A green approach towards utilization of Floral wastes for the extraction of Natural Colorants

 

Aardra V Kumar, Amal Raj, Amrutha Lakshmi, Nupur Ojha, Nilanjana Das*

Bioremediation Laboratory, Department of Biomedical Sciences, School of Bio-Science and Technology,

Vellore Institute of Technology, Vellore, 632014, Tamil Nadu, India.

 

ABSTRACT:

The present study is focused on the utilization of floral wastes for the extraction of natural colorants. Waste flowers were collected from various sites of temples, garden, domestic and decorative areas and identified as Rosahybrid, Tageteserecta, Spathodeacampanulata, Bougainvilleaglabra, and Euphorbiamili. Identification and characterization of extracted colorants was done using thin layer chromatography (TLC), UV Spectroscopy andFourier Transform Infra-red spectroscopy (FT-IR) analysis. Antimicrobial and antioxidant properties of extracted natural colorants were evaluated. Applicability of extracted colorants as natural dye and biofertilizer was also evaluated. It can be concluded that the natural colorants extracted from the floral wastes can be used in textile and pharmaceutical industries.In addition, contamination free natural colorants can also serve as safe biofertilizer.

 

 

 

INTRODUCTION:

Over the decades, more than 20 tons of flower wastes, collected from different public spots and community garbagehave beendumped everyday into the water bodies which indirectly leads to development of severe animals and human diseases. Eutrophication and imbalance in the ecosystem has also been reported by over dumping of waste flowers and domestic garbage. Improper flower wastes disposal has increased the development of organic pollution to various habitats, which caused several infectious as well as food-water borne diseases due to contaminated water resources¹. In addition to these problems, there are reports on synthetic dyes and colorants which have been suspected to release harmful chemicals which are allergic, carcinogenic and detrimental to human health².

 

 

 

 

Public concern about the safety of synthetic colorants in food industry, synthetic dyes in textile industry, chemical fertilizers, pesticides and insecticides in agricultural industry has led to increasing attention towards the development of natural colorants from natural sources like flowersasalternatives to overcome these problems³.

 

India has approximately 4,90,000 plant species of which about 17,500 are flowering angiosperms⁴. Coloring part of the flowering plants are due to various pigmentscontained in cells. Based on the chemical structures, flowers have been classified into four families viz., Tetra pyrroles (e.g. Chlorophyll), carotenoids (e.g. carotene), polyphenolic compounds (e.g. anthocyanins), and alkaloids (e.g. betalains)⁵. Many reports are available wherein flowers (Cassia Fistula, Hibiscus rosa-sinensis L, Tagetes erecta etc.) or their extracts have been shown to exhibit rich antioxidant and antimicrobial properties. The dried flowers have been reported for the treatment of hemorrhoids, liver diseases, piles, disorders of mucous membranes, dysentery, diarrhea, leucorrhoea, menorrhagia, ulcers,wounds, burning sensations, skin diseases and herpes⁷. Pigments such as anthocyanins, are a group of phenolic compounds widely existing in the flowering plants, present a spectrum from orange to blue in color in the natural world, satisfying the consumers demand as food and dye colorants³.They also possess known pharmacological properties and are used for therapeutic purpose⁸.

 

There are reports that everyday our skin comes in contact with environmental stresses such as pollution and radiation from the sun which leads to free radicals formation. Antioxidants are important in defending against free radicals and act as health protecting factors which diminished the chance for persistent diseases such as cancer and heart diseases⁷. These are highly reactive free radicals and unstable molecules that injure healthy skin cells and disrupt normal skin functions and leads to the premature signs of skin aging⁹. Flower such as pigments have been reported to possess antioxidant property, which helps in scavenging human free radicals and can be used in pharmaceuticals as a precaution medicine to various major diseases like emphysema and cataract. In addition, these pigments have also been reported as a source of various vitamins like carotenes (Tagetes erecta) having effective anti-oxidants properties¹⁰. Phytochemical studies of Euphorbia milii revealed the presence of β-sitosterol, β-amyrin acetate, triterpenes, phenols and flavonoids due to which it possesses antiarthritis, anticancer, anticonvulsant, antidiabetic, anti-eczema, anti-inflammatory and antimicrobial activities¹¹.

 

Natural dyes are eco-friendly having antiseptic and antioxidant property, for example turmeric and indigo, which are natural dyes, imparts antiseptic property and a cooling sensation in addition to dyeing property.Natural floral dyes obtained from flowers such as Woodfordia fruticosa, Rein wardtia, Butea monosperma, Clitoriaternatea, Nyctanthes arbortristis,Spathodea campanulata, Callistemon citrinus, AlceaRosea,Crocus Sativus Linn and Tagetes erecta have been reported as excellent sources for dyeing of textile materials¹². It provides color to the textile and fragrance in retaining the freshness of the textile material by keeping body odor away from the garment for a long period of time. Flower extracts were also reported as natural pH indicators as they remain pink in acidic, purple in neutral and greenish yellow in alkaline condition.

