Estimation of Some Phytochemical Compounds and Antioxidant Properties of Leaves from Different Mulberry Varieties Grown in Syria

The study was conducted to compare the chemical composition and bioactive compounds of leaves from different mulberry species grown in Syria (Morus alba, Morus nigra, Morus rubra, and Morus nigra sp). The leaves were collected in July 2022 and their proximate composition (moisture, ash, lipids, and protein), total polyphenol content (TPC), ascorbic acid, and antioxidant activity, as measured by ferric reducing antioxidant power (FRAP), were investigated. The results showed that the moisture, ash, and fat content ranged from 61.84 to 71.17%, 12.87 to 17.61%, and 3.82 to 9.68% dry weight (DW), respectively. The phenolic concentration of leaves from four different mulberry cultivars varied significantly, from 164.9 (Morus alba) to 461.5 gallic acid equivalents mg/100 g DW (M. nigra). The antioxidant activity of mulberry leaves ranged from 122 to 166.3 meq ascorbic acid/100 g DW and was arranged as follows: M. rubra > M. nigra sp > M. nigra > M. alba. According to the findings, mulberry leaves could be used to create new food supplements, functional foods, and medical applications.


Introduction
Te market for natural food products is one area of the health food sector that is currently expanding quickly. Tis is because the plant contains a wide variety of bioactive compounds with antioxidant properties which may help in the prevention of many chronic diseases [1].
Te mulberry tree is a member of the Moraceae family and is cultivated in many Asian countries [2]. Mulberry is a woody plant that grows in a variety of climatic, topographical, and soil conditions and can be found all over the world [3]. Today, it is grown in Southwest Asia and Southern Europe, and it is noted as one of the most important fruits in the Mediterranean [4]. Te most well-known species of the Morus genus are white mulberry (Morus alba), black mulberry (Morus nigra), and red mulberry (Morus rubra) [5]. Mulberry genotypes are very diverse in Syria, as they were sometimes obtained from seeds or cuttings in the past. Tis process has resulted in many landraces being adapted to various conditions and uses across the country. Tere are many local traditional accessions in Syria, but no named cultivars. Syrians distinguish and refer to the mulberry by using local names that are often based on the color of the fruit, such as "Abyad" (for a white mulberry), "Ahmar Baladi" (for a red mulberry), "Shami," and "al-Masry" (black mulberry).
Diferent uses have been created for this plant's various morphological parts (leaves, fruits, roots, and stems). Most of the information is focused on mulberry fruits and leaves, which have medicinal qualities and are frequently consumed as part of a typical diet [6].
Mulberry leaves are important to the sericulture sector because they are the only food source for the silkworm (Bombyx mori). Te leaves are also used in dairy animal feed because they increase milk production [7]. Mulberry leaves have been used in many countries as a medicine, nutritious beverage, and functional food [8]. For more than 3,000 years, mulberry leaves have been used in traditional Chinese medicine. Tey have long been used to treat many diseases such as colds and diabetes [9]. Modern research has shown that mulberry leaves contain polysaccharides, favonoids, alkaloids, volatile oils, and other active components [10]. Te active ingredients in mulberry leaves can be used to treat many diseases, including hypertension, diabetes, hyperlipidemia, Alzheimer's disease, and immunomodulation [11]. Te Ministry of Public Health of China has currently approved mulberry leaves as pharmaceutical/food resources [12]. Gupta et al. [7] confrmed that mulberry leaves have a higher protein content than other green leafy vegetables. Terefore, mulberry leaves can be used as an excellent source of nutrients and fght malnutrition in many underdeveloped areas of the world [13]. Te mulberry leaf's nutritional and functional content was signifcantly afected by cultivar, harvesting time, and degree of maturity. Also, the functional compounds of mulberry leaves vary between varieties, according to a previous study [14]. Terefore, it is important to choose the right mulberry leaves through activity studies based on variety. Iqbal et al. [15] evaluated the chemical composition and bioactive compounds in the leaves of three mulberry cultivars and found that there were signifcant variations in some nutrients and bioactive compounds between the various mulberry cultivars.
To date, no research has been carried out on the chemical composition and antioxidant activity of mulberry leaves cultivated in Syria. Terefore, the purpose of this study was to determine the proximate composition, total phenols, and antioxidant activity of leaves from four Syrian mulberry cultivars.

