Qualitative Screening of Phytocompounds and Spectrophotometric Investigations of Two Pumpkin Species ^†

Abstract

Pumpkin (Cucurbita maxima) is a fruit packed with vitamins and nutrients beneficial to human health with numerous therapeutic uses including antiparasitic, antioxidant, it helps to lower bad cholesterol, as an adjuvant in weight loss, improves cancer prevention, etc. Pumpkin is rich in beta-carotene, and contains significant amounts of lutein and zeaxanthin, antioxidants that can considerably prevent cataracts and macular degeneration. Worldwide, five pumpkin species are grown for their edible fruit and seeds. This paper describes the qualitative screening of phytocompounds and the quantitative determination of the main bioactive compounds found in two pumpkin species: Valenciano and Waltham Butternut. The qualitative screening of phytochemicals was based on the visual change in color of aqueous extracts upon adding known reactants. This allowed a preliminary evaluation regarding the presence of different bioactive compounds such as saponins, alkaloids, tannins, flavonoids, etc. In order to determine the specific amount of different phytocompounds (e.g., total content of polyphenols, total content of flavonoids, etc.) UV-Vis spectra were recorded in triplicate at well-established wavelengths, thus obtaining an average absorbance. For example, a method widely applied for the determination of total polyphenolic content is the Folin–Ciocalteu (FC) reaction, which is basically an antioxidant analyses that relies on electron transfer that measures the reductive ability of a specific antioxidant. Briefly, the FC reaction involved mixing 1 mL diluted aqueous extract with 5 mL FC reagent and adding 4 mL Na₂CO₃ after 8 min After 60 min incubation at room temperature, we recorded the absorptions at 765 nm, which corresponds to the gallic acid curve calibration standard. Also, the antioxidant activity was recorded by using the DPPH method for both aqueous extracts.

1. Introduction

Cucurbita maxima (pumpkin, squash, gourd) is an important representative of the Cucurbitaceae family and is among the top cultivated crops in the world, especially in temperate and subtropical climates [1]. The squash pumpkin has gained greater recognition in the past years, mainly due to the nutritional and health benefits of its seeds, which are packed with nutraceutical compounds such as polysaccharides, carotene, minerals and vitamins [2]. The health benefits of different squash pumpkin species have been reported previously, including that they can lower blood glucose [3] and can even prevent some types of diabetes and reduce their complications [4]. Squash pumpkin contains an impressive amount of vitamin A, that is, 245% of the reference daily intake (RDI), is low in calories as it contains over 94% water, and contains significant amounts of antioxidants (e.g., alpha-carotene, beta-carotene and beta-cryptoxanthin) that can neutralize the action of free radicals [5].

Plants contain multiple and different radical scavenging molecules that are therapeutically active, including alkaloids, amines, betalains, vitamins, phenolic acids, terpenoids. Most phytochemicals are antioxidant agents, which essentially reduce the damages caused in tissue during physiological processes. The pharmaceutic value of plants depends on their bioactive compounds, which exhibit different physiological effects on human health. Therefore, screening for bioactive compounds allows the detection of various components that can be used as a starting point for modern drugs that can treat different diseases [6]. These “metabolic chemicals” are better known as “secondary metabolites” [7] and they include alkaloids, flavonoids, coumarins, tannins, terpenes, terpenoids, phenols, polysaccharides and glycosides [8].

There are many known bioactive compounds, each with their own health action [9,10], including:

• antioxidant, meaning that it protects the human cells from the oxidative stress;
• hormonal, especially isoflavones that can imitate human estrogens;
• antibacterial;
• physical, bioactive compounds can physically attach to cell walls.

This paper describes the qualitative screening of phytocompounds and the quantitative determination of the main bioactive compounds found in two pumpkin species: Valenciano and Waltham Butternut. The fresh samples were air-dried at room temperature for 10 days under no direct sunlight followed by an aqueous extraction at a controlled temperature of 4 °C in the refrigerator. The qualitative screening for bioactive compounds involved the color-changing reaction that results from mixing different extracts with known reagents, while the quantitative screening for bioactive compounds was based on spectrophotometric determinations at specific and well-established wavelengths. The results are presented as a comparison of the two squash pumpkin species and clearly show that both Valenciano and Waltham Butternut are important sources of bioactive compounds with numerous health benefits.

