2.1 Chemicals and equipment
1,1-Diphenyl-2-picrylhydrazyl (DPPH) (HiMedia), ascorbic acid (HiMedia), gallic acid (Merk), Folin-Ciocalteu reagent (CDH), quercetin dihydrate (CDH), aluminum chloride (Merk), and all the chemicals were of high-purity analytical-grade reagents. Methanol (analytical grade), chloroform (analytical grade), and n-hexane (analytical grade) were throughout used for the phytochemical analysis, TLC, UV, antioxidant, and antibacterial activity. Milli-Q Ultra-pure water was obtained from a Millipore water purification system (Millipore, Milford, MA, USA). All other reagents were of analytical grade. The major equipment used were Soxhlet apparatus (Borosilicate Genuine, JSW) and UV-visible spectrophotometer (Shimadzu UV-Vis spectrophotometer-1800).
2.2 Plant materials and preparation of extract
The C. papaya flowers were collected as a fresh sample in the campus of IGNTU, Lalpur, and Amarkantak in the month of February–September (2017–2018). The plant was identified and authenticated by the botanist of the department of botany, IGNTU (Herbarium no. BT_C-20). The C. papaya flowers were washed, filled into Soxhlet apparatus and extracted with 150 ml methanol, chloroform, n-hexane, and aqueous solvent at a specific boiling point of the solvent for 3–4 h. The extracts were filtered through Whatman filter paper no. 1, and the filtrate was concentrated under reduced pressure at a specific temperature. The extracts were dried, weighed, and yield percentage were calulated for each extraction. Focused extracts were stored at 4 °C for further experimental use.
2.3 Phytochemical investigation
The analysis of C. papaya fresh flower extract in different solvents were carried out according to standard procedures [19] with minor modifications as follows.
2.3.1 Test for alkaloids
HCl was added in the plant extracts (3 ml) and then allowed to steam bath for a few minutes. Then few drops of Mayer regent were added to the mixtures. Turbidity indicates the presence of alkaloids.
2.3.2 Test for flavonoids
Few drops of diluted sodium hydroid solution were added to the stock solution of C. papaya extracts (0.5 ml). An intense yellow color appeared in the plant crude extract, which becomes colorless upon the addition of a few drops of diluted H2SO4 that show the presence of a flavonoid.
2.3.3 Test for saponins
A stock solution from each crude extract C. papaya flowers (0.5 ml) was diluted with distilled water (20 ml), and the test tube was shaken by hand for 15 min. The formation of foam layer on the top of the test tube showed the presence of saponin.
2.3.4 Test for steroids
The plant extracts (2 ml) were dissolved in chloroform (10 ml) and added concentrated sulfuric acid (1 ml) into the test tube by wall sides. The color of the upper layer turned red and the sulfuric acid layer turned yellowish with green fluorescence. This indicated the presence of steroids.
2.3.5 Test for tannins
Two milliliter of extracts were added in 2 ml of distilled water and stirred. Few drops of ferric chloride solution were added. The formation of green precipitate showed the presence of tannins.
2.3.6 Test for phlobatannins
Two milliliters of extracts were hydrolyzed with 1 ml HCl and the mixture was boiled for a few minutes. The deposition of red precipitation indicates the presence of phlobatannins.
2.3.7 Test for glycosides
Two milliliters of extracts were dissolved in chloroform and to the mixture 2 ml of acetic acid was added followed by few drops of sulfuric acid in the mixtures and observed color change from blue to green indicate the presence of glycosides.
2.4 Thin layer chromatography
Thin layer chromatography was carried out using solvent system chloroform: methanol (80:20) used as mobile phase. TLC runs were made in the laboratory conditions at room temperature (RT) and 60% relative humidity. The TLC plates (Merck-silica gel 60 F254) were placed in UV chamber (254 nm) for few minutes for visualizing different spot position of the compounds. The Rf value of plant extract was calculated using the standard formula [20].
2.5 UV-visible spectroscopy
The plant extracts were scanned using UV-Vis spectrophotometer (Shimadzu, UV-1800). Methanol, chloroform, n-hexane, and aqueous solvents were used as blank and plant extracts were prepared in specific solvents at final concentration of 0.10 mg/ml. The λmax was scanned at 800–200 nm range.
2.6 Determination of total phenolic content
The total phenolic contents of the C. papaya flowers were determined by the Folin-Ciocalteu procedure [21]. The Folin-Ciocalteu (F-C) reagent is sensitive to reducing compound, polyphenols and thus produces a blue color [22, 23]. Plant samples (2 ml, triplicates) were introduced into test tubes; 1.0 ml of (1:10) Folin-Ciocalteu’s reagent and 0.8 ml of sodium carbonate (7.5%) were added. The sample mixtures tubes were mixed and allowed to stand for 30 min. Absorption at 690 nm was measured (Shimadzu UV-Vis spectrophotometer). The standard curve of gallic acid solution (1, 1.5, 1.8, 3, and 3.4 mg/ml) was prepared using a similar procedure. The total phenolic content was expressed as gallic acid equivalents (GAE) in milligram per gram dry material. All the experiments were performed in triplicate.