 

Based on the literature survey, natural compounds extracted from flowering plants have been proved to possess health-promoting, antioxidant, antimicrobial, dyeing and eco-friendly properties. Flowers are found to be a reservoir of many pesticides, fertilizers and preservatives. Therefore, the present study is focused towards the utilization of flower wastes for the extraction of potential natural compounds and its application as antimicrobial, antioxidant, dyeing and biofertilizing agent to overcome the improper management of flower waste and to reduce the use of toxic synthetic chemicals in a sustainable and ecofriendly manner.

 

MATERIALS AND METHODS:

Collectionof floral wastes:

Flower wastes were collected in sterileplastic bags from temples, churches, marriage halls, garden wastes and auditoriums situated in local areas of Vellore, Tamil Nadu, India. Flowers with no physical, insect or microbial damage were separated out. Based on their anatomical and inflorescence characteristics, the morphologically distinct flowers were identified⁵. Later, the petals of distinct flowers were carefully removed, washed under tap water to remove dirt particles and dried under hot air oven at 50˚C for 48 h separately. The dried flowers were ground into powdered form (mesh size 30), covered with aluminum foil to avoid exposure to light and stored at 4˚C for conducting further experiments⁶.

 

Extraction of flower extracts:

Thefinely powdered flowers (2g) were packed into soxhlet column and extracted with 70% methanol (100ml) for 48 hours (64.5-65.5°C). The extract was filtered using muslin cloth, concentrated under reduced pressure (water bath 50°C) and then the dried extract was stored in air tight container separately for each flower samples for further use^13,14.

 

Separation and identification of compounds present the flower extracts:

The thin-Layer chromatography (TLC) technique was used for the separation and quantitative determination of natural pigments such as anthocyanins, flavonoids, carotenoids, and other pigment classes present in the flower extracts. TLC plates were preparedusing homogenous suspension of 30g of silica gel and 60ml distilled water¹⁵. The suspension was distributed over the plate, air dried until the transparency of the layer disappeared and were dried in hot air oven at 110°C for 30 min. After drying the TLC plates were used for further experiments. The solutions of different flower extracts were taken in capillary tubes and spotted on a TLC plate 2cm above its bottom. Later on, the adsorbent TLC plate was kept in the solvent in TLC glass chamber and allowed the mobile phase (acetone, isopropanol and petroleum ether in ratio of 2:2:1) to move through adsorbent phase up to 3/4^th of the plate. After the separation, the colored spots were visualized under UV illuminator and the retardation time (Rf values) for different spots for different flower extracts were determined using the formula given below:

 

 

                Distance travelled by the solute

Rf  =    ---------------------------------------------- × 100

                Distance travelled by the solvent

 

The natural pigments present in the flower extracts were identified using UV spectrophotometer. The UV absorption spectra for the different extracts were obtained in the range of 200–400 nm using UV-visible spectrophotometer and peaks were evaluated¹⁶.

 

Characterization of flower extracts:

The types of chemical bonds (functional groups) present in compoundsextracted from different flowers were characterized using Fourier transform infrared spectroscopy (FTIR). Dried powders of methanolic extracts of each flower were used for FTIR analysis. The translucence sample discs were prepared by encapsulation 10 mg of dried flower extract powder in 100 mg of KBr pellet for different flower extracts. The powdered sample of each flower specimen was loaded in FTIR Spectroscope (Shimadzu, IR Affinity1, Japan), with a scan range from 400 to 4000 cm^-1 with a resolution of 4 cm^-117.

 

Evaluation of Antibacterial Activity of the flower extracts:

Preliminary test:

The antimicrobial activities of floral extracts were evaluated against Candida tropicalis (fungi) and Staphylococcus aureus (bacteria) by well diffusion method. The culture of Candida tropicalis and Staphylococcus aureus were swabbed on yeast extract peptone dextrose (YEPD) and nutrient agar plates respectively, and then different concentrations (1.0, 2.0, 5.0, 10, 20, 50, 100, and 200 μg/ml) of each flower extracts were added into the wells bored in the YEPD and nutrient agar plates. A reaction mixture containing without flower extracts in the respective well of YEPD and nutrient agar plates were served as negative control. The discs of antibiotic vancomycin (10 µg)were placed at the center of the respective plates, serving as the positive control. All the testing plates were incubated at 37°C overnight and were observed to detect the presence of zone of inhibition against the test cultures by the samples^{6, 18}.

 

Minimum inhibitory concentration (MIC) test:

The antimicrobial activities of 5 different flower extracts were evaluated using MIC method. The antimicrobial effectiveness was determined against the Candida tropicalis and Staphylococcus aureus. Different concentrations (0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200 μg/ml) of each flower extracts were added in 50 ml of YEPD broth inoculated with Candida tropicalis cells(10⁶ CFU/ml). Similarly, different concentrations (1.0, 2.0, 5.0, 10, 20, 50, 100, 200 μg/ml) of each flower extracts were added in 50 ml of nutrient broth inoculated with Staphylococcusaureus (10⁶ CFU/ml) cells. The culture flasks were incubated at 37°C at 250 rpm for 24 h. Microbial concentrations were determined by measuring optical density (OD) at 600 nm (0.1 OD 600 corresponding to 10⁸ cells per ml). The effect of each flower extracts on the fungal and bacterial growth curve were also determined using same cultures (Candida tropicalis and Staphylococcus aureus) with the respective concentrations of flower extracts mentioned above¹⁸.