Leaf Samples Collection.
Te fresh leaves (young) of four mulberry species were collected in July 2022. Te leaves of Al-Abyad (Morus alba), Al-Masry (Morus nigra sp), and Ahmar Baladi (Morus rubra) were harvested from mulberry felds in Damascus city (Basatin Abu Jarash, 33°31′51.6″N 36°17′31.6″E), while the Al-Shami (Morus nigra) leaves were collected from Khan Arnabah (Al Qunaitra Governorate, southern Syria), Syria. Te largest glossy leaves from the tops of the branches were collected from several randomly selected trees of the same species after the fruits had reached full maturity. Te sampling was performed only once to avoid the diferent environmental and climatic conditions that afect the natural phytochemical compounds.
After collection, the leaves are removed from the stalk, washed with water, and shade dried to constant weight. Te samples were kept in a dry place, away from light, until they were analyzed.

Proximate Composition.
Te proximate composition of leaf samples (moisture, ash, lipids, and protein) was analyzed using the AOAC [16] method. Te moisture was determined by drying the samples in an oven drier at 105 ± 2°C until a constant weight was obtained. Te ash content was determined using the ashing method, which involved heating the sample in a furnace. A Soxhlet extractor was used to extract lipids from powdered samples. Te nitrogen content was estimated using the Kjeldahl apparatus, and the protein content was calculated as N × 6.25.

Total Soluble Carbohydrate (%).
Te total soluble carbohydrate in the leaf samples was determined according to the method of Xiao et al. [17] with some modifcations. Te mulberry leaf powder (1 g) was dissolved in distilled water at a ratio of 1 : 10 g/mL and extracted in a water bath for 20 min at 60°C. Te aqueous solution (1 mL) was mixed with 1 mL of the phenol solution (5%), followed by 5 mL of concentrated sulphuric acid. After 10 min, the reaction was thoroughly mixed with a vortex and placed in a water bath at 30°C for 20 min. Te absorbance was measured at 490 nm with a UV-VIS Spectrophotometer (PG Instruments T80+ UV/ VIS Spectrophotometer, UK). Te standard curve of Dglucose ranging from 10 to 100 μg/mL was prepared.

Determination of Ascorbic Acid Content.
Ascorbic acid was determined according to the method of Dinesh et al. [18] with some modifcations. A fne-dried powder (1 g) was extracted with 20 ml of oxalic acid (0.4%) and stirred at room temperature for 20 min. Te mixture was fltered through Whatman No. 4 flter paper. Te sample extract was titrated against a solution of 2,6-dichloroindophenol (0.02%) until the pink color of the solution was sustained for 10 s. Te dye solution was standardized with 10 mL of ascorbic acid (0.2 mg/mL). Te ascorbic acid content of the samples was expressed as mg/100 g of DW leaves.

Preparation of Phenolic Extract.
One Gram of the dried leaves was extracted with 15 ml methanol (70%) for 1 h at ambient temperature under continuous stirring in a magnetic stirrer. After fltration, the extracts were stored at −20°C until further analysis.

Determination of Total Polyphenol Content (TPC).
Te TPC was determined using the Folin-Ciocalteu calorimetric method described by Kostic et al. [19]. Te phenolic extract (1 mL) was combined with 0.5 mL Folin-Ciocalteu reagent and 2 ml sodium carbonate (20%). After 10 min of incubation at room temperature, the absorbance was measured at 765 nm. Te total phenolic of the extracts was calculated as mg of gallic acid equivalents/100 g dry leaves (mg GAE/100 g DW).