2.7 Determination of total flavonoid content
The total flavonoid contents in the examined flowers were determined using a spectrophotometric method, aluminum chloride (AlCl3) complex forming assay. C. papaya extracts (1 mL, triplicates) were introduced into test tubes and then added 0.30 ml (5% NaNO2). After that, wait for 5 min for the reaction, and 0.30 ml of 10% aluminum chloride solution was added and allowed to stand for later than 5 min, 2 ml solution of 1 M sodium hydroxide was added sequentially, and volume made up 10 ml with distilled water. The absorbance of this reaction mixture was recorded at 283 nm on UV spectrophotometer. The sample tubes were mixed and allowed to stand for 30 min. Absorption at 283 nm was measured (Shimadzu UV-Vis spectrophotometer-1800). The standard quercetin dihydrate solution in different concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mg/ml) was prepared in methanol. A calibration curve for quercetin dihydrate was drawn and determined as quercetin dihydrate equivalent.
2.8 DPPH radical scavenging assay
The antioxidant and free radical scavenging activities of the C. papaya flower were determined by using standard method 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay, as described earlier with some modification [24]. DPPH is able to oxidize by the decolorization of methanol solution gives deep violet color, and an antioxidant compound donates the electron causing its reduction and reduced from its color changes from deep violet to yellow [25]. The 0.1 mM DPPH solution was prepared in 95% methanol. The stock solution of the extracts was also prepared in 95% methanol. After that from the stock solution, 2 ml, 4 ml, 6 ml, 8 ml, and 10 ml were taken in five test tube and diluted with the same solvent to get a final concentration of 20 μl/mg, 40 μl/mg, 60 μl/mg, 80 μl/mg, and 100 μl/mg respectively. The sample extracts were taken 1 ml in each test tube and added 2 ml fresh DPPH solution each of these test tube. As a control, we used 95% methanol. After 30 min incubation in darkness at room temperature, the absorbance was recorded at 517 nm by using spectrophotometer. Ascorbic acids were used as a standard. The percentage inhibition of DPPH by extracts was calculated by using the following formula:
$$ \mathbf{DPPH}\ \mathbf{scavenging}\ \mathbf{effect}=\frac{\left[\mathbf{1}-\left(\mathbf{Abs}\ \mathbf{sample}-\mathbf{Abs}\ \mathbf{blank}\ \mathbf{Sample}\right)\right]\ }{\mathbf{Abs}\ \mathbf{Control}\kern0.5em }\times \mathbf{100} $$
2.9 Antibacterial activity
2.9.1 Bacterial strains
The antibacterial activity of the C. Papaya was tested individually on Gram-positive and Gram-negative bacterial strains. The well diffusion method of antibacterial test was performing on two bacterial strains Bacillus subtilis (Gram-positive) (MTCC code, 441) and E. coli (Gram-negative) (MTCC, 1687) both strains are purchased from Institute of Microbial Technology, Chandigarh, India. Bacterial strains were maintained on nutrient agar at 4 °C and sub-cultured every month in our laboratory.
2.9.2 Culture media and plates preparation
Culture media Muller Hinton (MH) was used in antibacterial activity as nutrition for bacterial growth. For the preparation of the media, we took powder (weight 3.4 g) and dissolved it in 100 ml distilled water. The media was autoclaved and after some time poured in Petri plates and left for solidification. Keep all plates in a cool, clean, and dry place when ready to use for experiments.
2.9.3 Culture preparation
Nutrient broth media for culture of B. subtilis strain
1.3 g of nutrient broth (NB) media was taken and mixed in 100 ml distilled water. The media was autoclaved and cooled. B. subtilis was added (powder form) and to 5 ml of NB media. The tubes were kept in an incubator for incubation at 35 °C (24 h).
LB media for culture of E. coli strain
3.4 g of LB media was taken and mixed in 100 ml distilled water. E. coli (lyophilized powder form) was taken in falcon tube in few amounts and added 5 ml LB media; the tubes were left for overnight incubation at 37 °C for optimum E. coli growth.
Standard preparation
The antibiotic kanamycin was used as the standard for the antibacterial activity. Five milligram powder was added to 10 ml distilled water in a 15-ml falcon tube and was used as a stock solution.
Antibacterial activity
The previously prepared Petri plates were used in the experiment. One hundred milliliter of overnight culture was taken and centrifuged, supernatant was discarded, and the pellet was re-suspended in 1 ml of media and spread on LB agar plate. Wells were punched in the LB agar plates and the samples were loaded in the plates. The Petri plates were sealed with parafilm and kept in an incubator for 12 h at 37 °C for E. coli and at 35 °C for B. subtilis.
2.10 Statistical analysis
Primary phytochemical analysis, total phenolic content, total flavonoid contents, and antibacterial activity were carried out in triplicate and expressed as an average of three analyses ± standard deviation. All experimental measurements were calculated and presented in graphs by the using of MS-excel and SPSS 20 statistical software.