Green synthesis of copper nanoparticals from the flowers of Pandanus tectorius

Introduction

The Research work in the field of Nanotechnology is increasing because it plays an important role in many fields. Nanoparticles are particles that exist on a nanometer scale (below 100nm in at least one dimension). Nanotechnology was founded by Richard Feynman. They possess physical properties such as uniformity, conductance or special optical properties that make them desirable in science and biology. Various methods of synthesis have been reported they are Biological, Physical, Chemical, Vacuum vapor deposition etc. Biological synthesis of Nanoparticles is the best way and it also reduces the toxic substance which affects human and environment. A Nanometer is one thousand millionth of a meter. In this study deals with the synthesis of copper nanoparticles by using Pandanus tectorius. It belongs to the family Pandanaceae and commonly known as Screw pine. This consists of palm-like often branched trees or shrubs with tristichous long leaves spinous at the margin, apex and often at the keel or back of the mid-rib. The flowers are minute and lack perianths. Male flowers contain numerous stamens with free or fused filaments. Female flowers have a superior ovary, usually of many carpels in a ring, but may be reduced to a row of carpels or a single carpel. It is otherwise called as Kewra in hindi and Thalampoo in tamil. Flower buds and inner parts of leaves are eaten as vegetable, Anthers are use to treat headache, earache.Oil is used for application to scalp to treat alopecia and improve the luster of hair. Oil of flower-Used as nasal drops to treat convulsions due to epilepsy, flowers used for making perfumes and used to make garland or wreath.

Materials and Methods

Collection of Pandanus tectorius

The flowers of PANDANUS TECTORIUS were bought from Koyambedu market. The flowers were washed, cut into strips and was shade dried for 2 to 3 weeks. Then it was powdered and stored in air tight containers.

Phytochemical Analysis

Cold Extraction: 5gms of flower powder (Pandanus tectorius) was dispensed in 30ml of solvents. Aqueous -6ml, Chloroform -6ml, Ethanol -6ml, N-hexane -6ml, Acetone -6ml. It was allowed to soak for 72 hrs in a plant tissue culture bottle. Then the extract was collected by filtering with filter paper.

Soxhlet Extraction: 5gms of flower powder (Pandanus tectoriusi) was taken and 250ml of solvents was added for each. Aqueous -50ml, Chloroform -50ml, Ethanol -50ml, N-hexane -50ml, Acetone 50ml.

The solvents were poured in a soxhlet reservoir and the temperature was set to 50 . c. The powder was added over the muslin cloth and was made into a bag and placed inside the thimble.

Qualitative Analysis

Quantitative Analysis

Estimation for Alkaloid: In 1 ml of sample 2 ml of dragendroff's reagent was added it was centrifuged at 10,000 rpm for 10

International Journal of Agriculture and Food Science minutes. 1ml of supernatant was taken. 3% (0.5ml) of Thiourea solution was added and the reading were taken in spectrophotometer at 435nm.

Estimation for Tannins:

Folin & Ciocalteu's Method. Estimation of Carbohydrates: Phenol sulphuric acid method Estimation of Terpenoids: 0.1g of sample powder was macerated with 5ml of Ethanol and filtered. To the filtrate 2.5ml of 5% of aqueous Phosphomolybdic acid solution was added and 2.5ml of Concentrated Sulphuric acid was gradually added and mixed. The mixture was left to stand for 30 minutes and then made upto 12.5ml with Ethanol. The absorbance was taken at 700nm.

Estimation of Proteins: Bradford's Method

Estimation of AMINO Acid: Ninhydrin Method Estimation of Cardiac Glycosides: Cardiac glycoside content in the sample was evaluated using Buljce's reagent. Powder sample (1mg) was soaked in 10ml of 70% ethanol for 24 hrs and then filtered. The extract obtained was then purified using lead acetate and di sodium hydrogen phosphate solution before the addition of freshly prepared Buljet's reagent. Samples gives the absorbance and propotional to concentration of the glycosides.

Estimation of steroids: 0.2ml of sample was taken 2ml of dilute Sulphuric acid was added, then 2ml of Ferric chloride was added. 0.5ml of Potassium hexagnoferate was added it was incubated in water bath for 30 minutes. Then 5.3 ml of distilled water was added. The absorbance was measured at 780nm. Estimation of phenol: 0.2 ml of sample was taken and 0.8ml of folin ciocalteau reagent was added. 75% of 2ml Sodium carbonate was added and 4ml of distilled water was added. It was kept in dark for 2 hours and the absorbance was measured at 765nm.