2. Materials and Methods

2.1. Materials

DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate), hydrochloric acid (HCl), sulfuric acid (H₂SO₄), copper sulphate (CuSO₄), copper acetate (Cu(CH₃COO)₂), silver nitrate (AgNO₃), aluminum chloride (AlCl₃), lead acetate (Pb(Ch3COO)₂), catechin standard, gallic acid standard, Folin–Ciocalteu reagent, ferric chloride (FeCl₃), glacial acetic acid (CH₃COOH), ammonium molybdate, Benedict and Millon reagents were purchased from Sigma-Aldricht. Ethanol (C₂H₅OH), methanol (CH₃OH), chloroform (CHCl₃) and sodium hydroxide (NaOH) were purchased from Scharlau. The distilled water was freshly prepared in the laboratory.

2.2. Preparation of the Aqueous Extracts from Cucurbita maxima

Both types of pumpkin (Valenciano and Waltham Butternut, Figure 1) used in the present research study were taken from a local homemaker in Gradistea, Giurgiu, Romania who grew them in an eco-friendly environment with no chemicals or additives.

All the component parts (e.g., shell, core and seeds and equal combinations of these parts) were carefully separated, thoroughly washed twice with tap water, thrice with freshly prepared distilled water, dried at room temperature for 10 days under no direct sunlight, finely ground and used to prepare the corresponding aqueous extracts. The protocol used to prepare the aqueous extracts was the same for both Cucurbita maxima squash pumpkin species and involved the following steps:

• 25 g dried squash parts (e.g., shell, core, seeds and equal mixtures of them) were weighed, transferred into a glass extractor and infused with 250 mL distilled water;
• the solutions were kept for 24 h in a refrigerator (4 °C) to infuse;
• the aqueous extracts were filtered until a clear liquid was obtained;
• the aqueous extracts were stable at 4 °C for more than 4 months.

2.3. Qualitative Screening for Bioactive Compounds

The qualitative screening for bioactive compounds uses standard analytical methods that rely on a color change reaction as a positive response [11].

Test for tannins: 2 mL of 5% FeCl₃ was added to 1 mL aqueous extract, and the formation of a dark blue or greenish black solution confirms the presence of tannins.

Test for saponins: 2 mL aqueous extract was mixed with 2 mL distilled water and vigorously shaken lengthwise, using a graduated cylinder, for 15 min The formation of a 1 cm foam layer confirms the presence of saponins.

Test for alkaloids:

(a)

Mayer test: 2 mL of concentrated HCl was added to 2 mL aqueous extract, followed by a few drops of Mayer reagent (potassium mercuric iodide). The formation of a green solution or white precipitate indicates the presence of alkaloids.

(b)

Wagner test: 1 mL Wagner reagent (iodine in potassium iodide) was added to 3 mL aqueous extract. A reddish-brown precipitate indicated the presence of alkaloids.

(c)

Hager test: 1 mL Hager reagent (saturated picric acid solution) was added to 3 mL aqueous extract, and if a yellow precipitate forms, then alkaloids are present.

Test for flavonoids: 2 mL aqueous extract was mixed with 1 mL 2N NaOH. This results in the formation of a yellow color that disappears after adding dilute HCl.

Test for proteins and amino acids:

(a)

Millon test: 1 mL aqueous extract reacts with 5–6 drops of Millon reagent and a white precipitate appears that changes its color to red at heating.

(b)

Biuret test: 3 mL 4% NaOH solution and few drops of 1% CuSO₄ were added to 3 mL aqueous extract, and a purple solution is formed.

(c)

Ninhydrin test: 3 drops of 5% Pb(CH₃COO)₂ were added to 3 mL aqueous extract and heated for 10 min A purple or blue color is a positive response.

(d)

Cysteine test: a few drops of 40% NaOH and 5% were added to 5 mL aqueous extract, and boiled for 5 min The solution turns purple or blue or a black precipitate of lead sulphate is formed.

(e)

Xantoprotein test: 1 mL conc. H₂SO₄ was added to 3 mL aqueous extract. First a white precipitate is formed, which turns yellow upon boiling and orange after adding 1 mL NH₄OH.

2.4. Quantitative Determinations of Bioactive Compounds

The quantitative determination of bioactive compounds was also carried out to determine the total content of tannins (TCF), total content of flavonoids (TCF) and total content of polyphenols (TCP) [12,13].

The antioxidant activity (AA, %) was also determined, and for that a 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) solution was prepared in C₂H₅OH and 0.5 mL aqueous extract was mixed with 1 mL 0.02 mg/mL DPPH solution. Then, the absorbance was recorded at 517 nm. A blank was also prepared: 0.5 mL distilled water was mixed with 1 mL 0.02 mg/mL DPPH solution. The antioxidant activity was calculated using the formula:

AA % = [(A_Control™ A_Sample)/A_Control] × 100

where A_Control is the absorbance of the blank DPPH solution and A_Sample is the absorbance of the aqueous extract mixed with 0.02 mg/mL DPPH solution.

