**Identification of Bioactive Phytochemicals in Leaf Protein Concentrate of Jerusalem Artichoke (***Helianthus tuberosus* **L.)**

**László Kaszás 1 , Tarek Alshaal 1,2,\* , Hassan El-Ramady 1,2, Zoltán Kovács <sup>1</sup> , Judit Koroknai <sup>1</sup> , Nevien Elhawat 1,3 , Éva Nagy <sup>1</sup> , Zoltán Cziáky <sup>4</sup> , Miklós Fári <sup>1</sup> and Éva Domokos-Szabolcsy <sup>1</sup>**


Received: 24 June 2020; Accepted: 13 July 2020; Published: 14 July 2020

**Abstract:** Jerusalem artichoke (JA) is widely known to have inulin-rich tubers. However, its fresh aerial biomass produces significant levels of leaf protein and economic bioactive phytochemicals. We have characterized leaf protein concentrate (JAPC) isolated from green biomass of three Jerusalem artichoke clones, Alba, Fuseau, and Kalevala, and its nutritional value for the human diet or animal feeding. The JAPC yield varied from 28.6 to 31.2 g DM kg−<sup>1</sup> green biomass with an average total protein content of 33.3% on a dry mass basis. The qualitative analysis of the phytochemical composition of JAPC was performed by ultra-high performance liquid chromatography-electrospray ionization-Orbitrap/mass spectrometry analysis (UHPLC-ESI-ORBITRAP-MS/MS). Fifty-three phytochemicals were successfully identified in JAPC. In addition to the phenolic acids (especially mono- and di-hydroxycinnamic acid esters of quinic acids) several medically important hydroxylated methoxyflavones, i.e., dimethoxy-tetrahydroxyflavone, dihydroxy-methoxyflavone, hymenoxin, and nevadensin, were detected in the JAPC for the first time. Liquiritigenin, an estrogenic-like flavanone, was measured in the JAPC as well as butein and kukulkanin B, as chalcones. The results also showed high contents of the essential amino acids and polyunsaturated fatty acids (PUFAs; 66-68%) in JAPC. Linolenic acid represented 39–43% of the total lipid content; moreover, the ratio between ω-6 and ω-3 fatty acids in the JAPC was ~0.6:1. Comparing the JA clones, no major differences in phytochemicals, fatty acid, or amino acid compositions were observed. This paper confirms the economic and nutritional value of JAPC as it is not only an alternative plant protein source but also as a good source of biological valuable phytochemicals.

**Keywords:** circular economy; green biorefinery; polyunsaturated fatty acids; phytochemicals; amino acids; food and feed; UHPLC-ESI-ORBITRAP-MS/MS

#### **1. Introduction**

The global protein demand continuously grows as the world population exponentially increases. In Europe, the increasing protein dependency particularly obtained from soybean has triggered an

urgent need for alternative production systems. Locally grown green biomass crops represent an alternative protein source. Due to high green biomass yield and regrown capacity, clover, alfalfa, and grasses are the most common and prospective plant species for leaf protein isolate. However, digestion of green biomass by monogastric animals is difficult because of its high fiber content [1]. Green biorefinery is a complex processing system with a dedicated goal of making a commercially viable production system of added-value protein based on green biomass [2]. Separating fresh green biomass into two fractions is a key step in the green biorefinery. The fibrous pulp contains insoluble and fiber-bound protein, while the other fraction (green juice) is soluble protein-rich [1]. Soluble proteins in green juice can be precipitated by different techniques. Recovered protein concentrate is separated from brown juice fraction by filtration. Moreover, the quality of leaf protein concentrate as a main product is very important. Based on extended qualitative and quantitative analysis, alfalfa leaf protein concentrate can be directed towards feed and/or food [3,4]. However, besides the well-known herbaceous species, a range of agro-industrial crops is constantly expanding, which can be utilized in the green biorefinery [5].

Jerusalem artichoke (JA), a perennial plant, belongs to the Asteraceae family. Cultivation of JA has many advantages as it is tolerant to biotic stress, i.e., pests and diseases [6]. It can grow normally in a wide range of soils including salt-affected soil, sandy soil, and marginal lands with nearly zero levels of fertilization [7–9]. Moreover, it showed potential resistance to drought, frost, and high temperatures [10]. It yields a huge green biomass almost 120 tons ha−<sup>1</sup> fresh mass [11]. These aspects are important when avoiding competition with food production on arable lands. The recognized nutritional value of JA is mainly due to the high inulin and fructose contents in its tubers, which additionally contain protein, nutrients, and vitamins [12]. Additionally, JA is well-known as multipurpose use crop where its aerial part has attracted the interest of many researchers, firstly, concerning bioenergy production due to its high lignocellulosic content, high biomass yield, and low inputs [6]. Among the phytochemicals, sesquiterpene lactones, phenolic acids, flavone glucosides (kaempferol 3-*O*-glucoside and quercetin 7-*O*-glucoside), chlorophylls, and carotenoids have been described by several authors in the whole plant or different organs such as tubers, leaves, or flowers [13–19]. These isolated phytochemicals are known as potential anticancer, antidiabetic, antioxidant, antifungal, and antimicrobial in addition to their other medical uses [13,17].

Despite green leafy shoot of JA can be utilized directly as fresh forage, silage, or food pellets for animal feeding [9,12], most of the animal species do not prefer it because of trichome-rich leaves and stems [8]. Considering its high green biomass, regeneration capacity, and chemical composition, leafy shoots of JA can be alternatively used in the green biorefinery practice; however, there is a shortage of knowledge in this area [20].

The objectives of the present work were to produce and characterize the biological value of JAPC originating from the green biomass of JA. We aimed to provide detailed insights into the extraction efficiency and biochemical composition of JAPC. Therefore, three clones of JA representing different climatic zones were grown under low input conditions in Hungary. In addition to total protein, amino acid composition, and fatty acids profile the biochemical composition and qualitative determination of phytochemicals in the JAPCs from these clones were measured using ultra-high performance liquid chromatography-electrospray ionization-Orbitrap/mass spectrometry analysis (UHPLC-ESI-ORBITRAP-MS/MS).