 

Antioxidant assay of the flower extracts:

Diphenyl picryl hydrazyl free radical (DPPH) is a well-known radical to monitor chemical reactions involving radicals which is widely used for antioxidant assay. The flower extracts were tested for the scavenging effect on DPPH radical following the method of Venugopalan and Revathy⁹. In this method, the solution of DPPH free radicals was prepared by dissolving free radicals inabsolute methanol to a concentration of 100 μl. Then the flower extracts (0.2 mL) were diluted with absolute ethanol of different concentrations (0.2, 0.5, 0.8 and 1.2 mg mL^-1). The methanolic solution of each samplewere added to 8 ml of 0.004 % (w/v) DPPH solution and the mixtures were left tostand for 20 min in the dark at room temperature. When a solution of DPPH having a strong absorption at 517 nm was mixed with a substance that can donate a hydrogen atom or a free radical, then it was converted into its reduced form which could be monitored by measuring the absorbance at 517 nm. The scavenging activity on the DPPH radical was determined by measuring the absorbance at 517 nm using UV- visible spectrometer. Lower the absorbance at 517 nm represents higher DPPH scavenging activity. The DPPH radical scavenging activity for each sample was calculated using the formula as follows:

 

                           (control absorbance-sample absorbance)

% DPPH radical = ------------------------------------------------------ x 100

scavenging effect                  control absorbance

 

Radical scavenging potential of the extracts were compared from inhibition (IC₅₀) value, which represents the sample concentration at which 50% of the DPPH radicals scavenged.

 

Evaluation of dyeing property of the flower extracts:

The flower extracts obtained through above mentioned method was filtered and used for dyeing. Cotton cloth pieces (10 × 10 cm) used for dyeing were boiled in 10 % sodium hydroxide (NaOH) solution for 15 minutes to remove starch from the cloth, then washed with cold distilled water. The cloth pieces were dipped in three different mordant solutions viz., potassium dichromate, copper sulphate and ferrous sulphate at a concentration of 3%, boiled at 80ºC for 30 min and left for another 30 min in the mordant solution. Then, the mordanted cloth pieces were rinsed, squeezed and dried at room temperature. The mordanted cloth pieces were immediately used for dyeing since the mordants are light sensitive. For dyeing, these mordanted samples were immersed in dye bath (10g flowers in 100 ml distilled water) separately for 2 h at a temperature range of 80˚C¹⁹. After completion of dyeing, the cloth pieces were treated with tepol (color fixative) and dried in sunlight for 2 h. The sun dried cloth pieces were further evaluated for its color, lightness and wash fastness. The wash fastness was tested by washing these samples with soap water (10% w/v) and heat resistance was tested by keeping the cloth pieces at various temperatures of 50, 60 and 70°C for 30 minutes in the oven without water²⁰.

 

Evaluation of flower extracts as biofertilizer:         

The extracts prepared from the collected flowers were evaluated for its activity as biofertilizer. Extracts was prepared by boiling 100 g of different flower in 100 ml of distilled water for 30 minutes and filtered. The filtrate was taken as 100% concentration of the respective flower extracts, from this, 20% concentration of each flower extracts were prepared using distilled water and wererefrigerated between 0 and 4ºC for further use²¹. The seeds of Pisum sativum was selected as crop plant seeds for conducting the experiment. The seeds of almost uniform size, colour and weight were selected for the experiment, soaked with 20 % concentration of each flower extracts for 24 h and then used for further experiment²¹. Soil was collected from VIT nursery and was sieved to remove unwanted wastes. To determine the activity of different flower extracts as biofertilizer, five sets of experiments were conducted in separate plastic containers containing 100 g of soil sowed with seeds of Pisum sativum. The experimental set of treatments were as follows: (C) control, seeds soaked with water, (1) seeds soaked with Spathodea campanulata extracts, (2) seeds soaked with Bougainvillea glabra extract, (3) seeds soaked with Euphorbia mili extract, (4) seeds soaked with Rose hybrid extract and (5) seeds soaked with Tagetes erecta extract. Seeds treated with different flower extracts were sowed in the respective container containing soil and were watered with tap water regularly. Test plant samples were taken out from each set on zeroth, first, second, third and fifth day and the growth parameters including fresh and dry weight, length of shoot were calculated.

