Phytochemical prospecting and biological activities of the floral extract from [ Impatiens walleriana (Hook.)] (Balsaminaceae)

Impatiens walleriana is a plant species that presents diversity and variety of colors among flowers. This plant species has wide distribution and is easily cultivable throughout the world including natural gardens, greenhouses and parks. This study aims at evaluating the floral extracts of I. walleriana in terms of their qualitative phytochemical constitution and antifungal activities on Sclerotinia sclerotiorum , Colletotrichum gloeosporioides and Colletotrichum acutatum , as well as on the reduction of DPPH free radical and on the inhibition of acetylcholinesterase (AChE). Flowers were collected from two cultivars Pink and White of I. walleriana and the extract produced by maceration. The phytochemical qualitative assay was carried out using different reagents for determination by precipitation or colorimetric alteration. The antifungal test was carried out in Petri dishes with different concentrations of floral extract on S. sclerotiorum , C. gloeosporioides and C. acutatum . The DPPH reduction assay was performed by percentage and as standard antioxidant ascorbic acid. The acetylcholinesterase inhibition test was performed in percentage using Electrophorus electricus . Positive results were observed for phenolics, anthocyanins, organic acids, alkaloids, catechins, tannins, carboxylic acids, hemolytic saponins and sesquiterpene lactones. Fungal inhibition effect was better observed for cv. Pink with 10-26%, 6% and between 9-11% on S. sclerotiorum , C. gloeosporioides and C. acutatum , respectively. The DPPH reduction activity showed satisfactory results for cv. Pink 49% followed by cv. White 41%. And for inhibition of AChE cv. Pink with 30% and cv. White with 27%. The floral extracts of Impatiens walleriana show good results for the biological activities tested, especially for cv. Pink .


Introduction
Several floristic plant species are being included in floriculture systems, ornamentation in parks and gardens. Within this broad floristic spectrum Impatiens walleriana Hook. f. popularly known as "maria-sem-vergonha, lollipop cherry red or write, and busy lizzie" is globally used due to its availability in varied color range.
Although being native to tropical East Africa it is widespread from North to South of America, the Pacific Islands, China in particular (Taiwan), Australia and New Zealand (Mandle et al., 2010).
The genus Impatiens L., family Balsaminaceae A. Rich., is described in studies with important biological activities such as antioxidant, antimicrobial, anti-inflammatory and anti-proliferative. The species Impatiens walleriana is herbaceous and perennial having diverse flower morphology and colors ranging from pink to white, with early flowering, resistant to diseases and extreme sunlight (Anderson, 2006;Lai, 2015;Ghanbari et al., 2019).
In recent days, studies that characterize and relate the composition of edible flowers along different bioactive properties have been taken up. This study aimed to evaluate the floral extracts of Impatiens walleriana var. pink and white in biological activities on the inhibition of phytopathological fungi Sclerotinia sclerotiorum, Colletotrichum gloeosporioides and Colletotrichum acutatum, antioxidant activity (DPPH) and on acetylcholinesterase inhibition (AChE). Immediately after harvesting, the flowers were washed thrice using water and dried with the help of absorbent paper. The petals were removed, stored, and separated according to their colour in plastic containers with 70% ethanolic solution. The extract was produced by maceration for 48 h. It was then filtered through qualitative filter paper and the filtrate was collected. The extract was reduced in a rotary evaporator and the samples prepared were frozen, freeze dried, and stored in airtight bottles protected from light in frozen at analysis.

Phytochemical prospection
Phytochemical analysis was performed for the main groups of special metabolites present; this was done using staining, and qualitative precipitation techniques. The floral extracts from I. walleriana were subjected to test for: alkaloids, anthocyanins, organic acids, reducing and non-reducing sugars, anthraquinones, catechins, depsides and depsidones, steroids, coumarins, tannins, phenols, flavonoids, polysaccharides, proteins, amino acids, purines, foaming and hemolytic saponins, organic acids, azulenes, and sesquiterpenolactones as proposed by (David et al., 2019).

Antifungal activity
The agar diffusion method was used to determine the antifungal activity on Sclerotinia sclerotiorum, Colletotrichum gloeosporioides and C. acutatum as described by Toigo et al. (2022) adapted. The strains used are: SS12-21, CG 16-21 and CA15-67 respectively, from the second author's mycological bank. The fungal strains were cultured at 20 °C for 10 days for S. sclerotiorum and 28 °C for 3-5 days for the other strains.
A mycelium disc with a diameter of 7 mm was transferred to the center of Petri dishes with a diameter of 10 cm containing sterile potato, dextrose and agar (PDA) medium. Different floral extracts concentrations were used, dissolved in 0.5% dimethylsulfoxide (DMSO) to render doses between 50-500 μL mL -1 , in each plate 500 μL of the concentration was pipetted. The plates were transferred to an incubator at 20 °C (10 days) and 28 °C (3-5 days), respectively. The diameter of the zone of inhibition was measured and recorded as an indicator of antifungal activity and expressed in percentage (%) using a digital caliper. The commercial reference fungicide Frowncide 500 SC was used as a positive control (dose of 10 μL mL -1 ).
The DMSO emulsifier was also evaluated at the lowest dose under investigation (100 μL mL -1 ). The agar diffusion assays applied against the three fungi were performed in quadruplicate. Mycelial growth was obtained daily until complete fungal growth separately on control plates. The experimental design was completely randomized.