#### **2. Materials and Methods**

#### *2.1. Experimental Installation*

A field experiment was conducted in 2016 at the Horticultural Demonstration garden of the University of Debrecen, Hungary (47◦33′ N; 21◦36′ E). Three different clones of JA (i.e., Alba, Fuseau, and Kalevala) were compared for their fresh aerial biomass, phytochemical content, and biochemical traits of the JAPC, under low input conditions. Tubers of JA clones representing three climatic zones

were obtained from different sources as follows: Alba was obtained from a Hungarian market; Fuseau was obtained from Ismailia, Egypt; and Kalevala was obtained from Helsinki, Finland. The experiment was set up in a randomized complete block design with six replicates. The area of the experimental plot was 0.8 × 0.6 m2; the row was 3.5 m in length and 0.8 m in width, and within-row spacing was 0.6 m. The cultivation of the JA clones started on 5 April 2016, using identically sized tubers (60–80 g/tuber). Neither irrigation nor fertilization was applied. The chemical characteristics of the experimental soil were as follows: total N (555 <sup>±</sup> 2 mg kg−<sup>1</sup> ); total P (6793 <sup>±</sup> 17 mg kg−<sup>1</sup> ); total K (1298 <sup>±</sup> 7 mg kg−<sup>1</sup> ); and humus (1.9% ± 0.02%).

#### *2.2. Harvest of Above-Ground Biomass*

Due to the ability of JA plants to regrow, the green biomass of the three clones was harvested twice during the growing season, when young shoots reached 1.3–1.5 m in height from the soil surface. The first harvest was conducted on 27 June 2016, and the second on 8 August 2016. The fresh yield of the aerial parts was measured.

#### *2.3. Fractionation of Harvested Green Biomass*

The harvest of JA plants was conducted early in the morning and they were immediately transferred to the laboratory in an icebox to prevent the chemical compounds from degrading. The plants were harvested 15–20 cm above the soil surface. A 1 kg harvest of green biomass was mechanically pressed and pulped using a twin-screw juicer (Green Star GS 3000, Toronto, ON, Canada) in three replicates. Thereafter, the green juice was thermally coagulated at 80 ◦C in one step to obtain the JAPC. The JAPC was separated from the brown-colored liquid fraction using cloth filtration. Both the fresh and dry masses of the JAPC were measured before it was lyophilized using an Alpha 1–4 LSC Christ lyophilizer.

#### *2.4. Biochemical Composition of JAPC*

#### 2.4.1. Crude Protein Content

The total protein content of the JAPC was measured as total N content using the Kjeldahl method [21]. Briefly, 1 g lyophilized sample was weighed in a 250 mL Kjeldahl digestion tube, then 15 mL concentrated sulfuric acid (99%, VWR Ltd., Debrecen, Hungary) and two catalyst tablets were added. The Kjeldahl digestion tubes were placed in a Tecator Digestor (VELT, VWR Ltd, Debrecen, Hungary) at 420 ◦C for 1.5 h. The total N content in the digested samples was measured by titration and calculated based on the weight of the titrated solution and the sample weight. The total protein content of the sample was calculated using the following equation: Total protein % = total N content × 6.25.

#### 2.4.2. Quantification of Amino Acid Composition in JAPC Using an Amino Acid Analyzer

Lyophilized and ground samples of JAPC were digested with 6 M HCl at 110 ◦C for 23 h. Since the digested sample was designed to contain at least 25 mg N, the measured weights of the samples were variable. Alternating application of inert gas and a vacuum using a three-way valve was conducted to remove air. Following hydrolysis, the sample was filtered into an evaporator flask and the filtrate was evaporated under 60 ◦C to achieve a syrup-like consistency. Thereafter, distilled water was added to the sample and evaporation was conducted twice more under the same conditions. The evaporated sample was washed with citrate buffer pH 2.2. For the analysis of amino acid composition an INGOS AAA500 (Ingos Ltd., Prague, Czech Republic) amino acid analyzer was used. The separation was based on ionic exchange chromatography with post-column derivatization of ninhydrin. A UV/VIS detector was used at 440/570 nm.

#### 2.4.3. Determination of Fatty Acid Composition in JAPC Using Gas Chromatography

The esterification of fatty acids in the JAPC fraction into methyl esters was conducted using a sodium methylate catalyst. Lyophilized homogeneous sample (70 mg) was weighed into a 20 mL tube; 3 mL of n-hexane, 2 mL of dimethyl carbonate and 1 mL of sodium methylate in methanol were added. The contents of the test tube were shaken for 5 min (Janke and Kunkel WX2) and then 2 mL of distilled water was added before the tube was shaken again. The samples were centrifuged at 3000 rpm for 2 min (Heraeus Sepatech, UK). A 2.0 mL sample of supernatant (hexane phase) was transferred into a container through filter paper, which contained anhydrous sodium sulfate. The prepared solution contained approximately 50–70 mg cm−3 fatty acid methyl ester (FAME) and was suitable for analysis by gas chromatography. Gas chromatography was performed using an Agilent 6890 N coupled to an Agilent flame ionization detector. A Supelco Omegawax capillary column (30 m, 0.32 mm i.d., 0.25 µm film thickness) was used to separate FAMEs. The oven temperature was 180 ◦C and the total analysis time was 36 min. An Agilent 7683 automatic split/splitless injector was used with an injector temperature of 280◦C and a 100:1 split ratio. The injection volume was 1 µL. The carrier gas was hydrogen with a flow rate of 0.6 mL min−1 and the makeup gas was N with a flow rate of 25.0 mL min−<sup>1</sup> . The components were identified from retention data and standard addition.

#### *2.5. Screening of Phytochemicals in JAPC by UHPLC-ESI-ORBITRAP-MS*/*MS*

#### 2.5.1. Sample Preparation

To prepare the hydro-alcoholic extracts, 0.5 g ground JAPC powder was extracted with 25 mL methanol:water solution. The mixture was stirred at 150 rpm for 2 h at room temperature. The hydro-alcoholic extracts were filtered using a 0.22 µm PTFE syringe filter.

