Comparative assessment optimization and characterization of bioactive constituents from Amarantus species: An indigenous lesser-known vegetables of India

Amaranthus Tricolor L. (red amaranth) and Amaranthus Viridis (Green amaranth) are Amaranthaceae members, widely cultivated in Asia and consumed as a leafy vegetable in many parts of the world. This study deal with the nutritional and functional, and biochemical characterization of A. Tricolor and A. viridis. Single-factor experiments and Box Behnken Design (BBD) were used to optimize the extraction process for the phenolic compound from A. Tricolor and A. viridis. The BBD shows that 11.87mg/g phenolic extract of A. Tricolor produced at the optimal condition of solid to water ratio (1:15), temperature (30°C), and time (15 min). Similarly, A. viridis isolated 18.36mg/g of phenolics at optimal condition solid to liquid ratio (1:30), temperature (30°C), and time (60 min). The radical scavenging activity of A. Tricolor and A. viridis shows 63.52% and 19.27%, respectively, by the DPPH method. The bioactive compounds 3,4,5Trihydroxystilbene and caffeic acid were found in A. Tricolor, and in A. viridis it showed caffeic acid, which was identified using LC-MS/MS.


Introduction
Small, frequently green or reddish flowers clustered in dense clusters, strongly coloured stems and leaves, and dry, indehiscent, one-seeded fruit define Amaranth spp. The genus Amaranthus contains around 60 species that are found throughout the world in temperate, subtropical, and tropical climates, with at least 17 species having edible leaves and three grain amaranths. Amaranthus is a widely grown plant with a high genetic and particular diversity that is used for food (as a pseudo cereal plant), feeding, and ornamentation. Despite the fact that some species are commonly considered weeds, amaranths are valued as green vegetables, grains, and ornamentals by people all across the world. Amaranthus species are widely distributed throughout the world's temperate and tropical regions even before man converted some of them into cosmopolitan weeds and domesticated others (Das 2016). Schröter et al. (2018) provided information about presence of secondary metabolites in six different species of Amaranth, which strongly recommended to consider crop for food consumption which delivers the health benefits associated with it.

Amaranthus Tricolor L. (red amaranth) and
Amaranthus Viridis (green amaranth) are leafy vegetables produced mainly in Africa and Asia, which belong to the family Amaranthaceae. Amaranthus tricolor L. (A. tricolor) leaves consist of health beneficiaries bioactive compounds such as antioxidants, polyphenols, and betacyanin. These could be a potential source for anti-inflammatory and antioxidant (Khandaker et al. 2008). These are also known for protein sources with a balance of essential amino acids, minerals (iron and calcium), and vitamin C (Islam et al. 2003).
Amaranthus Viridis (A. viridis ) reported bioactive properties such as antioxidant activity, phenolic acid, and improved nutritional quality due to the drought stress (Sarkar and Oba 2018). A. viridis is a good source of antioxidants, phenolic acids, and vitamin C (Datta et al. 2019). The extract of A. viridis has been utilized in the bread fortification to enrich with a polyphenolic group with increasing antioxidant properties (Alashi et al. 2018).
The various pharmaceutical, food, and beverage industries demanded natural antioxidant from plant sources, as demand increases as ingredient/ dietary supplements or functional food to avoid the effect of synthetic antioxidants on human health (Ilaiyaraja et al. 2015;Arnáiz et al. 2016). Polyphenols playing key role in the health benefits which contribute as antihypertensive, anti-obesity, anti-diabetic, anti-hyperlipidemic and antiinflammatory effects (Cherniac 2011). Polyphenols are thought to be good for your health, therefore they could be used in new ways to prevent diabetes and obesity issues (Mihaylova et al. 2018).
Solvent extraction is an efficient method widely used to isolate phenolic compounds from the fruits and vegetable sources with a maximum yield of target compounds with the highest quality and antioxidant activity (Spigno et al. 2007). The increased extraction efficiency for bioactive compounds is influenced by using a combination of solvents. Advanced techniques like pressurized liquid extraction are used nowadays to extract bioactive compounds from plant sources which are observed to more efficient (Li et al. 2002;Petkova et al. 2020;Trifonova et al., 2021). Water and ethanol and a combination of the two were the most popular solvents for extracting bioactive chemicals for food applications (Mihaylova and Lante, 2019). Lante et al. (2018) utilzed water as solvent for extraction of soy isoflavones. The various extraction parameters like solvent, temperature, time, particle size, and solid-to-liquid ratio impact extraction (Dent et al. 2013).

