Introduction

Infectious diseases caused by bacteria ,fungi, viruses and parasites are still risk to public health, due to evolvement of microbial resistance to synthetic drug.[4] About 80% people use tradition medicines, which compounds obtained from medical plant. Plant contains some organic compounds which show some physiological effect on human body. Phytochemical are secondary metabolites with obscure function .Phytochemical derived from any part of the plant

The family euphorbiaceae consists of 2000 species (Davis et al.,1988).The genus euphorbia is the largest genus of medical plant . E.milii is commonly known as “crown of thrown”.[1] The part of plants that grow above ground that used for make medicine. Fungi of the genus Aspergillus produce a toxic substance called aflatoxin, which contaminates crops (e.g., corn and peanuts) and causes human diseases. Aflatoxin has even been implicated as a contributing factor in liver cancer. Euphorbia milii flowers, when dried and processed as powder, inhibit the growth of Aspergillus. [1

Table 1: Taxonomical Classification

Kingdom Plantae
Division Magnoliophyta
Class Magnoliopsida
Order Malpighiales
Family euphorbiaceae
Genus Euphorbia
Species E.milii
Origin Madagascar
Flowering Yes
Fragrant Slightly
Growing case No extra care requires
Temperature 30-40⁰c
Vernacular names Crown of thrown, Christ plant, Christ thorn

Milin, an extract of Euphorbia milii latex, is a glycosylated serine protease (an enzyme that breaks down protein and has a sugar attached to it). Because it is more stable than most proteases, it will be useful to food processers and makers of detergents who have been using proteases in their operations. [1,3]

Phytochemical studies of E. milii revealed the Presence of Flavanoids, Terpenoids, Tannins.Flavanoids are yellow pigments,which occure in plant kingdom either in free state or as a glycosides or associated with tannins.These are known as anthoxanthins.[2].

Material and Methods

Collection of plat material and identification

Flower of Euphorbia milii was collected from Jahngirpara, Surat. Identification of plant material was done by Department of Botany, P. T. Science College, Athwalines,Surat

Aqueous extraction

20 grams of dried plant material was extracted in 100 ml water for 2 hour on rotary shaker. After 2 hour it was filtered through whattman filter paper-1 and centrifuged at 5000 rpm for 15 minute. The supernatant was collected and used for further phytochemical analysis.[4]

Ethyl acetate and petroleum ether extraction

20 grams of dried plant material was extracted in 100 ml of Ethyl acetate and Petroleum ether. The flasks were covered with aluminum foil and kept on rotatory shaker at 191 rpm for 24 hour at room temperature. After 24 hour the solution was filtered through whattman filter paper-1. The filtrates were collected and were concentrated up to dryness by keeping it in hot air oven at 35⁰C. The stock solution (0.5 mg/ml) of each extract was prepared in Dimethyl sulfoxide (DMSO).[1,4]

Methanol,n-hexen,chloroform fraction

10 grams of dried plant material was soaked in 25 ml methanol for 5 days and then subjected to repeated extraction with 25 3 until exhaustion of plant material. The obtained were then evaporate at below 55⁰c.crude methenolic extracts were suspended in water and successively partitioned with n-hexen and chloroform.[5,8]

Antimicrobial activity

Three strains of Gram-positive (Staphylococcus aureus(MTCC NO:737), Bacillus subtilis(MTCC NO:441), Bacillus megaterium(MTCC NO:2949), Entrococci(MTCC NO:439)) and three of Gram negative (Escherichia coli(MTCC NO:119), Proteus vulgaris(MTCC NO:426), Pseudomonas aeruginosa(MTCC NO:2297))bacteria were used[9], which were obtained from microbiology laboratory of Ramakrishna institute. Tests for susceptibility were determined using modified agar well diffusion method to test the antibacterial activity of the different solvent fractions. The nutrient agar was used as medium. All the cultures were taken in triplicates at incubation temperature of 37°C for 24 to 72 hours. In a sterile Petri-dish, test organism broth culture (0.1mL) was placed with sterile nutrient agar medium (20mL). Holes were bored in to the medium and each fraction was added (0.2mL). Amikacisn sulphate was the standard antimicrobial agent at a concentration of 2 mg /ml. it was incubated at 37°C for 24h. The diameter of the zone of inhibition of microbial growth in the plate was measured in millimeter (mm).[2,3,9]

Phytochemical screening

The chemical tests were performed on the water, hexane, chloform, ethyl acetate, petroleum ether extracts of E. milii followed by the standard procedures for identification of the chemical constituents.[1,5,10]

Test for alkaloids

Each fraction (0.2g) was warmed with 2% H₂SO₄ (2.0ml) for two minutes. The reaction mixture was filtered and few drops of Dragendrof’s reagent were added to filtrate. Orange red precipitation showed the presence of alkaloids moiety.

Test for tannins

Each extract in small quantity was mixed with water and heated on water bath and filtered. To the filtrate, few drops of FeCl₃ and a dark green solution was obtained which indicate the presence of tannins.