#### 2.5.2. UHPLC-ESI-ORBITRAP-MS/MS Analysis

Phytochemical analyses were performed using UHPLC-ESI-ORBITRAP-MS/MSwith a Dionex Ultimate 3000RS UHPLC system (Thermo Fisher, Waltham, MA, USA) coupled to a Thermo Q Exactive Orbitrap hybrid mass spectrometer equipped with a Thermo Accucore C18 analytical column (2.1 mm × 100 mm, 2.6 µm particle size). The flow rate was maintained at 0.2 mL/min and the column oven temperature was set to 25 ◦C ± 1 ◦C. The mobile phase consisted of methanol (A) and water (B) (both acidified with 0.1% formic acid). The gradient program was as follows: 0–3 min, 95% B; 3–43 min, 0% B; 43–61 min, 0% B; 61–62 min, 95% B; and 62–70 min, 95% B. The injection volume was 2 µL.

#### 2.5.3. Mass Spectrometry Conditions

A Thermo Q Exactive Orbitrap hybrid mass spectrometer (Thermo Fisher, Waltham, MA, USA) was equipped with an ESI source. The samples were measured in both positive and negative ionization modes separately. The capillary temperature was 320 ◦C and spray voltages were 4.0 kV in positive ionization mode and 3.8 kV in negative ionization mode, respectively. The resolution was 35,000 for MS1 scans and 17,500 for MS2 scans. The scanned mass interval was 100–1500 *m*/*z*. For the tandem MS (MS/MS) scans, the collision energy was set to 30 nominal collision energy units. The difference between measured and calculated molecular ion masses was less than 5 ppm in each case. The data were acquired and processed using Thermo Trace Finder 2.1 software based on own and internet databases (Metlin, Mass Bank of North America, *m*/*z* Cloud). After processing, the results were manually checked using Thermo Xcalibur 4.0 software (ThermoFisher, Waltham, MA, USA).

#### *2.6. Quality Assurance of Results*

The glass- and plastic-ware used for analyses were usually new and were cleaned by soaking in 10% (v/v) HNO<sup>3</sup> for a minimum of 24 h, followed by thorough rinsing with distilled water. All chemicals were analytical reagent grade or equivalent analytical purity. All equipment was calibrated, and uncertainties were calculated. Internal and external quality assurance systems were applied at the Central Laboratory of the University of Debrecen, according to MSZ EN ISO 5983-1: 2005 (for Total N), and the Bunge Private Limited Company Martf ˝u Laboratory, according to MSZ 190 5508: 1992 (for fatty acid composition).

#### *2.7. Statistical Analysis*

Before the ANOVA test, Levene's Test for Equality of Variances was performed. The Levene's test for different variables at all treatments was negative, *p* < 0.05, showing homogeneity of the variances. The experimental design was established as a randomized complete block design with six replicates. The data obtained from the experiments were subjected to one-way ANOVA by 'R-Studio' software and the means were compared by Duncan's Multiple Range Test at *p* < 0.05 [22].

#### **3. Results**

#### *3.1. Green Biomass of Jerusalem Artichoke Clones*

The yield of the aerial fresh biomass of different JA clones is presented in Table 1. Clones displayed almost the same fresh biomass yield. Hence, no significant differences among the clones (i.e., Alba, Fuseau, and Kalevala) were noticed, especially during the first harvest. The harvest time largely influenced the yield. The average fresh biomass yield was approximately 5.3 kg m−<sup>2</sup> for the first harvest, while for the second harvest the yield was significantly reduced to 2.4 kg m−<sup>2</sup> (Table 1). The total aerial fresh biomass yield—as an average—was estimated to be 7.7 kg m−<sup>2</sup> .

**Table 1.** Aerial fresh biomass, dry mass, and total protein content of Jerusalem artichoke leaf protein concentrate (JAPC) isolated from green biomass of different clones.


Means followed by different letters in the same column show significant differences according to Duncan's test at *p* < 0.05.

#### *3.2. JAPC Yield*

The yield of JAPC, extracted using thermal coagulation, from 1 kg fresh green biomass of the JA clones is displayed in Table 1. No significant differences were seen between the JA clones in either the first or the second harvests. The JAPC yield ranged from 28.3 (Fuseau) to 32.3 (Kalevala) g kg−<sup>1</sup> fresh biomass for the first harvest, while for the second harvest it varied from 28 (Kalevala) to 30.4 (Alba) g kg−<sup>1</sup> fresh biomass (Table 1). However, the results showed that the average JAPC dry yield from the first and second harvests was 30.8 and 29.1 g kg−<sup>1</sup> fresh biomass, respectively. Therefore, 1 kg of green biomass of JA was estimated to yield approximately 30 g JAPC dry mass as an annual average.

#### *3.3. Total Protein Content of JAPC*

The total protein content (m/m%) of JAPC generated from fresh green biomass of JA clones ranged between 33.3 m/m% (Fuseau) and 35.3 m/m% (Alba) in the first harvest, while in the second, it varied from 31.6 m/m% (Alba) to 35.2 m/m% (Fuseau). Statistically, no significant differences were calculated either between the clones or harvests (Table 1). The average total protein content in the first harvest was 34.1 m/m% and 33.4 m/m% in the second based on the dry weight. The annual average total protein content of the JAPC extracted from the JA fresh biomass was estimated to be 33.8 m/m% (Table 1).

#### *3.4. Amino Acid Composition of JAPC*

The amino acid composition of the JAPC obtained from the green biomass of the JA clones is presented in Table 2. Essential amino acids (i.e., lysine, histidine, isoleucine, leucine, phenylalanine, methionine, threonine, and valine) play a major nutritional role in feed; therefore, they are of special interest. Among the investigated JA clones, Kalevala displayed the highest content of five essential amino acids (i.e., phenylalanine, histidine, isoleucine, threonine, and valine). Additionally, the content

of aspartic acid, glycine, glutamic acid, proline, and serine was the highest in Kalevala, with values of 4.23, 2.13, 4.82, 2.20, and 1.90 m/m%, respectively (Table 2). Lysine is particularly important in animal feed and its content in Alba, Fuseau, and Kalevala ranged between 2.19 and 2.32 m/m% in the first harvest. Lysine content in the clones was similar regardless of the harvest time with higher value in the second harvest (2.35–2.54 m/m%) than the first harvest. Methionine is another limiting essential amino acid. The methionine content in Alba and Fuseau clones ranged between 0.82 and 0.95 m/m% in both harvests (Table 2). A reduction in methionine content was found in the second harvest for all clones except Fuseau.