A. tricolor and A. viridis powder preparation
A. tricolor and A. viridis were collected from APMC market, Navi Mumbai, India (a local market) in one lot. The washed and clean leaves of vegetables were dried overnight at 60°C. These leaves ground and powder were stored in polyethylene bags at 4°C to carried out research uniformly (Sonawane and Arya 2015).

Proximate analysis of A. tricolor and A. viridis
The AOAC (1995) methods estimate moisture, fat, protein, fat, and mineral from A. tricolor and A. viridis .

Analysis of moisture content
The samples were analyzed for their moisture content by heating at about 105ºC in a hot air oven for 5 h (AOAC Official method 931.04 -31.1.02).

Analysis of fat content
The crude lipids were estimated by the Soxhlet extraction method (using instant Soxhlet apparatus -Socs Plus, Pelican equipment, Chennai, India). The fat was extracted using petroleum ether (b.p. 60-80ºC) as solvent (AOAC Official method 920.39 -4.5.01).

Analysis of ash
The samples were analyzed for their mineral content by heating it in a muffle furnace at about 570ºC for 5 h (AOAC Official method 972.15 -31.1.04). The charred mass left behind stands for the ash content of samples.

Analysis of protein
The protein content of samples was analyzed by determining the nitrogen content by Kjeldahl method and then using a conversion factor of 6.25 (AOAC Official method 970.22 -31.1.08).

Carbohydrate content
Finally, the carbohydrate content of samples was calculated on a dry weight basis by a difference as follows.

Experimental design for extraction of polyphenols from A. tricolor and A. viridis
The extraction of polyphenols from A. tricolor and A. viridis to obtain from preliminary data (not shown) with a suitable range for temperature (30 to 50ºC), time range for A. tricolor (15 to 45 min), and for A. viridis it ranges from 60 to 120min, solid to solvent ratio A. tricolor (1:10 to 1:30) and for A. viridis it ranges from for (1:30 to 1:50).
In preliminary data, water was suitable solvent to extract maximum amount of polyphenols. A threelevel-three-factor BBD (Box-Behnken design, Design-Expert1 6 software, Stat-Ease Inc., Minneapolis, USA) was employed to enhance extraction parameters for polyphenols from A. tricolor and A. viridis. These designed used various combinations between temperature (A, ᵒC), time (B, min), and the ratio of solid to liquid (C, w/v)) to enhance the extraction of polyphenols from A. tricolor and A. viridis. These provide 17 combinations for the experiment using the above extraction parameters to keep total phenolic content, as shown in Table 1.
The experimental data obtained were fitted by the following regression equation: Whereas Y is the total phenolic content (predicted response), β0, β1, β2,……… β33, where the regression coefficient, A-temperature, B-time, Csolid to liquid.

Total phenolic content of A. tricolor and A. viridis extract
The phenolic content of extracts from A. tricolor and A. viridis was investigated using the Folinciocalteau method described by Singh et al. 2002;Sonawane and Arya, 2013;. The results were reported in mg of GAE/g of a sample.

Total flavonoid estimation in A. tricolor and A. viridis extract
The estimation of total flavonoid content in the extract of A. tricolor and A. viridis was carried out using Parimala and Shoba (2013). The extract (0.5mL) mixed with methanol (4.5mL) with addition of 10% aluminium chloride (0.1mL).
Further, potassium acetate (0.1mL) was added to the mixture and kept in the dark for 30min incubation, and absorption was measured at 415nm. The results expressed as quercetin equivalent (mg/g).

