Many devices and materials were used to complete the requirements of this study, including Power Sonic-405 (DAIHAN LABTECH(LTD), Namyangju-si, South Korea), Soxhlet apparatus (Daihan scientific, Wonju, South Korea), High performance liquid chromatography (HPLC) (Sykam, Eresing, Germany), and Spectrophotometer (Jenway, Germany). We also used many Materials including Quercetin (manufactured by the American company Microingredients), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (manufactured by Sigma-aldrich, Steinheim, Germany), Snailase enzyme (manufactured by Solarbio, Beijing, China).

Table 1 (Ref. [12, 13, 14, 15, 16, 17, 18]) shows all the plants used in the study, their cultivation areas, and harvest periods.

Fresh plants were obtained at the time of their harvest and availability in the markets, such as spinach, dill, and chemri (digalkhasab chemri, and leelwi chemri). They were then cleaned and washed, and the pits and funnels were removed from the chemri. After that, they were dried in the sun. As for the dry plant matter, such as onion peels, sumac, and Pistacia eurycarpa, which are available throughout the year in markets, these were purchased and then individually ground into a fine powder using an electric grinder. The powder was then stored in sterile glass containers with airtight seals and kept in a freezer until used in the experiments.

The content of total flavonoids was estimated using the aluminum chloride method with quercetin as a standard according to the method followed by Dewanto et al. [19]. First, 0.1 mL of the extracts was mixed with 0.9 mL of distilled water in test tubes, after which 75 µL of NaNO₂ (5%) was added. After 6 minutes, 150 µL of aqueous aluminum chloride (AlCl₃·5H₂O 10%) was added and left for 5 minutes, then 0.5 mL of NaOH (1 M) was added. Next, 2.5 mL of distilled water was added and shaken well, immediately after which the absorbance was measured at a wavelength of 510 nm using a spectrophotometer.

A standard solution of quercetin was prepared according to the content of flavonoids in plant extracts at different concentrations (20–140 mg/mL). The absorbance was measured at a wavelength of 510 nm, and the content of flavonoids was calculated based on the graphical relationship between the concentration of quercetin and the absorbance.

The antioxidant activity of both standard and purified quercetin was estimated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical capture method [22], with some modifications. The method included preparing several concentrations (5, 10, 15 mg/mL) using water once and ethanol again by mixing 2 mL of different concentrations with 2 mL of (DPPH) solution concentration of 0.1 mM prepared in methanol, while the control sample was prepared by mixing 2 mL of distilled water instead of the sample. After that, the industrial antioxidant butylated hydroxytoluene (BHT) was used for comparison. This was prepared at a concentration of 0.01 mg/mL in methanol and kept in the dark at a temperature of 25 °C for 30 minutes, after which the absorbance was measured at a wavelength of 517 nm according to the following equation:

(2)$$\%\text{of antioxidant activity}=[(\mathrm{Ac}-\mathrm{As})÷\mathrm{Ac}]\times 100$$

where Ac is the control reaction absorbance and As is the testing specimen absorbance.

Statistical analysis of the data was conducted using completely randomized designs (CRD) for factorial experiments, and the least significant difference (LSD) test was used to calculate the significant differences between the averages of the coefficients at the 0.05 probability level using SPSS version 26 (IBM SPSS statistics, Chicago, IL, USA).

Plant extracts obtained from a wide range of plants are of great importance as they contain many biologically active compounds. These resources have received much scientific interest due to their positive effects on health, demonstrating nutritional, economic, and medicinal importance. These extracts can be sourced from fruits, vegetables, medicinal herbs, spices, and so forth, and their consumption is closely linked to improving the health of the digestive system and reducing the risk of many diseases, as there is an inverse relationship between the consumption of fruits and vegetables and many diseases. Therefore, choosing an effective extraction method and the appropriate extraction solution to ensure the optimal concentration and high performance are obtained is the most important step in the extraction of these products [23, 24]. In the present study, quercetin was extracted and quantified from the plants under study based on the standard curve shown in Fig. 1.