Estimation of saponins:

Test extract were dissolved in 80% methanol, 2ml of vanillin in ethanol was added, mixed well and 2ml of 72% sulphuric acid solution was added, mixed well and heated on a water bath at 60Oc for 10 minutes. Absorbance was measured at 544nm against reagent blank. Diosgenin was used as a standard material.

Synthesis of copper nanoparticles: 5 grams of flower powder of Pandanus tectorius was added to 20ml deionized water and extracted using filter paper. 2ml of extract was added to 8ml of copper sulphate solution kept under constant stirring using magnetic stirrer at 50℃ for 3 hours. At the end of the centrifugation process the pellet was obtained and dried in hot air oven at 99℃ for 3 hours. Finally the dried powder was stored. This same were carried out with cold and soxhlet extraction extracts.

Characterization of copper nanoparticles:

The samples of Pandanus tectorius with nanoparticles were given for U-V spectrophotometer and Fourier transform infrared spectroscopy. The powdered samples with copper nanoparticles was characterized by Scanning electron microscopy and X-ray diffraction spectroscopy.

Antimicrobial activities of copper nanoparticles:

The antimicrobial activity of copper nanoparticles Were done by agar well diffusion method and was tested against five different bacterial isolates like E. coli, Streptococcus, Staphylococcus, Pseudomonas, Bacillus subtilus. The agar plates were incubated at 37℃ for 48 hours, and the antifungal activity was tested against four fungal isolates like Aspergillus flavus, Candida albicans, Penicillium and Aspergillus fumigates.

Anti-coagulation assay: Determination of Pt (Prothrombin time): 10ml of blood samples was drawn from healthy volunteers by making vein puncture. To the 9µl of blood 1µl volume of 3.8% tri sodium citrate was added to avoid natural coagulation process. Immediately centrifuge for 15 minutes at 3000rpm to separate blood cells from plasma and to obtain pure platelet plasma (PPP). PPP was used for PT test. Negative control: 0.2ml plasma+0.1ml of 0.9%saline water+0.3ml of 25ml Cacl 2 , Positive control: 0.2ml plasma+ 0.1ml of 50mg/ml EDTA+ 0.3ML Cacl 2 (0.5g/ml), Plant extract: 0.2ml plasma+ 200ul of plant extract (stem, leaf) = 0.3ml Cacl 2 All tubes were titled at an angle of 45, for every 30 seconds to measure the clotting time. This is Prothrombin time.

Thrombolytic activity: Whole blood was drawn from healthy volunteers without a history of contraceptives or anticoagulant therapy. 1ml of blood was transferred to the sterile white tile and was allowed to form clots. Then 1ml of sample was added and allowed to break the clot.

Anti-Larval Activity: Larvae was taken in a crucible and 50µl of sample was added. This activity was watched continuously in every 5 minutes' interval, To report killed time of larvae.

Anti-Diabetic Activity:

The antidiabetic activity was done by alpha amylase activity.

Anti-inflammatory Activity: Prepare various concentration of extract in buffer solution. Take 1ml of extract in buffer + 1ml of RBC suspension and mixed gently. Duplicate into 2 sets. One set was incubated at 54℃ for 20 minutes. Another set was incubated at 10℃ for 20 minutes. Centrifuge at 3000 rpm/ 3 minutes at haemoglobin content in the supernatant was measured in Colorimeter at 540nm. Percentage inhibition of haemoglobin by the extract was calculated.

In vitro Assay for cytotoxicity activity (MTT ASSAY):

The Invitro assay for cytotoxicity activity was done by MTT assay method. The % cell viability was calculated using the formula: % Cell viability = A570 of treated cells/ A570 of control cells x 100.

Hardness of water by edta titration: Pipette 100ml of tap water into a conical flask. Add 2 cm 3 buffer solution followed by 3 drops of Eriochrome Black T indicator solution. Titrate with 0.01M EDTA until the solution turns from wine red to sky blue with no hint of red. This is used as control. Pipette 50ml of tap water into a conical flask. Add 2ml of sample followed by 3 drops of Eriochrome Black T indicator solution. Titrate with 0.01M EDTA until the solution turns from wine red to sky blue with no hint of red. Nanoparticle Synthesis 3gms flower powder of Pandanus tectorius was taken and 15ml of distilled water was added to the powder. The extract was collected using filter paper. 1.25gms of Copper sulphate was weighed followed by adding 8ml of distilled water, it was added to 2ml of samples. The samples were kept in Magnetic stirrer at 50 . C for 3 hours and were centrifuged at 10,000rpm for 15 minutes (Fig 12). The pellet was collected and dried in hot air oven at 99 . C for 3 hours and the nanoparticles were obtained. Similar work has been done by (S. Rajeshkumar et al, 2018) [4] .