Estimation of tannin content in A. tricolor and A. viridis extract
The estimation of tannin in A. tricolor and A. viridis was carried out using Mohan et al. (2016). Initially, 0.2g of A. tricolor and A. viridis was taken with 70% aqueous acetone (10 mL) for 30ºC for two hours in an ice bath shaker to extract tannin. The supernatant was collected by using a centrifuge at 8000 RPM. The 0.2mL of extract+0.8ml of distilled water+0.5ml Folin-ciocalteu's reagent+2.5ml of sodium carbonate (20%) was incubated for 40 min, and absorbance measured at725 nm.

Antioxidant activity estimation in A. tricolor and A. viridis extract
Antioxidant capacity of A. tricolor and A. viridis extract were measured by using DPPH (radical scavenging activity) reported by Sonawane and Arya (2015) and Sahreen et al. (2012), and results are expressed as % radical scavenging activity by using the following formula: where

Statistical analysis
All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation. The SPSS (Statistical Package for Social Sciences) for Windows version (16.0) was used to analyze the data (SPSS Inc., Chicago, IL). Statistical significance was considered at p<0.05.

Response surface methodology
The effect of extraction parameters such as temperature (A), time (B), and solid-to-liquid ratio (C) on phenolic material extraction from A. tricolor and A. viridis leaves was investigated using a BBD design. The one factor that helps in designing an experiment based on total phenolic content as a response. Table 1 shows the experimental outcomes obtained by design. The various combinations of extraction parameters led to the extraction of phenolic contents varying from 7.55 to 12.65 mg/g for A. tricolor and 13.03 and 20.09 mg/g for A. viridis.

Model fitting
Furthermore, in our experiment, efforts were made to investigate interaction of temperature, time, and solid to liquid ratio on the total phenolic content using BBD. The ANOVA of the polyphenolic extract is shown in Table 2 which shows that model is fitted for the extraction parameters. The best explanatory model provided in Eq. (1) is given in Eq. (2) and (3) (3) The lack of fit test for A. tricolor was an insignificant p-value of 0.1184) which indicates data in the experiment fitted well and acceptable to describe observed data. The results are presented in Table 3. The R 2 = 0.9838 suggested that the fitted model can explain 98.38% of the variation in the data.   Also, a high correlation was observed between Adj R 2 (0.9629) and Pred R 2 (0.8022). If the value of R 2 is more or equal to 0.80 shows that the model is suitable for directing design space (Guan and Yao 2008). This model had a CV (coefficient of variation) of 2.71 %, suggesting an excellent precision and high reliability of the experiment achieved. Similarly, for A.viridis, the lack of fit test shows that the model was insignificant (p-value was 0.2239), indicating data in the analysis fitted well and acceptable to describe observed data (shown in Table 2). The R 2 = 0.9888 suggested that the fitted model can explain 98.88% of the variation in the data. Also, a high correlation was observed between Adj R 2 (0.9744) and Pred R 2 (0.8810). This model had a CV (coefficient of variation) of 2.39%, suggesting an excellent precision and high reliability of the experiment achieved.

Interpretation of the response surface model
The models' three-dimensional response surface and contour plots are shown by graphical representation in Figure 1 and Figure 2 which shows the interaction between each parameter to the corresponding response of the phenolic content of A. tricolor and A.viridis. Figure 1 (a) shows that the highest solid to solvent ratio and extraction time of A. tricolor shows the lowest amount of phenolic content was extracted. Also, as solid to liquid ratio concentration increases, phenolic extraction increases up to a certain level and further decreases as the solid to liquid ratio increases. Figure 1 (b) shows the interactive effect of extraction temperature and solid to liquid ratio. The temperature above 40ºC led to a decrease in the phenolic content as solid to liquid ratio increases above 1:20. But, in the case of Figure  1 (c), interactive plots show that higher extraction time and temperature lead to degradation of phenolic content in A. tricolor. A similar kind of response of phenolic extraction for the interaction of solid to solvent ratio, extraction time, and the temperature observed in Solanum macrocarpon (Famuwagun et al. 2017). In A.viridis, Figure 2 (a) shows that total phenolic concentration decreases as an increase in the solid to liquid ratio and time. In Figure 2 (b), interactive plots, solid to liquid ratio versus temprature shows that, as solid to liquid ratio increas, which decrease the phenolic content, but increase in the temprature leads to enhancement of the phenolic extraction. But, time and temprature interation in Figure 2(c) shows that if it is increases which lead to decrease in the phenolic content.