Antioxidant activity
The antioxidant assay was performed using the methodology as described by David et al. (2019) adapted, in the reduction of the free radical 2,2-diphenyl-1-picryl-hydrazyl (DPPH). DPPH stock methanolic solution, was prepared with a concentration of 40 µg mL-1 and kept refrigerated at -8 °C in a place free of light. The floral extracts were diluted in methanol in a 1:5 ratio (v/v). Then, an aliquot of 0.8 mL of the solution was added to 0.2 mL of the DPPH stock solution. The mixture was then kept at rest protected from light for 30 min in a refrigerator at -4 °C. After this time, the sample was read in a UV-Vis spectrophotometer at a wavelength of 555 nm. Distilled water was used as a negative control, and a standard ascorbic acid solution was used as a positive control. The ability to reduce DPPH was expressed in percentage (%) of sequestration according to equation 1. AA(%) = 100 -{[(Abs sample -Abs blank/Abs control)100]/Abs control} Eq. 1 Where: AA(%) = Abs absorbance of the sample; Abs absorbance of the blank, and Abs absorbance of the control.

Acetylcholinesterase inhibition
The colorimetric method for determining AChE inhibition followed as Where: A, B, C and D are the absorbances of the maximum enzymatic activity, reaction blank, enzymatic activity in the presence of the floral extract sample and the color of the sample solutions, respectively. The AChE assay was performed in triplicate.

Statistical analysis
The results are presented as mean ± standard. Tukey's test was applied to assess significant differences between samples, throughout the work, the level of significance is 5%.

Results and Discussion
Floral extracts from I. walleriana have been observed to contain an interesting number of special metabolic groups with positive qualitative results for phenols, anthocyanins, organic acids, cathequins, tannins, carboxylic acids, hemolytic saponins and sesquiterpenolactones (Table 1). Pandey & Tripathi (2014) and Shaikh & Patil (2020) consider that, after prior analysis of the qualitative composition of vegetables, it is necessary to quantify these groups of compounds by chromatography such as: gas chromatography, liquid chromatography, high-performance liquid chromatography, high-performance thin layer chromatography, etc.
For examples, flavonoids and tannins are phenolic compounds and plant phenolics are a major group of compounds that act as primary antioxidants or free radical scavengers (Bhandary et al., 2020); saponins have hypotensive and cardiodepressant properties; glycosides are naturally cardioactive drugs used in the treatment of congestive heart failure and cardiac arrhythmia (Brian et al., 1985;Olaleye, 2007). Reducing and non-reducing sugars provide protection against solar irradiance; organic acids show bacterial and fungicidal activity; alkaloids activities, anticholinergic (Table 3), antihypertensive, antimalarial, antitumor, antitussive, antiviral, etc. (Duarte et al., 2014). The floral extract of I. walleriana cv. Pink showed a maximum inhibitory activity of 26% on S. sclerotiorum (Table 2) while cv. White with only 8%. S. sclerotiorum showed some sensitization activity, this is possibly due to the special metabolites compound groups described in (Table 1). For the anthracnose strains C. gloeosporioides and C. acutatum growth inhibition was only observed for cv. Pink. The commercial fungicide showed 100% inhibition of fungal growth evaluated at a concentration of 10 μL mL -1 .
It is suggested that the phytocompound groups of both floral extracts of I. walleriana must be separated by high performance liquid chromatography (HPLC) and tested separately in future studies inorder to verify which or which groups present antifungal activity on the tested phytopathogens. The floral extracts of I. walleriana demonstrate reducing activity on the free radical DPPH (Table 3) however, lower than the standard of ascorbic acid (vitamin C) with 96.48% a . Menezes Filho et al. (2021) describe for the floral extract of Tabebuia impetiginosa potential activity with an inhibition concentration (IC50) = 21.18 µg mL -1 , which is a promising result, mainly in the production of cosmetic or food products with the ability to reduce free radicals. Kakimori et al. (2019) also obtained promising results from the flora extract of banana (Musa paradisiaca) with a 43.03% reduction equivalent to IC50 = 0.2765 mg mL -1 . In the AChE enzyme inhibition, both extracts showed promising results, especially for cv. Pink (Table 2). Important results were also described by Marques et al. (2013) evaluating the ethanolic extract of the flowers of Bellis perennis (Asteraceae f.) between 38.13 to 55.83%. Results greater than 30% are considered moderate and greater than 50% potent AChE inhibiting agents.

Conclusions
The floral extracts of Impatiens walleriana cv. Pink and White have potentially able phytochemical groups having wide importance in agriculture, food, pharmacology and in bio-industrial use. Significant effects were observed mainly in the antifungal activities on the phytopathogens Sclerotinia sclerotiorum and Colletotrichum acutatum, as an antioxidant in the reduction of free radical DPPH and inhibition of acetylcholinesterase, especially for cv. Pink. However, future studies should be carried out by evaluating and elucidating by means of chromatography; the quantitative determination of each compound within the groups of phytocompounds observed and thus analyzing separately that which compounds are responsible for such activities.