**Table 2.** Amino acid profile (m/m%) of Jerusalem artichoke leaf protein concentrate (JAPC) extracted from green biomass of different clones.


‡ Standard deviation. Means followed by different letters in the same row and same harvest show significant differences according to Duncan's test at *p* < 0.05.

#### *3.5. Qualitative Analysis of JAPC Fatty Acid Composition*

Both saturated (SFA) and unsaturated fatty acids (UFA) were detected in the JAPC. Polyunsaturated fatty acids (PUFA) including linoleic acid (C18:2ω –6) and linolenic acid (C18: 3ω –3) predominated (66%–68%) in all of the JA clones (Figures 1 and 2). Among these fatty acids, linolenic acid (38.6%–42.7%) exhibited a narrow range of content that was present in the highest amount regardless of harvest time or clone. Linoleic acid was present in the second-highest concentration, at a minimum of 23.4% in the first harvest JAPC of Kalevala and a maximum of 26.9% in the second harvest JAPC of Alba. All of the analyzed JAPC samples exhibited a low concentration of unknown fatty acid, which comprised 0.3–0.6% of the total fatty acid content (Figure 1). Among the monounsaturated fatty acids (MUFAs), oleic acid (C18:1ω–9) was detected at a high value (6.6–11.6%), whereas the content of palmitoleic acid (C16:1ω–7) was significantly lower and ranged from 0.7% to 1.1% (Figure 1). The saturated fatty acids (SFA), myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0) were also identified. Palmitic acid was the most abundant saturated component with no significant differences (16.4–17.9%) either between clones or time of harvest. The percent composition of myristic acid (2.5–6.9%) and stearic acid (1.5–1.8%) in the JAPC fractions were markedly lower than that of palmitic acid. Opposing tendencies were found for the oleic and myristic acid contents between the first and second harvests. The myristic acid content in JAPC was higher in the first harvest in all the three JA clones, while the oleic acid content was higher in the second harvest JAPC of Alba and Kalevala (Figure 1).

*Plants* **2020**, *9*, x FOR PEER REVIEW 7 of 17

**Figure 1.** Fatty acid composition (%) of Jerusalem artichoke leaf protein concentrate (JAPC) extracted from the green biomass of three clones (Alba, Fuseau, and Kalevala). **Figure 1.** Fatty acid composition (%) of Jerusalem artichoke leaf protein concentrate (JAPC) extracted from the green biomass of three clones (Alba, Fuseau, and Kalevala). **Figure 1.** Fatty acid composition (%) of Jerusalem artichoke leaf protein concentrate (JAPC) extracted from the green biomass of three clones (Alba, Fuseau, and Kalevala).

**Figure 2.** Distribution of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and **Figure 2.** Distribution of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) in Jerusalem artichoke leaf protein concentrate (JAPC) extracted **Figure 2.** Distribution of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) in Jerusalem artichoke leaf protein concentrate (JAPC) extracted from the green biomass of three clones (Alba, Fuseau, and Kalevala).

polyunsaturated fatty acids (PUFA) in Jerusalem artichoke leaf protein concentrate (JAPC) extracted

#### from the green biomass of three clones (Alba, Fuseau, and Kalevala). from the green biomass of three clones (Alba, Fuseau, and Kalevala). *3.6. Screening JAPC Phytochemicals Using UHPLC-ESI-ORBITRAP-MS*/*MS*