3. Results and Discussions

3.1. Preparation of the AQUEOUS Extracts from Pumpkin Squash Species

The protocol used to prepare the aqueous extracts was the same for both Cucurbita maxima squash pumpkin species. For both species of squash pumpkin, five different aqueous extracts were prepared from three different parts of squash pumpkin as follows:

• three simple aqueous extracts from only one part, e.g., core, shell and seeds;
• two combined aqueous extracts from the core and shell in equal amounts, and the core, shell and seeds in equal amounts.

The resulted aqueous extracts were kept 24 h in a refrigerator (4 °C) to infuse, filtered until a clear liquid was obtained and kept in the refrigerator for more than 4 months.

3.2. Qualitative Screening for Bioactive Compounds

The qualitative screening for tannins clearly showed that they are absent in both squash pumpkin species, and the same conclusion was drawn for steroids and triterpenoids.

The qualitative screening for flavonoids showed a positive response only in the case of the aqueous extract prepared from the seeds of Valenciano pumpkin while in the case of Waltham Butternut pumpkin the presence of flavonoids could be visually observed for the seeds and the complex aqueous extract prepared from all three constituents.

Proteins are the main components of protoplasm and they are involved in all the natural processes that happen in all living cells. In nature, all proteins are colloidal and are irreversibly coagulated at a temperature higher than their boiling point. Proteins are not soluble in neutral salts (e.g., NaCl, MgSO₄) and only solubilize once the salts are diluted. On the other hand, the majority of amino acids are soluble in water.

The qualitative analysis of amino acids refers to a color change, precipitation or even ring formation that appears as a result of a modification in the structural configuration upon reacting with a reagent. The results for the qualitative screening of carbohydrates in both squash pumpkin species are detailed in

Table 1. Qualitative screening of proteins and amino acids in Valenciano pumpkin.

Phytochemical Test	Valenciano Shell	Valenciano Core	Valenciano Seeds	Valenciano Core and Shell	Valenciano Core, Shell and Seeds
Millon	+	++	+++	+++	+++
Biuret	+	-	+++	++	++
Xantoprotein	-	+++	-	++	+++
Ninhydrin	-	-	-	-	-
Cysteine	-	-	+	-	+

note: “+” present; “-” absent.

for Valenciano pumpkin and

Table 2. Qualitative screening of proteins and amino acids in Waltham Butternut pumpkin.

Phytochemical Test	Waltham Butternut Shell	Waltham Butternut Core	Waltham Butternut Seeds	Waltham Butternut Core and Shell	Waltham Butternut Core, Shell and Seeds
Millon	+++	+++	-	++	+++
Biuret	+	-	-	-	+
Xantoprotein	+	+	-	+	+
Ninhydrin	-	-	-	+	+
Cysteine	-	-	-	-	-

note: “+” present; “-” absent.

for Waltham Butternut pumpkin.

The qualitative screening for proteins and amino acids in Waltham Butternut pumpkin squash clearly reveals that cysteine, a non-essential sulfur-containing amino acid, is absent from all the aqueous extracts while in the case of Valenciano pumpkin squash, cysteine can be found only in the seeds and the complex mixture of core, shell and seeds. The results in Table 2, and specifically for the aqueous extract prepared from the seeds of Waltham Butternut pumpkin squash, clearly show that they do not contain protein or amino acids.

Alkaloids can be analyzed as a bioactive compound by using three different qualitative tests and the results are presented in

Table 3. Qualitative screening of alkaloids for Valenciano pumpkin.

Phytochemical Test	Valenciano Shell	Valenciano Core	Valenciano Seeds	Valenciano Core and Shell	Valenciano Core, Shell and Seeds
Mayer	++	+	+++	+++	+++
Wagner	+	+	+	+	+
Hager	+	++	+++	+++	+++

note: “+” present.

for Valenciano pumpkin squash and

Table 4. Qualitative screening of alkaloids for Waltham Butternut pumpkin.

Phytochemical Test	Waltham Butternut Shell	Waltham Butternut Core	Waltham Butternut Seeds	Waltham Butternut Core and Shell	Waltham Butternut Core, Shell and Seeds
Mayer	+	+	+	+	+
Wagner	+	+	+	+	+
Hager	++	+++	++	++	+++

note: “+” present.

for Waltham Butternut pumpkin squash.

The results detailed in Table 3 and Table 4 prove that, whatever the qualitative test used to screen for alkaloids, they are present in all ten aqueous extracts and the only difference is the intensity of the resulting solution.

Saponins represent another bioactive compound that was screened for in all the aqueous extracts and the results show that they are present in 8 out of 10 of the aqueous extracts, the exception being the seeds of both pumpkin squash species.