 

RESULT AND DISCUSSION:

Morphological identification of flowers and its extracts:

Based on the anatomical and inflorescence characteristics, five morphologically distinct flowers were identified viz. Spathodea campanulata, Bougainvillea glabra, Tagetes erecta, Euphorbia milii and Rosa hybrid^5,22. The collected Spathodea campanulataflower petals werereddish-orange or crimson in colour commonly known as african tulip, belongs to genus Spathodea and familybignoniaceousas shown in figure 1(a). The flowers of Bougainvillea glabra were pinkish in colour commonly known as paperflower, belongs to genus Bougainvillea and family nyctaginaceaeas shown in figure 1(b). The collected Tagetes erecta flowers were orange-yellowish in color commonly known as mexican or african marigold, belongs to genus Tagetes and family asteraceae as shown in figure 1(c). Flowers of Euphorbia mili baredreddish bracts with yellowish centre, commonly known as Christ thorn, belongs to genus Euphorbia and family euphorbiaceae as shown in figure1(d). The collected flowers of Rosa hybrid were red in colour commonly known as rose, belongs to genus Rosa and family rosaceae as shown in figure1(e). Methanolic flower extracts and its dried powdered form prepared from flowers of Spathodea campanulata, Bougainville glabra, Euphorbia mili, Tagetes erecta and Rosa hybrid using solvent extraction method.

 

 

Figure 1:  Flowers collected from different sites (a) African Tulip (Spathodea campanulata); (b) Bougainvillea (Bougainville glabra), (c) Marigold (Tagetes erecta), (d) Euphorbia (Euphorbia mili), (e) Rose (Rosa hybrid)

 

Separation and identification compounds present in flower extracts:

The thin Layer Chromatographytechnique was usedfor separating the natural compound present in different flower extracts. The retardation factor (R_f value) (0.26-0.97) of the thin layer chromatogram for Spathodea campanulata, Bougainville glabra, Euphorbia mili, Tagetes erecta and Rosa hybridextract were calculated as shown in Table 1, which indicated the presence of moleculeswhich were corresponding to flavonoids, carotenoids, lutein, anthocyanin and pheophytin pigments.

 

Table 1:Separation and identification of pigments present in the floral extract through thin layer chromatography and

UV visible spectrophotometer analysis

Floral extracts	Retention time  (Rf  value)	Pigments color	Absorbance bands (nm)	Tentative pigments
Spathodea campanulata	Rf1 = 0.26	Pale yellow	210, 252, 330	Flavonoids, β carotene
	Rf2 = 0.37	Ivory
	Rf4 = 0.96	Orange
Bougainville glabra	Rf1 = 0.27	Pale Yellow	220, 360, 280	Flavonoids, Anthocyanin
	Rf2 = 0.36	Blue
	Rf3 = 0.87	Reddish yellow
Euphorbia mili	Rf1 = 0.26	Pale Yellow	220, 250, 350	Flavonoids, Neoxanthin, Lutein
	Rf2 = 0.38	Ivory
	Rf3 = 0.42	Yellow
Tagetes erecta	Rf1 = 0.10	Yellow	210, 340, 257	Laricitrin-di-hexoside (flavonoid), Lutein, Carotene
	Rf3 = 0.42	Yellow
	Rf2 = 0.97	Orange Yellow
Rosa hybrid	Rf1 = 0.27	Pale Yellow	220, 480, 650	Chlorophyll a, Anthocyanin, Pheophytin b
	Rf2 = 0.36	Blue
	Rf3 = 0.52	Olive green

 

The UV-Visible spectrum of flower extract of Spathodea campanulata shown in figure 2(a), showed very strong absorption peaks at 210, 252 and 330 nm which resembled the presence of flavonoids and β carotene compounds in the sample¹⁶. The absorption peaks at 220, 360 and 280 nm shown in figure 2(b), by Bougainville glabra extracts were corresponding to the presence offlavonoids and anthocyanin compounds¹⁴. In figure 2(c), the strong absorption peaks at 220, 250 and 350 nm by Euphorbia mili showed the presence of compound like flavonoids, neoxanthin and Lutein²³. The extract of Tagetes erecta shown in figure 2(d), showed absorption peaks at 210, 340 and 257 nm which resembled the presence of laricitrin-di-hexoside (flavonoid), Lutein, and carotene constituents in the sample¹⁰. Also, the UV- Visible spectrum of Rosa hybrid extract shown in figure 2(e),  showed prominent peaks at 220, 480 and  650 nm which were corresponding to the constituents of chlorophyll a, anthocyanin and pheophytin²⁴ Table 1.

 

 

Figure 2: Identification of compounds present in the extracts of (a) Spathodea campanulata; (b) Bougainville glabra; (c) Euphorbia mili; (d) Tagetes erecta; (e) Rosa hybrid through UV visible spectrophotometer analysis

 

Characterization of flower extracts:

FTIR spectra and the functional group of compounds present in the powdered flower extracts of Bougainville glabra, Euphorbia mili, Tagetes erecta, Rosa hybrid and Spathodea campanulata were shown in figure3(a-e) and Table 2, respectively. Spectral analysis of the flower extract of Bougainville glabra, Euphorbia mili, Tagetes erecta, Rosa hybrid and Spathodea campanulata revealed the major functional groups of the compounds which were corresponding to polysaccharides, suberin, lipids and glycogen. In Euphorbia mili and Rosa hybrid the spectral peaks were slightly differed from  Bougainville glabra, Tagetes erecta, and Spathodea campanulata with some additional peaks at 1442.75 cm^_1 (lipid origin),asymmetric C–O–C vibration, Suberin at 1197.79 and 1192.01 cm^-1 and alkenyls and aromatic p- disubstituted at 869.90     and 869.90 cm^-1respectively (Table 2). FTIR spectra of all the samples showed peaks in the range of 3219.19 to 3286.70 cm^-1 and 2920.23-2927.94cm^-1 which were corresponding to the OH group of the phenolic groups and asymmetric C–H vibration, suberin compounds present in the samples^{25, 26}. In addition to, FTIR spectra of all the samples also showed peaks in the range of 1701.22-1724.36 cm^-1 and 511.14-590.22 cm^-1 which resembled the presence of ester COO stretch, lipids, triglycerides and glycogen constituents in the samples⁶.