revealed a compound with a [M−H]<sup>−</sup>

revealed a compound with a [M−H]<sup>−</sup>

*3.6. Screening JAPC Phytochemicals Using UHPLC-ESI-ORBITRAP-MS/MS*  The profiles of the phytochemicals in the JAPCs isolated from the JA clones Alba, Fuseau, and Kalevala, exhibited negligible differences between them. Up to 61 phytochemicals were defined, based on specific retention time, accurate mass, isotopic distribution, and fragmentation pattern, and by screening the following MS databases: Metlin, mzCloud, MoNA-MassBank of North America, and our own database. Table 3 indicates that phenolic compounds comprised a significant component of the compounds identified. Regardless of JA clones, three caffeoylquinic acid isomers: chlorogenic acid (3-O-caffeoylquinic acid), neochlorogenic acid (5-O-caffeoylquinic acid), and cryptochlorogenic acid (4-O-caffeoylquinic acid), respectively, were identified in the JAPCs with a characteristic [M−H]<sup>−</sup> ion at m/z 353.0873. Considering the area of the peak of extracted ion chromatogram of isomers the 3-O-caffeoylquinic acid is the dominant one, while a lower ratio of neochlorogenic acid (5-Ocaffeoylquinic acid) and cryptochlorogenic acid (4-O-caffeoylquinic acid) were detected (Figure 3). Additionally, three di-O-caffeoylquinic acid isomers ([M−H]<sup>−</sup> ion at m/z 515.1190), four coumaroylquinic acid isomers ([M−H]<sup>−</sup> ion at m/z 337.0924), and a 5-O-feruloylquinic acid ([M−H]<sup>−</sup> *3.6. Screening JAPC Phytochemicals Using UHPLC-ESI-ORBITRAP-MS/MS*  The profiles of the phytochemicals in the JAPCs isolated from the JA clones Alba, Fuseau, and Kalevala, exhibited negligible differences between them. Up to 61 phytochemicals were defined, based on specific retention time, accurate mass, isotopic distribution, and fragmentation pattern, and by screening the following MS databases: Metlin, mzCloud, MoNA-MassBank of North America, and our own database. Table 3 indicates that phenolic compounds comprised a significant component of the compounds identified. Regardless of JA clones, three caffeoylquinic acid isomers: chlorogenic acid (3-O-caffeoylquinic acid), neochlorogenic acid (5-O-caffeoylquinic acid), and cryptochlorogenic acid (4-O-caffeoylquinic acid), respectively, were identified in the JAPCs with a characteristic [M−H]<sup>−</sup> ion at m/z 353.0873. Considering the area of the peak of extracted ion chromatogram of isomers the 3-O-caffeoylquinic acid is the dominant one, while a lower ratio of neochlorogenic acid (5-Ocaffeoylquinic acid) and cryptochlorogenic acid (4-O-caffeoylquinic acid) were detected (Figure 3). Additionally, three di-O-caffeoylquinic acid isomers ([M−H]<sup>−</sup> ion at m/z 515.1190), four coumaroylquinic acid isomers ([M−H]<sup>−</sup> ion at m/z 337.0924), and a 5-O-feruloylquinic acid ([M−H]<sup>−</sup> ion at m/z 367.1029) were identified in the hydro-alcoholic extracted JAPC. The investigation also The profiles of the phytochemicals in the JAPCs isolated from the JA clones Alba, Fuseau, and Kalevala, exhibited negligible differences between them. Up to 61 phytochemicals were defined, based on specific retention time, accurate mass, isotopic distribution, and fragmentation pattern, and by screening the following MS databases: Metlin, mzCloud, MoNA-MassBank of North America, and our own database. Table 3 indicates that phenolic compounds comprised a significant component of the compounds identified. Regardless of JA clones, three caffeoylquinic acid isomers: chlorogenic acid (3-*O*-caffeoylquinic acid), neochlorogenic acid (5-*O*-caffeoylquinic acid), and cryptochlorogenic acid (4-*O*-caffeoylquinic acid), respectively, were identified in the JAPCs with a characteristic [M−H]<sup>−</sup> ion at *m*/*z* 353.0873. Considering the area of the peak of extracted ion chromatogram of isomers the 3-*O*-caffeoylquinic acid is the dominant one, while a lower ratio of neochlorogenic acid (5-*O*-caffeoylquinic acid) and cryptochlorogenic acid (4-*O*-caffeoylquinic acid) were detected (Figure 3). Additionally, three di-*O*-caffeoylquinic acid isomers ([M−H]<sup>−</sup> ion at *m*/*z* 515.1190), four coumaroylquinic acid isomers ([M−H]<sup>−</sup> ion at *m*/*z* 337.0924), and a 5-*O*-feruloylquinic acid ([M−H]<sup>−</sup> ion at *m*/*z* 367.1029) were identified in the hydro-alcoholic extracted JAPC. The investigation also revealed a compound with a [M−H]<sup>−</sup> ion at *m*/*z* 299.0767 in all of the JAPC extracts. The ion scan

ion at m/z 299.0767 in all of the JAPC extracts. The ion scan

experiment of this ion showed corresponding fragment ions at *m*/*z* values of 137.0233; 113.0229; 93.0331; 85.0281; and 71.0122. After comparison with the databases, this compound was identified as salicylic acid 2-*O*-β-d-glucoside.

**Table 3.** Chemical composition of Jerusalem artichoke leaf protein concentrate (JAPC) extracted from green biomass.


<sup>3</sup>Betaine (Trimethylglycine)

<sup>5</sup>Nicotinic acid

8 Salicylic acid-2-O-

Neochlorogenic acid (5-O-Caffeoylquinic acid)

Chlorogenic acid (3- O-Caffeoylquinic acid)

Cryptochlorogenic acid (4-O-Caffeoylquinic acid)

Vanillin (4-Hydroxy-3 methoxybenzaldehy de)

5-O-(4- Coumaroyl)quinic acid

<sup>11</sup>4-O-(4-Coumaroyl)

7

9

10

12

13

C5H11NO 2

4 Malic acid C4H6O5 1.33 133.013

(Niacin) C6H5NO2 1.51 124.039

6 Citric acid C6H8O7 1.73 191.019

glucoside C13H16O8 13.56 299.076

quinic acid C16H18O8 16.14 337.092

<sup>C</sup>8H8O3 16.22 153.055


**Table 3.** *Cont.*

**Figure 3.** Extracted ion chromatogram of chlorogenic acid structural isomers. **Figure 3.** Extracted ion chromatogram of chlorogenic acid structural isomers.

**Table 3.** Chemical composition of Jerusalem artichoke leaf protein concentrate (JAPC) extracted from green biomass. **No . Compound Formula Retention Time Measured Mass (m/z) Fragment s 1 Fragment s 2 Fragment s 3 Fragment s 4 Fragment [M + s 5 H]<sup>+</sup> [M − H]- <sup>1</sup>**γ-Aminobutyric acid C4H9NO2 1.25 104.071 Among flavonoids, isorhamnetin-3-*O*-glucoside with *m*/*z* 477.1033, kaempferol 3-glucuronide (kaempferol 3-*O*-β-d-glucopyranosiduronic acid) with *m*/*z* 461.0720, and astragaline (kaempferol 3-*O*-β-d-glucopyranoside) with *m*/*z* 447.0927 was found in the JAPC. However, to our knowledge, this is the first time glucuronide derivatives of isorhamnetin (isorhamnetin-3-*O*-glucuronide) and isoquercetin (quercetin 3-*O*-β-d-glucopyranoside) with *m*/*z* 463.0877 (Table 3 and Figure 4) have been identified. In addition to flavonols, most of the identified flavonoids belonged