Test for anthraquinone

Each extract (0.5g) was boiled with 10% HCl for few minutes. The reaction mixtures was then filtered and allowed to cool. Equal volume of CHCl₃ was added to each filtrate along with few drops of 10% NH₃ and heated. Rose-pink color formation was obtained which indicate the presence of anthraquinones.

Test for glycosides

Each extract (0.6g) was hydrolyzed with HCl and neutralized with NaOH solution and few drops of Fehling’s solution A and B were added. Red precipitate were formed which indicate the presence of glycosides.

Test for reducing sugars

Extract was treated with Benedict’s reagent and boil for 5 minutes in water bath .Formation of an orange red precipitate indicated the presence of reducing sugars.

Test for saponins

Each extract (0.2g) was shaken with distilled water (5.0mL) and boiled. Frothing (appearance of creamy miss of small bubbles) was observed showed the presence of saponins.

Test for flavonoids

Each extract (0.2g) was dissolved in diluted NaOH and few drops of HCl were added. A yellow solution turned into colorless which indicate the presence of flavonoids.

Test for phlobatanins

Each extract (0.5g) was dissolved in distilled water and filtered. The filtrate was then boiled with 2% HCl solution. Red precipitates were obtained which showed the presence of phlobatanins.

Test for steroids

Acetic anhydride (2.0mL) was added to mixture of extract (0.5g) and H₂SO₄ (2.0mL) of each extract. The change of color from violet to blue or green in some samples showed the presence of steroids.

Test for terpenoids

Each extract (0.2g) was mixed with CHCl₃ (2.0mL) and carefully concentrated H₂SO₄(3.0mL) was added to form a layer.

Test for phenol

Treat the extract with 3ml of 10% lead acetate solution. A bulky white precipite indicate the presence of phenolic compounds.

Results

Phytochemical analysis

The primary Phytochemical screening of different extraction was done and ascertain bioactive compound are presence. The result show in Table-2.

Table 2: Phytochemical screening of n-hexane, chloroform, ethyl acetate, petroleum ether and water extraction

Phytochemical n-hexen Chloform Ethyl acetate Petroleum ether Aqueous
Alkaloids
Tannis
Antraquinone + + + + +
Reducing sugar
Sapponis
Flavonoids + + + + +
Phlobatanins
Steroids
Terpenoids + + + + +
Phenol + + + + +

‘+’= presence and ‘-’=absence

Antimicrobial activity

Preliminary antibacterial screening was done against some gram positive and gram negative organism using agar well diffusion method. The various zones of inhibition obtained are show in Table-3.

Table 3: Inhibition Zones produced by sample on both gram positive and gram negative organisms

Sr.No Name of bacteria Sample Inhibition zone(mm)
1 Escherichia coli hexen 19mm
Chloroform 15mm
Ethyl acetate 17mm
Petroleum ether 15mm
Aqueous 19mm
2 Proteus vulgaris hexen 15mm
Chloroform 14mm
Ethyl acetate 10mm
Petroleum ether 10mm
Aqueous 6mm
3 Pseudomonas aeruginosa hexen 18mm
Chloroform 13mm
Ethyl acetate 13mm
Petroleum ether 14mm
Aqueous 20mm
4 Staphylococcus aureus hexen 17mm
Chloroform
Ethyl acetate 16mm
Petroleum ether 14mm
Aqueous 20mm
5 Bacillus subtilis hexen
Chloroform
Ethyl acetate
Petroleum ether
Aqueous
6 Bacillus megaterium hexen
Chloform
Ethyl acetate
Petroleum ether
Aqueous
7 Entrococci hexen 10mm
Chloform 12mm
Ethyl acetate 16mm
Petroleum ether 16mm
Aqueous 6mm

‘-‘ = ”no zones of inhibition”.

Image 1

Figure 1: n-hexen , chloform and ethyl acetate fraction

Image 2

Figure 2: Petroleum ether and aqueous extraction

Image 3

Image 4

Discussion

The present study reveals that the phytochemical constituents of flower extract of Euphorbia milii include antraquinone,flavonoids,trepenoids,phenol which are similar to the Rauf .A.,et al.(2014) except steroids and protein.result of antimicrobial activity of different solvent extraction against Escherichia coli, Staphylococcus aureus, Proteus vulgaris gave greter activity capare then the result of the devanaboyina .N.,et.al.(2015).Bacillus subtlis has not get antimicrobial activity from any solvent extraction as similar to the Dr Nizzam.U.,et,al.(2017)

Conclusion

The present study revealed that slovent extraction shows antimicrobial activity.Tested extract are directly attributed to presence of various phtoconstituent.The activities might be due to the prensence of bioactive compunds or certain inhibitory compund or due to the synergist effect of bioactive compund.Current investigation suggests that reserch needed to isolate these bioactive molecules responsible for the antimicrobial activity of the Euphorbia milii.