Ferric Reducing Antioxidant Power (FRAP) Assay.
Te Mekonnen and Desta [20] method was used to determine the FRAP assay. One ml of the phenolic extract was mixed with 2.5 mL of a phosphate bufer solution (pH � 6.6) and 2.5 mL of potassium ferricyanide (1%). After the mixture was incubated at 50°C for 20 min, 2.5 ml of trichloroacetic acid (10%) was added. Ten, 2.5 mL of the mixture was taken, and 2.5 mL of water and 0.5 ml of ferric chloride (0.1%) were added. Finally, the absorbance was measured at 700 nm against a blank. Te L-ascorbic acid (0.5-2.5 μg/mL) was used to prepare the calibration curve. Te results were expressed as mg of ascorbic acid/100 g dry sample (mg AA/ 100 g DW).

Statistical Analysis
Te results were expressed as mean ± standard deviation (SD). Te statistical analysis was carried out using GenStat software 12 and a one-way analysis of variance (ANOVA) followed by a Fisher's least signifcant diference (LSD) test to determine the signifcant diference between the treatments at the 5% level. Table 1 illustrates the mean compositional values for the leaves of four diferent mulberry species.

Proximate Composition.
Te moisture content varied between 61.84% in M. nigra and 71.17% in M. rubra. According to the data presented by several authors, the average moisture of mulberry leaves ranges between 60% and 75% [21,22]. Low moisture content may contribute to leaf roughness [15].
Te moisture content in dried leaf powder ranged from 2.66% to 6.33%. Low moisture content in mulberry leaves enhances the extraction of bioactive compounds and helps protect the product from microbial attack and spoilage [23]. Te total mineral content is refected in the total ash content, and a high ash content indicates that plant material has high inorganic nutrients [24]. Te ash content of dried mulberry leaves varied signifcantly in all experimental types except between M. alba and M. rubra. Te ash content in mulberry leaves was lowest in M. nigra. Srivastava et al. [13] have achieved similar results.
Te protein content of mulberry leaves can be used to determine the leaf's quality. Te trend of protein concentration in leaves from all the cultivars studied was as follows: M. nigra > M. alba > M. nigra sp > M. rubra, with signifcant diferences between them (p < 0.05). Sanchez-Salcedo et al. [21] found slightly lower levels (13.4% to 19.4%) in leaves of M. alba and M. nigra, while Srivastava et al. [13] found higher values in leaves of six genotypes of mulberry in India. Yu et al. [8] illustrated that mulberry leaves contain high-quality proteins which are used to fortify food in the subcontinent.
Estimating lipids is one of the most important aspects of any material's nutritional evaluation [25]. In the four analyzed mulberry leaves, the fat content ranged from 3.82% dw (M. alba) to 9.68% DW (M. nigra sp). Te ranges for fat in mulberry leaves given by specifc literature are 4.24-6.75% [15] and 2.09-6% [13].
Mulberry leaves' total soluble carbohydrate was analyzed (Table 1). Te fnding showed that the investigated mulberry extracts had a total soluble carbohydrate content that ranged from 1.77 to 3.57% DW. M. alba and M. rubra had the highest soluble carbohydrate, while Morus nigra had the lowest concentration. Our fndings are lower than that of the previously reported data [13].