<sup>81</sup>59.0737 58.0659

<sup>86</sup>96.0450 80.0501 78.0347

1.28 118.086

<sup>C</sup>16H18O9 10.14 353.087

<sup>C</sup>16H18O9 14.83 353.087

<sup>C</sup>16H18O9 16.11 353.087

<sup>C</sup>16H18O8 17.38 337.092

<sup>16</sup>87.0446 86.0607 69.0342 58.0658

<sup>57</sup>173.0447 171.0289 127.0388 93.0331 85.0280

<sup>70</sup>115.0024 89.0230 87.0075 72.9916 71.0123

<sup>18</sup>173.0082 129.0182 111.0075 87.0073 85.0280

<sup>70</sup>137.0234 113.0229 93.0331 85.0280 71.0123

<sup>26</sup>191.0556 179.0344 173.0443 161.0234 135.0441

<sup>26</sup>191.0555 179.0344 173.0447 161.0232 135.0441

<sup>35</sup>191.0555 173.0447 163.0390 119.0489 93.0331

<sup>35</sup>191.0556 173.0447 163.0391 119.0490 93.0332

<sup>17</sup>125.0600 111.0445 110.0366 93.0341 65.0393

<sup>26</sup>191.0557 179.0344 173.0448 135.0441

**4. Discussion** 

to the flavones. As far as we are aware, none of these has been identified previously in JAPC. For instance, we identified two dimethoxy-trihydroxyflavone isomers ([M − H]<sup>−</sup> ion at *m*/*z* 329.0661), dimethoxy-tetrahydroxyflavone ([M − H]<sup>−</sup> ion at *m*/*z* 345.0611), dihydroxy-methoxyflavone ([M − H]<sup>−</sup> ion at *m*/*z* 283.0607), and trihydroxy-trimethoxyflavone ([M − H] − at *m*/*z* 359.0767). Hymenoxin (5,7-dihydroxy-3′ ,4′ ,6,8-tetramethoxyflavone) at *m*/*z* 375.1080 and nevadensin (5,7-hydroxy-4′ ,6,8-trimethoxyflavone) at *m*/*z* 317.1389 were identified in positive ESI mode (Table 3). Within flavonoids, Butein (2′ ,3,4,4′ -tetrahydroxychalcone) and kukulkanin B (3′ -methoxy-2′ ,4,4′ -methoxychalcone) which related to chalcones subgroup were identified. Finally, liquiritigenin (4′ ,7-dihydroxyflavanone; [M − H]<sup>−</sup> at *m*/*z* 255.0657) was the only flavanone found in this study (Figure 4). *Plants* **2020**, *9*, x FOR PEER REVIEW 11 of 17

**Figure 4.** Extracted Ion Chromatograms (XIC) and MS spectra of selected phytoconstituents from Jerusalem artichoke leaf protein concentrate: (**A**): quercetin- 3-O-glucuronide; (**B**): 7-Hydroxy-6 methoxycoumarin (Scopoletin); (**C**): 1,3-dihydroxy-3,5,5-trimethylcyclohexylidene-4-acetic acid lactone (Loliolide); and (**D**): 4',7-Dihydroxyflavanone (Liquiritigenin). **Figure 4.** Extracted Ion Chromatograms (XIC) and MS spectra of selected phytoconstituents from Jerusalem artichoke leaf protein concentrate: (**A**): quercetin-3-*O* -glucuronide; (**B**): 7-Hydroxy-6-methoxycoumarin (Scopoletin); (**C**): 1,3-dihydroxy-3,5,5-trimethylcyclohexylidene -4-acetic acid lactone (Loliolide); and (**D**): 4′ ,7-Dihydroxyflavanone (Liquiritigenin).

The protein content of JAPC is influenced by plant type and also by the processing method. The average total protein content of the JAPC produced from Alba, Fuseau, and Kalevala was 33.4 m/m%; however, most of the isolated protein was found in the leaves, as these organs contain 3-fold higher total protein than the stem [23]. The JAPC comprised parenchyma tissues (80–87%) containing easily released cytoplasmic and chloroplast proteins such as Rubisco, which is of high nutritional value [24].

In addition to polyphenols, three different terpenes consistently appeared in the JAPC of the JA clones. Loliolide (1,3-dihydroxy-3,5,5-trimethylcyclohexylidene-4-acetic acid lactone) is a C<sup>11</sup> monoterpenoid lactone, which was observed with a [M + H]<sup>+</sup> ion at *m*/*z* 197.1178 (Figure 4). Dihydroactinidiolide as a volatile monoterpene with a [M + H]<sup>+</sup> ion at *m*/*z* 181.1229, and 7-deoxyloganic acid isomer, an iridoid monoterpene with a [M − H]<sup>−</sup> ion at *m*/*z* 359.1342, were recognized. Several proteinogenic amino acids were also identified (Table 3). In terms of vitamins, vitamin B molecules such as nicotinic acid (niacin; [M + H]<sup>+</sup> ion at *m*/*z* 124.0399) and riboflavin ([M + H]<sup>+</sup> ion at *m*/*z* 377.1461) were seen, while organic acids, i.e., malic acid and citric acid, and plant hormones such as indole acetic acid, were also identified in the JAPC.

#### **4. Discussion**

One important aspect of the biorefinery to become a competitive process is to produce at least one product of high value. The quantitative analysis of crude protein content of JAPC is a priority. The protein content of JAPC is influenced by plant type and also by the processing method. The average total protein content of the JAPC produced from Alba, Fuseau, and Kalevala was 33.4 m/m%; however, most of the isolated protein was found in the leaves, as these organs contain 3-fold higher total protein than the stem [23]. The JAPC comprised parenchyma tissues (80–87%) containing easily released cytoplasmic and chloroplast proteins such as Rubisco, which is of high nutritional value [24]. The time of harvest is critical to the quantity and quality of the JAPC produced from the aerial parts of the JA. Rashchenko [25] reported that the N content of older leaves is ~50% less than that in young leaves and Seiler [26] reported that the total protein content fell by 32.6% between the vegetative and flowering stages of JA growth. Knowing this, the shoots were harvested at the point of the maximum green leaf; ahead of senescence and before the bottom leaves turn dry. Ultimately, there was no significant difference in protein content between the two harvests.

In terms of an ideal protein source, the amino acid profile cannot be ignored, because among the 20 proteinogenic amino acids, nine cannot be synthesized by most animal species [20]. The content of these essential amino acids is, therefore, of particular interest. Among the green biomass fractions, the JAPC, as a dedicated protein enriched product for feed, was examined thoroughly. Several indispensable amino acids, i.e., lysine, isoleucine, leucine, methionine, and threonine, were present in high concentrations in the JAPC. However, even higher amino acid contents were found in JAPC by Rawate and Hill [27]; this may be attributed to different extraction methods and varieties. Additionally, the amino acid profiles exhibited minor differences between the two harvests, which may be due to differences in weather and plant age, as has previously been documented [11,25,26].