 

 

 

Figure 3: Identification of functional groups present in the natural colorants extracted from the floral extracts of (a) Bougainville glabra;(b) Euphorbia mili;(c) Tagetes erecta;(d) Rosa hybrid;(e) Spathodea campanulata through FTIR analysis.

 

 

Table 2: Identification of functional groups of the colorant compounds present in the floral extracts through FTIR analysis

Samples/ Absorption peaks (cm -1)	Bougainville glabra	Euphorbia milii	Tagetes erecta	Rosa hybrid	Spathodea campanulata	Functional groups
1	3269.34	3219.19	3286.70	3281.70	3236.55	Phenol O–H stretch, Polysaccharides
2	2926.01	2920.23	2926.01	2926.01	2927.94	Asymmetric C–H vibration, Suberin
3	-	2850.73	-	-	-	Symmetric C–H vibration, Suberin
4	1701.22	1720.50	1724.36	1720.50	1722.43	Ester C‚O stretch, lipid, triglycerides
5	-	1602.85	1600.92	1604.77	1602.85	Diketone
6	1597.06	-	-	-	-	Isopropyl group
7	-	1442.75	-	1442.75	-	Lipids
8	-	1350.17	1386.60	1325.10	1369.46	CH2 wagging band progression
9	1251.80	1286.52	1282.66-1201.65	-	1201.65	Aromatic amines
10	-	1197.79	-	1192.01	-	Asymmetric C–O–C vibration, Suberin
11	1026.13	1068.56-1029.99	1022.27	1031.92	1064.71-1022.27	C–O valence vibration, Polysaccharide
12	-	869.90	-	869.90	-	Alkenyls and Aromatic p- disubstituted
13	771.53	742.59	-	748.38	-	Aromatic (C-H Stretch), Acid chlorides (C-Cl)
14	-	-	-	-	659.66	Halogen compounds, C-S linkage
15	572.86	514.99	514.99	590.22	511.14	Glycogen

 

 

 

 

Application of extracted natural colorants:

Antibacterialactivity:

For the assessment of the antimicrobial effects of different flower extracts, Candida tropicalis and Staphylococcus aureus were used in the present study. The effect was investigated by growing the test cultures, Candida tropicalis and Staphylococcus aureus on YEPD and nutrient agar plates respectively, supplemented with different concentration of 5 different flower extracts. The yeast and bacterial growth were completely inhibited in the presence of Euphorbia mili, Tagetes erecta and Rosa hybrid extracts, however, a slight zone of inhibition was observed in case Bougainville glabra and Spathodea campanulata extracts as shown in figure4(a-b) and Table 3on the YEPD and nutrient agar plates. The inhibition solely depended upon the phytochemicals present in the flower extracts and its concentration. The flower extracts (100µl) ofEuphorbia mili, Tagetes erecta and Rosa hybrid had shown a clear zone of inhibitionagainst C. tropicalis and S. aureuson YEPD and nutrient agar plates respectively as shown in figure4(a-b) which indicated that these flowers contained specific antimicrobial phytochemicals like flavonoids and phenols which inhibited the microbial growth¹¹.

 

Table 3: Antibacterial activity of the floral extract

Sample’s concentration (100 µl)	Inhibition Zone (in mm) against Candida tropicalis	Inhibition Zone (in mm) against Staphylococcus aureus
Vancomycin (control 10 µl)	20	20
Spathodea campanulata	0.8	0.5
Tagetes erecta	19	18
Euphorbia mili	17	11
Rosa hybrid	17	14
Bougainville glabra	0.5	0.4

 

To study the antimicrobial effectiveness of different flower extracts, a yeast and bacterial concentration at high CFU (10⁶/ml) with varying concentrations of different flower extracts from 1.0 to 200 μg/ml were tested. The results shown in figure4(c-d), indicated that on increasing the concentration of Euphorbia mili, Tagetes erecta and Rosa hybridextracts, the yeast and bacterial cell counts were found to be decreased. At concentration of 100 μg/ml of Euphorbia mili, Tagetes erecta and Rosa hybrid extracts, the growth of Candida tropicalis and Staphylococcus aureus was completely inhibited, which indicated that the minimum inhibitory concentration was 100 μg/ml for these three flower extracts. However, a minorantimicrobial activity was observed in case of Bougainville glabra and Spathodea campanulata extracts at high concentration (500 µg/ml) which indicated that the tested microbes are sensitive to these flower extracts at low concentration(figure 4(c-d). Higher concentration (above 500 µg/ml) of Bougainville glabra and Spathodea campanulata extracts was required for complete inhibition of the microbial growth.