3.3. Quantitative Determination of Bioactive Compounds

The quantitative determination of bioactive compounds was carried out to determine the total content of tannins (TCT), flavonoids (TCF) and total content of polyphenols (TCP) (

Table 5. Quantitative methods for the determination of bioactive compounds.

Assay	Reagents	Reaction Parameters	Recordings
TCT	0.5 mL extract+3 mL 4% vanillin-MeOH and 1.5 mL HCl	15 min incubation	Absorbance at 500 nm
TCF	1 mL extract+4 mL distilled water and 0.3 mL 5% NaNO2; after 5 min: 0.3 mL 10% AlCl3; after other 5 min: 2 mL 1 M NaOH and 2.4 mL distilled water	30 min incubation	Absorbance at 510 nm (catechin curve calibration standard)
TCP	1 mL diluted extract and 5 mL Folin–Ciocalteu reagent; after 8 min: 4 mL Na2CO3	60 min incubation	Absorbance at 765 nm (gallic acid curve calibration standard)

).

The number of total tannins and the total content of flavonoids (Figure 2) are described as mg catechin/L and the ten aqueous extracts were analyzed in triplicate. The total content of polyphenols uses gallic acid as a standard calibration curve. The results are detailed in the charts below.

Based on the detailed charts that represent the total content of flavonoids (TCF) for both pumpkin squash species it can be concluded that in the case of Valenciano pumpkin, the TCF was highest in the aqueous extract prepared from the core, followed by the mixture of core and shell, and the core and shell and seeds while seeds have the lowest concentration of TCF. In the case of Waltham Butternut pumpkin squash, the highest concentration was obtained from the core, followed by the mixture of the core and shell.

The total content of tannins (TCT) was investigated as mg catechin/L and the results are presented in Figure 3.

By analyzing the charts in Figure 3, it can be concluded that the highest concentration of TCT was found in the core of both Valenciano and Waltham Butternut pumpkin squash (373.1946 mg/L for Valenciano and 500.244 mg/L for Waltham Butternut).

The total content of polyphenols (TCP) uses gallic acid as a standard calibration curve and was investigated as mg gallic acid/L and are detailed in Figure 4.

The antioxidant activity (AA %) was measured using DPPH as the standard:

AA % = [(A_Control − A_Sample)/A_Control] × 100,

where A_Control is the absorbance of the blank DPPH solution and A_Sample is the absorbance of the aqueous extract mixed with 0.02 mg/mL DPPH solution. The results are presented in

Table 6. Antioxidant activity for pumpkin squash species.

AA (%)	Core	Shell	Seeds	Core and Shell	Core, Shell and Seeds
Valenciano	37.62	54.52	42.65	23.54	13.48
Waltham Butternut	37.22	54.12	41.85	21.73	12.87

as a comparison between the two pumpkin squash species.

From this table it can be concluded that the highest values were obtained for the shell aqueous extracts prepared from the shell of the two pumpkin squash species.

4. Conclusions

This research study describes two different species of squash pumpkin, Valenciano and Waltham Butternut, which were investigated by means of qualitative and quantitative phytochemical screening to determine the bioactive compounds. The qualitative screening for bioactive compounds involves a color change reaction that appears when mixing different extracts with known reagents and the quantitative screening for bioactive compounds is based on spectrophotometric determinations at well-established wavelengths.

The qualitative screening for tannins clearly showed that they are absent in both squash pumpkin species. The qualitative screening for proteins and amino acids in Waltham Butternut pumpkin squash clearly revealed that cysteine was absent in all the aqueous extracts. Alkaloids were also screened for and it was found that they were present in all ten aqueous extracts.

The quantitative determination of bioactive compounds was carried out to determine the total content of tannins (TCF), total content of flavonoids (TCF) and total content of polyphenols (TCP). In the case of Valenciano pumpkin, the TCF was highest in the aqueous extract prepared from the core (162.1772 mg/L), followed by the mixture of core and shell (95.8984 mg/L), and the core and shell and seeds (66.1759 mg/L) while seeds had the lowest concentration of TCF (22.3224 mg/L). In the case of Waltham Butternut pumpkin squash, the highest concentration was obtained for the core, followed by the mixture of core and shell.

The highest concentration for total content of tannins (TCT) was found in the core of both Valenciano and Waltham Butternut pumpkin squash (373.1946 mg/L for Valenciano and 500.244 mg/L for Waltham Butternut). From the results obtained for the antioxidant activity it can be concluded that the highest values were found in the shell aqueous extracts prepared from the shell of the two pumpkin squash species (54.52716% for Valenciano pumpkin and 54.12475% for Waltham Butternut).

The results presented clearly show that both Valenciano and Waltham Butternut are important sources of bioactive compounds with numerous health benefits.