Considering the scientific literature about phytoconstituents of different JA organs, it was assumed that the green biomass-originated JAPC can be more than an alternative protein source. Qualitative analysis of phytochemicals in JAPC was performed by UHPLC-ESI-MS in both negative and positive ESI modes. The negative mode was used to identify flavonoid and phenolic acid (hydroxycinnamic acid and benzoic acid) derivatives, as it provided better sensitivity. The easy protonation of N in the positive mode made it suitable for identifying terpenes, amino acids, coumarins, and coumaroylquinic acids.

Phenolic compounds are one of the largest groups of plant secondary metabolites. Among them, phenolic acids are an important subgroup and their presence is characteristic of the Asteraceae family. The most revealed phenolic acids are the mono- and di, and even tri-hydroxycinnamic acid (p-coumaric, caffeic, and ferulic acids) esters of quinic acids in the tuber and shoot organs of JA [15,17,19]. Our measurements confirmed 13 different "phenolic acids" from green biomass originated hydro-alcoholic extracted JAPC. The three structural isomers of caffeoylquinic acid were identified with a similar degree of ionization, and the same molecular weight and fragmentation pattern. Hence, the area of the peak of extracted ion chromatogram of isomers is comparable and the 3-*O*-caffeoylquinic acid seemed to be the dominant one (Figure 3). However, neochlorogenic acid (5-*O*-caffeoylquinic acid) displayed the lowest ratio. Chlorogenic acid (3-*O*-caffeoylquinic acid) is known as the most abundant isomer in plants, whereas cryptochlorogenic acid (4-*O*-caffeoylquinic glucoside were also in detectable amounts.

acid) and neochlorogenic acid (5-*O*-caffeoylquinic acid) are present in much lower concentration [27]. Yuan et al. [15] cited 3-*O*-caffeoylquinic acid and 1,5-dicaffeoylquinic acid in high concentrations in JA leaves. However, Liang and Kitts [28] mentioned that 5-*O*-caffeoylquinic acid is the predominant isomer in fruits and vegetables. The presence of these phenolic acids is interesting from the aspect of both humans and animals, as several biological roles are attributed to caffeoylquinic acid isomers including antioxidant and antibacterial activities, hepato- and cardio-protection, anti-inflammatory and antipyretic activities, neuroprotection, anti-obesity, antiviral, and anti-hypertension activities, and central nervous system stimulation. Additionally, these compounds modulate lipid metabolism and glucose levels in both genetic metabolism-related disorders and healthy people [15,29]. Based on their health-promoting effects, caffeoylquinic acid isomers are increasingly recommended as natural and safe food additives, in place of synthetic antibiotics and immunity boosters. activity, but extensive conjugation of free hydroxyl groups to flavones results in low oral bioavailability; hence, they undergo rapid sulfation and glucuronidation in the small intestine and liver by phase II enzymes. Consequently, conjugated metabolites, but not the original compounds, can be found in plasma [34]. However, if one or more hydroxyl groups are capped by methylation, the substitution of a methoxy group by the hydroxyl group induces an increase in metabolic stability and improves transport and absorption. Considering the biological properties and chemical characteristics of hydroxyl and methoxy groups together, the hydroxylated methoxyflavones combine many advantages from both functional groups, improving their potential for application in human health [34]. Therefore, the presence of several hydroxylated methoxyflavones such as dimethoxy-trihydroxyflavone isomers, dimethoxy-tetrahydroxyflavone, dihydroxymethoxyflavone, trihydroxy-trimethoxyflavone, hymenoxin, and nevadensin, increase the value of

has significant importance. One side the hydroxyl groups of flavones have free radical scavenging

*Plants* **2020**, *9*, x FOR PEER REVIEW 13 of 17

also shown potent antiadipogenic activity against the preadipocyte cell line in vitro assay systems [31]. Within non-flavonoid phenolics, two salvianolic acid derivatives and a salicylic acid-2-O-

Flavonoids are widespread secondary metabolites that occur as part of the phenolic constituents of plants. However, only a few of these have been described in the aerial part of JA including isorhamnetin glucoside, kaempferol glucuronide, and kaempferol-3-O-glucoside [14]. Based on the present qualitative analysis, 18 flavonoid compounds were revealed in the JAPC as green biomass originated product (Figure 5). Generally, cell vacuoles are the main storage places for soluble flavonoids. The JPAC is mostly made up of content released from the cytoplasm and vacuoles cell fractions, which may be the reason for the relatively high proportion of identified flavonoids. Within flavonoids, five flavonols were detected in the JAPC, in which all of them occurred as glycosides. Primarily, the solubility of flavonoids is due to their sugar substitutions. Among the sugars, glucose and glucuronic acid at a single position are probably the most common substituents [32]. The importance of flavonoid glucuronides is related to their health-promoting activities such as the antiinflammatory and neuroprotective activities of quercetin-3-O-glucuronide [33]. Most of the identified flavonoid compounds belong to the flavones (Figure 5C). All of the flavone compounds were

The four different coumarins have also been revealed in the JAPC. Coumarins are widely distributed non-flavonoid polyphenols in the plant kingdom (Figure 5B). However, "simple coumarins" as coumarin subgroup is mainly present in the Asteraceae family. Therefore, each coumarin subclass-related compounds are used for the chemotaxonomic approach, too. Scopoletin and ayapin were already described in tubers of JA and assumed the presence of them in aerial part as in the case of *Helianthus annuus* [30]. Our measurement confirmed the presence of scopoletin along with isoscopoletin, 6-methyl coumarin, and fraxidin from green biomass originated product of JA. Some of simple coumarins are known as phytoalexins. At the same time, fraxidin and scopoletin have also shown potent antiadipogenic activity against the preadipocyte cell line in vitro assay systems [31]. Within non-flavonoid phenolics, two salvianolic acid derivatives and a salicylic acid-2-*O*-glucoside were also in detectable amounts. JAPC. From minor flavonoids, two chalcones were detected in JAPC. Butein (2',3,4,4'- Tetrahydroxychalcone) is one of them which is widely biosynthesized in plants; however, no reference has been found citing it in JA. Based on preclinical studies, butein exhibits significant therapeutic potential against various diseases. In vitro and in vivo studies support that butein can suppress proliferation and trigger apoptosis in various human cancer cells with no or only minimal toxicity inducing in normal cells [35]. Liquiritigenin (4',7-dihydroxyflavanone) as the only flavanone was measured in JA flowers by Johansson et al. [13]. Our results confirmed the presence of liquiritigenin in JAPC, too. Liquiritigenin is known to be a promising active estrogenic compound and is a highly selective estrogen receptor β agonist, which may be helpful to women who suffer from menopausal symptoms [36].