Heavy metals removal by copper nanoparticles

Note: Ept is Pandanus tectorius X-Ray Diffraction Studies: XRD was performed for copper nanoparticles, using powder X-ray diffractometer instrument. In Ept one peak was observed at 25 º correspond to 75, the sample was noisier. The average diameter of the copper nanoparticles is calculated and found to be in the range of 42-90nm. (S.Rajesh kumar et al 2018) [4] Fig 15: XRD for Ept

Antimicrobial Activity

The agar well diffusion method was used to enumerate the antimicrobial activity of the test organisms but measuring the zone of inhibition. Different type's organisms were used in antimicrobial activity. In 50μl concentration 27 mm and 25mm of

Antifungal Activity

Anti-Oxiant Activity

The copper nanoparticles are continuously used for advanced biomedical applications. DPPH has been used extensively as a stable free to evaluate reducing substances and its useful reagent for investigation free scavenging activity of the component. Extracts with Pandanus tectorius with nanoparticles has the highest anti-oxidant activity (Yosie Andriani et al 2015).

Thrombolytic Activity

Thrombolytic activity test was done by nanoparticles. On a tile 1drop of blood was added and allowed to clot then 1ml of sample was added. It was allowed to break the clot within 45 minutes.

On comparing the cold with nanoparticles, soxhlet with nanoparticles and extract with nanoparticles, the extract with nanoparticles showed this activity as in Figure 26. (Mohammad Shahriar et al, 2013).

In vitro cytoxicity activity (MTT) assay

Removal of Heavy Metal

The results were observed in the nanoparticles for its heavy metal removal. The extract samples with nanoparticles had the ability to remove heavy metals from Sewage samples. On comparing the values, the concentration of heavy metals in treated sewage samples were lesser compared to the concentration of heavy metals in untreated sewage samples. (Ming Hua et al, 20112), Heavy metals were removed using effluents. (MA Barakat 2011)

Water Hardness Removal

The activity of Water hardness removal was not shown by the nanoparticles (Figure 29). This nanoparticle does not contain the ability of water hardness removal. (Handout for student activity)

Conclusion

 The flowers of Pandanus tectorius were washed and cut into strips then it was shade dried and ground into powder.  5 different solvents (acetone, chloroform, n-hexane, ssethanol and aqueous) were used for both cold and soxhlet extraction.  The phytochemical screening was done for Pandanus tectorius and Mimusops elengi. In the qualitative analysis it was confirmed the presence of glycosides, steroids and saponins in cold extraction. In soxhlet all the 10 were there.  In quantitative analysis of samples revealed the cold extraction samples has more of alkaloids, flavonoids and saponins. The soxhlet extraction showed more amount of tannins, steroids and phenols.  Synthesis of nanoparticles were done for all the 6 samples.  UV was done for all the six samples in the range from 300 to 700nm  FTIR was done with extract with nanoparticles  SEM was done with extract powder with nanoparticles.  XRD was done with extract powder with nanoparticles.  Antibacterial activity was studied against 5 bacterial organisms such as B. subtilis, S. aureus, P. aeruginoceae, E. coli, S. pneumonia .The cold extracts with nanoparticles and the extracts with nanoparticles has maximum zone of inhibition.  Antifungal activity was studied against 4 fungal organisms such as Candida albicans, A. niger, A. flavus and Penicillium SPS  Antioxidant activity was found highest in Pandanus tectorius extracts with nanoparticles.  Anti-inflammatory activity was not present in all the samples  Anti-diabetic activity was absent in all the samples.  Thrombolytic activity was present in the extracts containing the nanoparticles.  Anti larvicidal activity was done in which the larvaes died in10 minutes with the addition of sample  In vitro cytotoxicity was absent so the normal cells viability increased after the incubation period with the samples.  The anticancer activity was absent so the cancer cells viability was increased after the incubation period with the samples.  The removal of water hardness property was not present.

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