Fig. 1.

Standard curve for determination of flavonoids.

Figs. 2,3 show the extraction of quercetin from plants (spinach, dill, Onion Skin, sumac, Pistacia eurycarpa, digalkhasab chemri, and leelwi chemri) by the chemical method (Soxhlet) and the physical method (ultrasound), respectively, using three extraction solutions (water, ethanol, and chloroform). The results of the statistical analysis (p 0.05) show that there were significant differences among all plants and all extraction solutions in terms of their quercetin content. It was found that the best extraction solution in both methods and for all plants was ethanol, then chloroform, followed by water, and this was attributed to the polarity of quercetin, which makes it soluble in ethanol and chloroform and slightly soluble in water [25].

Fig. 2.

Concentration of quercetin extracted from plants using the chemical method (Soxhlet).

Fig. 3.

Concentration of quercetin extracted from plants using the physical method (ultrasound).

These results agree with those of Awang et al. [26], who extracted quercetin from the leaves of the Melastoma malabathricum plant with Soxhlet using three extraction solutions (ethanol, ethyl acetate, and hexane), finding that ethanol was the best extraction solution. The present study’s results also agree with the findings of Sharifi et al. [9], who extracted quercetin from the leaves of radish (Raphanus sativus) using a variety of extraction solutions and methods, reporting that ethanol was better than chloroform and than water in extracting quercetin. In addition, our results agreed with those of Sambandam et al. [20], who extracted quercetin from the leaves of the fenugreek plant (Trigonella foenum-graecum) by soaking for 48 hours using ethanol, hexane, and ethyl acetate, also finding that ethanol was the best solution for extraction.

It is clear from the results in Fig. 2 that the concentration of quercetin extracted from the plants using the chemical method ranged from 3.271–84.037 mg/g, and that Pistacia eurycarpa yielded the highest concentration of quercetin, which was 84.037 mg/g. On the other hand, Fig. 3 shows that the concentration of quercetin extracted from plants using the physical method ranged from 10.691–58.827 mg/g, and that sumac yielded the highest concentration of quercetin (58.827 mg/g), which was close to the concentration found by Khanam and Anis [27] when they extracted quercetin from the golden trumpet plant, Allamanda cathartica, using ultrasound (51.39 mg/g). The present study’s concentration was also higher than that obtained by Jin et al. [28] when they extracted quercetin from onion peels using ultrasound under different conditions of ethanol concentration, wave intensity, and time period, which ranged between 1.74–4.34 mg/g.

Also more than what Jang et al. [29] obtained when extracting onion peels using ultrasound technology using aqueous ethanol and under specific conditions such as 59% ethanol and the extraction temperature 49 °C, which led to obtaining a total quercetin content of 11.08 mg/g of dry weight of onion solid waste. And less than what Wei et al. [30] found when they carried out the extraction process of flavonoids, especially quercetin, from the leaves and bark of the Abies nephrolepis plant, with the help of ultrasound technology and by determining the optimal conditions for the extraction process in terms of temperature, frequency, and time. The total yield of quercetin reached 69.59 mg/g.

Fig. 4 shows the extraction of quercetin from plants under study using the enzymatic method. Enzymes are biological catalysts that accelerate chemical reactions in living organisms [31] and are considered one of the modern methods increasingly used to extract active ingredients from natural products such as plants. The plant cell wall is a dense structure consisting of polysaccharides and proteins that can prevent the release of active ingredients from the plant. Therefore, enzymes can be used to facilitate the release of these ingredients, as they work to break down the structure of the cell wall and thus accelerate the disintegration of the active ingredients in plant cells. This contributes to the dissolution of the active ingredients, which improves the extraction efficiency [32], as has been demonstrated for the enzyme snailase.

Fig. 4.

Concentration of quercetin extracted from plants using the enzymatic method.