 

 

 

Figure 4: Antibacterial activity shown by fresh extracts of (1) Spathodea campanulata; (2) Tagetes erecta;(3) Euphorbia mili;(4) Rosa hybrid;and (5) Bougainville glabra. (a) Zone of inhibition shown by the respective flower extracts (100 µg/ml) against yeast Candida tropicalis; (b) Zone of inhibition shown by the respective flower extracts (100 µg/ml) against bacteria Staphylococcus aureus; (c) Minimum inhibitory concentration (MIC) test by the respective flower extracts against yeast Candida tropicalis; (d) Minimum inhibitory concentration (MIC) test by the respective flower extracts against bacteria Staphylococcus aureus; (e) Effect of respective flower extracts on yeast (Candida tropicalis) growth; (f) Effect of respective flower extracts on bacterial (Staphylococcus aureus) growth.

 

 

The slope of the yeast and bacterial growth curve was continuously decreased (2.4 to 0.0 O.D) with increasing concentration (0.1 to 500 µg/ml) of Euphorbia mili, Tagetes erecta and Rosa hybrid extracts figure 4(e-f). This indicated that at low concentration of these flower extracts (0.1 – 50 µg/ml), the growth of microbial cells was delayed and at higher (100 µg/ml & above) concentration, growth was completely inhibited. In case of Bougainville glabra and Spathodea campanulata extracts, higher (500 µg/ml and above) concentration was required for complete inhibition of Candida tropicalis and Staphylococcus aureus growth. The test organisms were found to be sensitive with these two flower extracts and a slight decrease in the growth was observed at concentration range of 0.1 - 500 µg/ml flower extracts but no complete inhibition was observed (figure 4(e-f). So it can be concluded that Euphorbia mili, Tagetes erecta and Rosa hybrid extracts have a microbiostatic effect at low concentration and microbicidal effect at high concentration which will be suitable for preventing microbial contamination. These findings may lead to valuable contributions in various fieldslike antimicrobial systems as well as medical devices in the future.

Antioxidant activity:

The antioxidant activity was determined based on the overall scavenging effects of DPPH radical by the flower extracts of Rose hybrid, Spathodea campanulata, Euphorbia mili, Tagetes erecta and Bougainvillea glabraas shown in Fig. 5In the present study, methanolic extract of Tagetes erecta (87.06 %) exhibited highest scavenging effect on DPPH followed by Rose hybrid (83.44 %), Spathodea campanulata (77.86 %), Euphorbia mili (73.72 %) and Bougainvillea glabra (69.76 %). The rich radical scavenging activity of all the flower extracts attributed the presence of high phenolics, tannins or flavonols in the sample extracts¹¹.

 

 

Figure 5: Antioxidant activity of the respective flower extracts by DPPH radical scavenging assay. Highest antioxidant activity was shown by Tagetes erecta extract followed by Rose hybrid, Spathodea campanulata, Euphorbia mili and Bougainvillea glabra

 

Evaluation of dyeing property:

A different shade of colors were obtained from the flower extracts of Rose hybrid, Bougainvillea glabra, Euphorbia mili, Spathodea campanulata andTagetes erectaas shown in figure6(d) and Table 4. Effect of colorants with modarants (K₂Cr20₇, CuSO₄ and FeSO₄) were observed on the cotton cloth pieces as shown in figure 6(e-g), which showed best colorations (dark yellowish-brown, chocolate brown and blackish brown) respective to flower extracts with different mordant combinations. The intensity of color produced on cloth pieces by dyeing without mordanting was found slightly less than that obtained for mordants and flower colorants used successively. Dyed cotton cloth pieces showed attractive shades of yellow, brown, dark brown and blackish brown color with the treatment of potassium dichromate mordant and colorant extracted from the different flowers as tabulated in Table 4, shown in figure6(g). Similarly, yellowish brown, muddy brown, grey color shades were observed with ferrous sulphate and copper sulphate in figure 6(f). Similar trends were reported where flower extract of Woodfordia fruticose with different mordants were used for dyeing silk, jute and cotton cloth pieces²⁰. 