Characterization of a polyphenolic extract of A. tricolor and A.viridis
The polyphenolic composition of an extract of A. tricolor and A. viridis is shown in Table 3. The extract of A. tricolor and A. viridis contained 11.87 mg/g and 18.36 mg/g of total phenolic content, and 63.53 and 19.87% DPPH activity, respectively. The total flavonoids content (7.23 and 14.11 mg/g) and tannin content (0.57 and 0.97 mg/g) were found in the extract of A. tricolor and A.viridis. Sarkar and Oba (2020) was obsrved the variation in total phenolic content 72.69 to 153.08 μg/g FW basis, total flavonoids content 30.89 to 50.95 μg/g FW basis in red amaranth. Iqbal et al. (2012) reported that 10.3 µg/g of phenolic and 27.8 µg/g of flavonoid extracted from A. viridis which corresponds to 14.25 DPPH, (μg/mL). Amaranth is a rich source of antioxidants in vegetables, ranging from 2.95 -3.75 GAE, mg/100 g (Pasko et al. 2009). viridis) to 54.7 (A. tricolor), respectively (Bang et al. 2021).

Identification of compounds in extract of A. tricolor and A. viridis by using LC-ESI-Q-TOF-MS/MS
The compounds present in the extract of A. tricolor and A. viridis are presented in Table 4. Aspartic acid, 3,4,5-Trihydroxystilbene, and caffeic acid were found in A. tricolor, whereas A. viridis shows the presence of aspartic acid n-acetyl-dgalactosamine and caffeic acid. 3,4,5-Trihydroxystilbene, known as a phytoalexin, showed its presence in grape skin and was generated by many plants. Schröter et al. (2018) found novel hydroxycinnamic acid derivatives in secondary metabolite profile of Amaranth, leaves, which confirmed by Random Amplification of Polymorphic DNA (RAPD)-PCR fingerprinting. A. tricolor was found very high hydroxycinnamic acid concentrations (>20 mg·g−1 DW) (Schröter et al., 2018). According to Perrone et al. (2017), 3,4,5-Trihydroxystilbene possesses antiviral, antimicrobial, anticancer, anti-inflammatory, immunomodulatory, cardioprotective and neuroprotective properties.
Caffeic acid falls in the class of polyphenol derivatives, and an intermediate in the biosynthesis of lignin represents an antioxidant. Different fruit and vegetable species are a rich source of these compounds (Jakobek et al. 2007).

Conclusions
In conclusion, A. tricolor and A. viridis were good sources of nutritious and non-nutritive characteristics. In extraction, water was a suitable solvent for extracting maximum polyphenolic extract from A. tricolor and A. viridis. The extraction parameter like a solvent, solvent to solid ratio, temperature, and time was useful in extracting phenolic content in A. tricolor 10.47mg/g and A. viridis 13.13mg/g by using one factor. The BBD design obtained 11.87mg/g at optimal condition solid to water ratio (1:15), temperature (30°C), and time (15 min) for A. tricolor and in case of A.viridis, it extracts 18.36mg/g at optimal condition solid to liquid ratio (1:30), temperature (30°C) and time ( 60 min). A. tricolor shows the highest antioxidant activity against DPPH compared to A.viridis. Caffeic acid is an active polyphenolic derivative found in both A. tricolor L. and A. viridis by using LC-ESI-Q-TOF-MS technique. The extracts thus obtained shall find a wide range of application in food fortification practices including sherbets, fruit leathers, fruit bars.