The results of the statistical analysis (p 0.05) for the present study showed that there were significant differences among all the plants used in terms of their quercetin content. It is noted from Fig. 4 that Pistacia eurycarpa yielded the highest concentration of quercetin (30.864 mg/g), while digalkhasab chemri yielded the lowest concentration (10.679 mg/g). These results were higher than the results obtained by Manzoor et al. [7] when extracting quercetin by the enzymatic method (using cellulose, xylanase, and $\beta$-glucosidase) from the leaves of the guava plant (Psidium guajava), where the concentration of quercetin reached 2.589 mg/g.

We conclude that the best of the three methods used in extracting quercetin is the chemical method. This method has many major advantages, including that it is possible to manipulate temperature to increase the extraction of medium to low-soluble compounds using this technology, and that it does not require filtration after completing the extraction process [33, 34].

These results agreed with Zhang et al. [35] who found that the Soxhlet method was the best method for extracting quercetin from the stems of the Euonymus alatus plant, as it produced the highest concentration of quercetin compared to ultrasound and microwave methods. The results in Table 2 also showed that the best-quercetin-yielding plant was Pistacia eurycarpa, so it was chosen to complete the subsequent purification steps to obtain pure quercetin.

Natural antioxidants have gained increasing attention recently due to their essential roles in maintaining human health and preventing and treating various diseases by preventing oxidation chain reactions [36].

Figs. 8,9 show the results of the antioxidant activity assay of quercetin to capture free radicals using DPPH compared to BHT, which involved using different concentrations (5, 10, 15 mg/mL) of purified and standard quercetin dissolved in hot water and ethanol, respectively. For purified and standard quercetin dissolved in hot water, this reached 70%, 79%, and 86% and 80%, 86%, and 92%, respectively. As for purified and standard quercetin dissolved in ethanol, it reached 66%, 75.1%, and 82.3% and 71.3%, 79.1%, and 86.9%, respectively, compared to BHT (98%). The antioxidant activity of quercetin is due to its structure, which contains hydroxyl groups capable of giving an electron to the free radical and converting it into stable products—the higher the hydroxyl number, the greater the antioxidant activity—and the double bond C2 = C3 in the C ring, which contains a hydroxyl group attached to carbon 3, preventing oxidation and resulting in strong antioxidant activity by quercetin [37, 38].

Fig. 8.

Antioxidant activity 2,2-diphenyl-1-picrylhydrazyl (DPPH) of quercetin dissolved in hot water.

Fig. 9.

Antioxidant activity 2,2-diphenyl-1-picrylhydrazyl (DPPH) of quercetin dissolved in ethanol.

We also noted from Figs. 8,9 that the antioxidant activity of standard quercetin was higher than that of purified quercetin, and that antioxidant activity increased with increasing quercetin concentration. This agreed with previous estimations [39] of the antioxidant activity DPPH for different concentrations of quercetin.

The results also agreed with Tian et al. [40] who demonstrated the antioxidant activity of a group of flavonoids, namely quercetin, apigenin, luteolin, and kaempferol, against DPPH radicals, showing that these compounds exerted stronger antioxidant activity than BHT, with the order of DPPH radical scavenging activity as follows: quercetin $>$ luteolin $>$ kaempferol $>$ apigenin.

These results were similar to what Geetha et al. [39] found when estimating the antioxidant activity of both quercetin and ascorbic acid. The results showed that quercetin has a significant effect in scavenging free radicals, reaching 71.94%. It also showed that the effectiveness of quercetin is two times higher than ascorbic acid.

The results of the present study also came close to the findings of Diao et al. [41], where the antioxidant activity of quercetin reached 93% when estimating the activity of DPPH radical capture, indicating the high activity that quercetin possesses against free radicals. Furthermore, the results show that the antioxidant activity of quercetin dissolved in hot water was higher than that dissolved in ethanol for all concentrations and for both extraction types (purified and standard). The solubility of quercetin in water increases with increasing temperature. This is because the rise in temperature leads to an increase in the kinetic energy of the quercetin molecules and their release from the attractive forces that keep them together, and this in turn leads to their dissolution in water [42].