 

 

 

Figure 6: Evaluation of dyeing property (a) Methanolic extracts of (1) Rose hybrid;(2) Bougainvillea glabra;(3) Euphorbia mili; (4) Spathodea campanulata;(5) Tagetes erecta. (b) Mordents used were Potassium dichromate (K₂Cr₂0₇); copper sulphate (CuSO₄) and ferrous sulphate (FeSO₄). (c) White cotton cloth. (d) Dyeing of white cotton clothes using flower extracts without mordant. (e) Dyeing of white cotton clothes using flower extracts with mordents FeSO_4. (f) Dyeing of white cotton clothes using flower extracts with mordents CuSO_4. (g) Dyeing of white cotton clothes using flower extracts with mordents K₂Cr₂0₇

Table 4: Effect of different mordants with colorant extracted from different flowers on the cotton cloth

Samples	Mordants	Color obtained	Color fastness
Rose hybrid	Without	Beige	Slight
	FeSO4	Yellowish	Slight
	CuSO4	Brown	Slight
	K2Cr207	Dark brown	Slight
Bougainvillea glabra	Without	Light yellowish	Slight
	FeSO4	Muddy yellowish	Slight
	CuSO4	Light yellowish	Slight
	K2Cr207	Brown	Slight
Euphorbia mili	Without	Sandy yellowish	Slight
	FeSO4	Beige	Slight
	CuSO4	Light brown	Slight
	K2Cr207	Chocolate brown	Slight
Spathodea campanulata	Without	Dark beige	Slight
	FeSO4	Brown	Slight
	CuSO4	Light brown	Slight
	K2Cr207	Beige	Slight
Tagetes erecta	Without	Light yellowish	Slight
	FeSO4	Brown	Slight
	CuSO4	Grey	Slight
	K2Cr207	Blackish brown	Slight

 

 

Evaluation of flower extracts as biofertilizer:

The highest shoot length (5.65 cm), fresh and dry weight (1320 mg, 294 mg) were observed by Tagetes erecta soaked seeds followed by Rose hybrid, Euphorbia mili, Bougainvillea glabra and Spathodea campanulataon fifth day respectively as shown in Table 5 and figure7. No growth was observed in the control which indicated that the improvement in the germination and growth of the test plants was only due of the presence of soaked flower extracts that supplied nutrients to it. Development of small leafs were observed after second and third day of experiment in all the containers except the control as shown in figure 7(c-d). A significant growth of the Pisum sativum plants in each of the containers after five days of incubation were observed as shown in figure 7(d). However, there was no growth found in the control (C). This behavior clearly indicated the presence of some natural growth promoting hormones like auxins, gibberellins, cytokines, trace elements, vitamins and amino acid in lower concentrations of flower extracts which enhanced the plant growth.

 

 

Figure 7: Evaluation of biofertization property of different flower extracts using Pisum sativum seeds soaked with water as control (C);(1)Spathodea campanulata extract; (2)Bougainvillea glabra extract; (3)Euphorbia mili extract ; (4)Rose hybrid extract;and (5)Tagetes erecta extract. Growth of seeds under different sets of conditions on (a) Zeroth day of germination; (b) After first day of germination; (c) After second day of germination; (d) After third day of germination; (e) After fifth day of germination

 

Table 5: Effect of flower extracts on growth of Pisum sativum

Treatments	Shoot length (cm)					Fresh weight (mg)					Dry weight (mg)
	0th	1st	2nd	3rd	5th	0th	1st	2nd	3rd	5th	0th	1st	2nd	3rd	5th
Control	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Spathodea campanulata	-	0.0	0.0	0.5	0.78	-	0.0	0.0	40	90	-	0.0	0.0	0.07	1
Bougainvillea glabra	-	0.0	0.0	0.7	1.89	-	0.0	0.0	50	210	-	0.0	0.0	3	22
Euphorbia milii	-	0.0	0.1	1.4	3.00	-	0.0	9	100	850	-	0.0	1	87	154
Rose hybrid	-	0.0	0.3	2.9	4.05	-	0.0	10	200	990	-	0.0	1	23	199
Tagetes erecta	-	0.0	1.0	3.8	5.65	-	0.0	200	900	1320	-	0.0	20	96	294

No growth (-)

 

 

CONCLUSION:

The present study has demonstrated the extraction of natural colorant compounds from waste flowers ofRosa hybrid, Bougainvillea glabra, Euphorbia mili, Spathodea campanulata and Tagetes erecta and its applicability as antimicrobial, antioxidant, dyeing and biofertilizing agent. The methanolic extracts of Euphorbia mili, Tagetes erecta and Rosa hybrid were found to serve as excellent antimicrobial and antioxidant agent compared to Spathodea campanulata and Bougainvillea glabra. The presence of compounds like flavonoids, anthocyanin, carotenoids, and lutein in these flower extracts had enhanced the scavenging and antimicrobial activity. These natural colorants can be potentially applied in pharmaceutical and medical fields in a cost effective way. The effect of colorants with different mordants had enhanced the color intensity of the cloth pieces reflecting the excellent dyeing property which can be useful towards the development of cost effective ecofriendly dye for the textile industry. Moreover, the supplementation of flower extracts had shown enhanced growth and development in the test crop plant which indicated that these flower extractscan also be used as biofertilizing agent in the agriculture field in a sustainable and ecofriendly way. Thus, it can be concluded that the waste flower extracts of Rose hybrid, Bougainvillea glabra, Euphorbia mili, Spathodea campanulata and Tagetes erecta could be exploited as valuable sources of antimicrobial, antioxidant, dyeing and biofertilizing agents in pharmaceutical, textile and agricultural industry.