Total Phenolic Compounds (TPC).
Te concentration of TPC in the diferent types of mulberry leaves studied is presented in Table 2. Te concentration of phenols in the four mulberry cultivars' leaf extracts ranged from 164.9 to 461.5 mg/100 g DW. Furthermore, the data indicate that Morus nigra had a signifcantly higher TPC than other cultivars. Tis study's fndings agreed with a report on mulberry leaves from Pakistan and Italy [6,15], which also indicated that black mulberry had higher total phenolic compound values than white mulberry. Polumackanycz et al. [6] reported that the TPC for 19 varieties of mulberry leaf ranged from 11.17 to 31.14 mg GAE/g. While Yu et al. [8] found that TPC levels in mature leaves of diferent mulberry varieties grown in China ranged from 27.63 to 37.36 mg/ 100 g DW. Tis variation could be explained by the difference between extraction procedures and analytical methods used in each study. Furthermore, the phenolic compounds in leaves change in response to environmental factors such as drought, temperature changes, pollution, UV light, and pathogen attacks, among others [26].
Te ascorbic acid content in leaves ranged from 1.66 (M. nigra sp) to 3.84 mg/100 g DW (M. nigra) ( Table 2). Tis result is consistent with the fndings of Flaczyk et al. [27]. Srivastava et al. [13] found higher levels of ascorbic acid in the leaves of some mulberry (M. alba) genotypes, ranging from 100 to 200 mg/100 g DW. Iqbal et al. [15] observed that the highest concentration of ascorbic acid was found in the leaves of M. alba followed by M. nigra while the minimum was found for M. rubra. Te variations in nutritional compound levels between leaves of mulberry varieties or within the same variety can be attributed to pedoclimatic factors (soil type, sun exposure, and precipitation), genetic factors (variety), and agricultural variables (organic farming, tree fruit production, the state of maturation, growing area, fertilization, and irrigation) [28].

Antioxidant Activity.
Te antioxidant activity of food products is an important indicator of their health benefts [29]. Te FRAP assay was used to determine the total antioxidant activity of mulberry leaves. Tis method depends on the extract's capacity to reduce the ferric (Fe 3+ ) to the ferrous (Fe 2+ ) state. Te results of antioxidant activity in Evidence-Based Complementary and Alternative Medicine Figure 1 showed statistically signifcant diferences between the mulberry varieties (p < 0.05) and Morus rubra which had the highest reducing capacity (166.3 mg ascorbic acid equivalent/100 g DW). According to Cartea et al. [30], the chemical structures of compounds, particularly the number and positions of aromatic and hydroxyl groups, have a signifcant impact on the antioxidant activity of phenolics. Te antioxidant activity is also correlated with the degree of hydroxylation. A previous study showed that the FRAP values of white mulberry aqueous extracts were higher than those of black mulberry. Furthermore, no signifcant differences in antioxidant activity were observed between the white and black mulberry alcoholic extracts [6]. According to Wang et al. [2], there are many factors that can afect the antioxidant activities of plant samples, including extraction method, extraction solvent, and assay system. Table 3 shows the correlation coefcient between the experimental parameters investigated for the diferent mulberry leaf varieties. A positive correlation (r � 0.61) was found between TPC and antioxidant capacity. Tis suggests the important role of phenolic compounds as antioxidant     [6,8]. According to Yu et al. [8], the polyphenolic compounds in mulberry leaves are chlorogenic acid, rutin, isoquercitrin, and astragalin, and they are primarily potential antioxidants. Protein correlated negatively with all other parameters observed except ascorbic acid (r � 0.40).

Correlation between Diferent Experimental Parameters.
Our results agree with those reported by [8], who found a signifcantly negative correlation between TPC and crude protein. Te high correlation between fat and FRAP (r � 0.87) is possibly due to the presence of α-linolenic acid, linoleic acid, palmitic acid, and oleic acid in mulberry leaves [31]. Te FRAP assay was found to be negatively correlated with ascorbic acid (r � −0.35). Iqbal et al. [15] revealed a weak correlation between ascorbic acid and antioxidant activity in mulberry leaves.

. Conclusions
For the frst time, some chemical parameters, phenolic compounds, and antioxidant activity of Syrian mulberry leaves were investigated in this study. According to the fndings, Morus leaves are a good dietary source of nutritional and bioactive compounds. Tis study suggests that the leaves of Al-Shami mulberry had the highest protein and phenol content (p < 0.05), whereas the leaves of Al-Ahmar Baladi (Morus rubra) had signifcantly higher antioxidant activity compared to the other mulberry species. Our fndings indicated that Syrian mulberry leaves might be a promising source of natural antioxidants for the food, pharmaceutical, and cosmetic industries due to the presence of phenolic compounds. Further research is needed to isolate and identify the antioxidant components in mulberry leaves.

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.