. 11, 12

Traditional medicinal uses

Classical medicinal uses

Determination of minimum inhibitory concentration (MIC)

The extract were screened for their antibacterial activity in vitro by disc diffusion method 13 using S. capitis, S. mutans, P. mirabilis and B. fragillis as test organism. Agar cultures of the test microorganisms were prepared. Three to five similar colonies were selected and transferred to 5 ml broth with a loop and the broth cultures were incubated for 24 h at 37^oC and suspension was checked to provide approximately 10 10 colony forming units per ml. 0.1 ml of organism’s suspension were spread evenly on the agar plates. For screening, sterile 3 mm diameter disc (Whatman filter paper No. 1) were impregnated with different concentration till saturation, dried and placed in inoculated plates of Muller Hinton agar medium. DMSO solvent was used as negative control. The plates were incubated at 37^oC for

24 h. After incubation for 24 h, the results were recorded by measuring the zones of inhibition surrounding the disc and the lowest concentration of each extract which is showing i nhibition of growth of bacteria was determined as MIC. Penicillin (10µg/ml) was used as standard for bacteria.

Total resin content

A weighed quantity of sample (5 gm) is refluxed in acetone (3×200 ml) for six h. The extract was filtered and evaporated to dryness. The residue was dissolved in distilled water, warmed and filtered; the residue left was dissolved in solvent ether (2×200 ml). The solvent is evaporated and dried to constant weight at 105°C.

Total tannin content

The powdered sample (2 gm) was extracted with 100 ml distilled water for about 24 h. at room temperature. After 24 h. the mixture was filtered, followed by addition of 5 ml of saturated lead acetate. The addition of lead acetate to the mixture precipitates the tannins as lead tannate. The precipitate was washed with water and dried. Lead tannate obtained was suspended in ethanol, warmed and decomposed by bubbling in H₂S gas. Black precipitate of PbS was removed by filtration and the filtrate concentrated under reduced pressure. The residue obtained was then dissolved in 50 ml water followed by addition of 25 ml of 1% cupric acetate solution. The precipitate thus obtained was washed, dried and incinerated in a muffle furnace keeping all the material in silica crucible and weighed as cupric oxide. The tannin content was calculated by following formula (Ansari, 2010)

% Tannin =   (B-A) × 305 × 100/Weight of drug Where, A= Weight of silica crucible

B= Weight of silica crucible and material

Total fat content

Accurately weighed quantity of sample (3 gm) was extracted with anhydrous ether in a Soxhlet apparatus for 6 h. The extract was filtered into a clean dry previously weighed china dish. The extraction flask was rinsed with small quantity of ether, filtered and added to the weighed china dish. The solvent was evaporated and dried to constant weight at 105°C (Ansari, 2004).

Total alkaloid content

Accurately weighed 5 gm of the chloroform extract was mixed with 200 ml of 10% acetic acid in ethanol in 250 ml of a beaker and covered and allowed to stand for 4 h. This was filtered and extract was concentrated on a water bath to one quarter of original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the precipitation was complete. The whole solution was allowed to settle and the precipitates were collected and washed with dilute ammonium hydroxide and then filtered. The residue was the alkaloids, which was dried and weighed. 16, 17, 18, 19, 20

Saponin content

Saponin content of the sample was determined by double solvent extraction gravimetric method. 2 gm of the powdered sample was mixed with 50 ml of 20% aqueous ethanol solution. The mixture was heated with periodic agitation in water bath for 90 minutes at 55°C. It was filtered through Whatman filter paper. The residue was extracted with 50 ml of the 20% aqueous ethanol and both extracts were pooled together. The combined extract was reduced to about 40 ml at 90°C and transferred to a separating funnel where 40 ml of diethyl ether was added and shaken vigorously. Separation was done by partition during which the ether layer was discarded and the aqueous layer reserved. Re- extraction by partition was done repeatedly until the aqueous layer become clear in colour. The saponins were extracted with 60 ml of normal butanol. The combined extract were washed twice with 10 ml of 5% aqueous NaCl solution and evaporated to dryness in a pre-weighed evaporating dish. It was dried at 60°C in the oven and reweighed. Percentage saponins were calculated.

% Saponins= (W2 – W1) × 100/ Weight of sample

Where, W₁ = Weight of evaporating dish; W₂ = Weight of dish + sample.

Total carbohydrates content

The total Carbohydrates contents of the plant extract were determined according to the DNS procedure method (Miller, 1959) by some modification that is addition of 15 ml distilled water. Reagents: Potassium sodium tartrate (40% w/v) and Dinitrosalicylic acid (DNS) reagent were prepared, Prepared dilutions for glucose (standard) of 50, 100, 150….., 500 μg/ml.

Sample Preparation: 100 mg. of powder were prepared with 80% ethanol twice (5 ml each time). Collected supernatant and evaporated it. Then added 10 ml of distilled water to dissolve the sugars. 3 ml of solution was taken. To this added 3 ml of 3,5- Dinitrosalicylic acid (DNS reagent) and heated in boiling water bath for 5 min. 21, 22, 23, 24, 25, 26

Preparation of standard: 0-500μg/ml of standard dilutions of glucose was taken and added water to make final volume 3 ml. To this added 3 ml of DNS reagent and heated in boiling water bath for 5 minutes. Added 1 ml of sodium potassium tartrate followed by 15 ml distilled water. Kept for cooling and absorbance was taken at 510 nm.

Blank solutions: 3 ml of distilled water and 3 ml of DNS reagent. Heated in boiling water not Heat bath for 5 minutes. Added 1 ml of sodium potassium tartrate followed by 15 ml distilled water. Kept for cooling and absorbance was taken at 510 nm.

After taking the absorbance of standard dilutions, calibration curve was plotted. Reducing sugar content in drug was calculated by using standard calibration curve.

Crude fibre content

2 gm of the powdered drug was accurately weighed and extracted with petroleum ether. Then 200 ml of 1.25% of sulphuric acid was added to the marc and the whole mixture was boiled for 30 minutes under reflux. Then, the mixture was filtered and residue was washed with boiling water until free from acid. The residue was rinsed back with 200 ml of 1.25% of sodium hydroxide solution. The mixture was boiled for 30 minutes under reflux. The liquid was filtered and residue washed with boiling water until neutral, dried at 110°C to constant weight and then, incinerated to constant weight. The difference between the weight of the dried residue and incinerated residue was calculated with reference to the dried sample (Mukherjee, 2002).