 

ACKNOWLEDGEMENT:

The authors are grateful to VIT, Vellore for providing necessary laboratory facilities.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

REFERENCES:

1.     Kulkarni, Bodake, et al. “ Extraction of Natural Dye from Chili (Capsicum Annum) for Textile Coloration”, UJERT, 2011, 1(1)

2.     Wang, Lin, Chu, et al. “Protective effect of Hibiscus anthocyanins against tertbutylhydroperoxide-induced hepatic toxicity in rats”, Food Chem Toxicol, 2000, 38(5),411-6.

3.     Kant R,“Textile dyeing industry an environmental hazard”, Natural science, 2012, 4(1), 22-6

4.     Binapani, Pankaj et al. “Exploration of plant derived natural dyes in Assam”, AJST, 2014, 9(1), 17-20.

5.     Cronquist A, “The evolution and classification of flowering plants”, The evolution and classification of flowering plants, 1968.

6.     Mak, Bhat et al. “Antioxidant and antibacterial activities of hibiscus (Hibiscus rosasinensis L.) and Cassia (Senna bicapsularis L.) flower extracts”.JKSUES, 2013, 25(4), 275-82.

7.     Voon H C, Bhat R et al.“Flower extracts and their essential oils as potential antimicrobial agents for food uses and pharmaceutical applications”, COMPR REV FOOD SCI F., 2012, 11(1):34-55.

8.     Vankar P S and Srivastava J, “Evaluation of anthocyanin content in red and blue flowers”, IJFE, 2010, 6(4).

9.     Venugopalan P and K. T, Revathy, “Antioxidant Activity of Cassia Fistula Flower Extracts”. J. Pharm. Appl. Chem., 2016, 2(2), 77-80

10.   HaddenW L, et al.“Carotenoid composition of marigold (Tagetes erecta) flower extract used as nutritional supplement”, J Agric Food Chem, 1999, 47(10), 4189-94.

11.   Kamurthy H andDontha S, “Phytochemical Screening on Euphorbia milii Red Flowers â Isolation of Terpenoids, Flavone and Phenols”.AJEthno, 2015, 2(6)

12.   Singh R and Srivastava S, “Exploration of flower based natural dyes-a review”, Research Journal of Recent Sciences, 2015, (4) 6-8.

13.   Sumathi R and Anuradha R, “FT-IR Spectroscopic Studies on Flowers of Allamandaneriifolia” Hook. Int. J. Curr. Microbiol. App. Sci. 2016, 5(6):287-91

14.   Neha S and Jyoti S, “Phytochemical Analysis of Bougainvillea GlabraChoisy by FTIR and UV-VIS Spectroscopic Analysis”, Int. J. Pharm. Sci. Rev. Res., 2013, (33): 96-198.

15.   Chakraborty D D, Ravi V, Chakraborty P, “Phytochemical evaluation and TLC protocol of various extracts of Bombaxceiba Linn.” IJPSR, 2010, 1(8):66-73.

16.   Patil V V, Patil S B et al. “Study of methanolic extract of flower of Spathodea campanulata L. as an anti-solar”, IJGP, 2009, 3 (3).

17.   Nithyadevi J and Sivakumar R, “Phytochemical screening and GC–MS, FT-IR analysis of methanolic extract leaves of Solanum torvum” Sw. Int. J. Res. Studies in Biosci. 2015, 3(9), 61-6

18.   Maiti S, Krishnan D, et al. “Antimicrobial activities of silver nanoparticles synthesized from Lycopersiconesculentum extract”, JAST, 2014, 5(1), 40.

19.   Jain N, “Extraction and application of Natural dye by utilizing temple floral waste Tegeteserecta. L”., IJETSR, 2017, 4(3)

20.   Grover N, and Vidya P, “Extraction and application of natural dye preparations from the floral parts of Woodfordiafruticosa (Linn.)”Kurz, 2011, IJNPR, 2011,  2(4):403-408

21.   Bindhu K B, “Effect of Azolla extract on growth performance of Pisum sativum”, IRJBS, 2013, 2(10), 88-90.

22.   Takhtajan and Armen L, Outline of the classification of flowering plants (Magnoliophyta), The botanical review, (1980), 46(3), 225-359.

23.   Navarro-González I, González-Barrio R, et al. “Nutritional composition and antioxidant capacity in edible flowers: characterization of phenolic compounds by HPLC-DAD-ESI/MSn”, Int J Mol Sci, 2014, 16(1), 805-22.

24.   Domenici V, Ancora et al. “Extraction of pigment information from near-UV vis absorption spectra of extra virgin olive oils”, J Agric Food Chem, 2014, 62(38), 9317-25.

25.   Shurvell H F, “Spectra-structure correlations in the mid- and far-infrared. In: Chalmers, J.M., Griffiths, P.R. (Eds.)”, Handbook of Vibration Spectroscopy. John Wiley and Sons Ltd., UK, 2002, 1783–1816.

26.   Tejado A, Penaa, et al.“Physico-chemical characterization of lignins from different sources for use in phenol-formaldehyde resin synthesis”, Bioresour Techno, 2007, 98, 1655–1663.

 

 

 

 

Accepted on 29.10.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(1): 269-279.