**Figure 5.** Identified phenolic compounds from Jerusalem artichoke leaf protein concentrate: (**A**) ratio of flavonoid and non-flavonoid phenolic compounds; (**B**) number of identified compounds within non-flavonoid phenolics subgroup; and (**C**) number of identified compounds within flavonoid phenolics subgroup.

Flavonoids are widespread secondary metabolites that occur as part of the phenolic constituents of plants. However, only a few of these have been described in the aerial part of JA including isorhamnetin glucoside, kaempferol glucuronide, and kaempferol-3-*O*-glucoside [14]. Based on the present qualitative analysis, 18 flavonoid compounds were revealed in the JAPC as green biomass originated product (Figure 5). Generally, cell vacuoles are the main storage places for soluble flavonoids. The JPAC is mostly made up of content released from the cytoplasm and vacuoles cell fractions, which may be the reason for the relatively high proportion of identified flavonoids. Within flavonoids, five flavonols were detected in the JAPC, in which all of them occurred as glycosides. Primarily, the solubility of flavonoids is due to their sugar substitutions. Among the sugars, glucose and glucuronic acid at a single position are probably the most common substituents [32]. The importance of flavonoid glucuronides is related to their health-promoting activities such as the anti-inflammatory and neuroprotective activities of quercetin-3-*O*-glucuronide [33]. Most of the identified flavonoid compounds belong to the flavones (Figure 5C). All of the flavone compounds were hydroxylated methoxyflavones, which contain one or more methoxy groups instead of a hydroxyl group on a flavone framework. The substitution of a methoxy group for a hydroxyl group in flavones has significant importance. One side the hydroxyl groups of flavones have free radical scavenging activity, but extensive conjugation of free hydroxyl groups to flavones results in low oral bioavailability; hence, they undergo rapid sulfation and glucuronidation in the small intestine and liver by phase II enzymes. Consequently, conjugated metabolites, but not the original compounds, can be found in plasma [34]. However, if one or more hydroxyl groups are capped by methylation, the substitution of a methoxy group by the hydroxyl group induces an increase in metabolic stability and improves transport and absorption. Considering the biological properties and chemical characteristics of hydroxyl and methoxy groups together, the hydroxylated methoxyflavones combine many advantages from both functional groups, improving their potential for application in human health [34]. Therefore, the presence of several hydroxylated methoxyflavones such as dimethoxy-trihydroxyflavone isomers, dimethoxy-tetrahydroxyflavone, dihydroxy-methoxyflavone, trihydroxy-trimethoxyflavone, hymenoxin, and nevadensin, increase the value of JAPC.

From minor flavonoids, two chalcones were detected in JAPC. Butein (2′ ,3,4,4′ -Tetrahydroxychalcone) is one of them which is widely biosynthesized in plants; however, no reference has been found citing it in JA. Based on preclinical studies, butein exhibits significant therapeutic potential against various diseases. In vitro and in vivo studies support that butein can suppress proliferation and trigger apoptosis in various human cancer cells with no or only minimal toxicity inducing in normal cells [35].

Liquiritigenin (4′ ,7-dihydroxyflavanone) as the only flavanone was measured in JA flowers by Johansson et al. [13]. Our results confirmed the presence of liquiritigenin in JAPC, too. Liquiritigenin is known to be a promising active estrogenic compound and is a highly selective estrogen receptor β agonist, which may be helpful to women who suffer from menopausal symptoms [36].

Three terpenes consistently appeared in the tested JAPC from all the JA clones. Loliolide, a C<sup>11</sup> monoterpenoid lactone, is considered to be a photo-oxidative or thermally degraded product of carotenoids [37]. Similarly, we identified dihydroactinidiolide, a volatile monoterpenoid, which is a flavor component of several plants such as tobacco and tea. According to Yun et al. [38], thermal treatment induces the formation of dihydroactinidiolide from β-carotene. Kaszás et al. [8] confirmed that the green juice of the JAPC contains a marked number of carotenoids, which may be able partially to convert to loliolide or dihydroactinidiolide, causing the number of detectable terpenes to increase. Studies have confirmed that loliolide inhibits growth and germination, while also being phytotoxic, repelling leaf-cutter ants and having antitumor and antimicrobial activities in animals and microorganisms [37,39]. Dihydroactinidiolide has a carbonyl group that can react with nucleophilic structures in macromolecules, providing high potential reactivity to the molecules. It also shows cytotoxic effects against cancer cell lines [38]. The 7-Deoxyloganic acid isomer is the third terpene, which is known to be an intermediate in the secoiridoid pathway in plants.

The fatty acid and lipid contents of JA tubers have been reported by several authors [11,40]; however, little information is available about the fatty acid composition of its leaves and JAPC [41]. Recently, rapidly growing interest is for PUFAs, because humans and other mammals are incapable of synthesizing omega-6 and omega-3 PUFAs, due to the lack of ∆12 and ∆15 desaturase enzymes, which insert a cis double bond at the n-6 and n-3 positions [42]. Hence, linolenic and linoleic acids are essential nutrients converted from oleic acid in the endoplasmic reticulum of plant cells. Linolenic acid is the precursor of longer-chain PUFAs such as eicosapentaenoic acid (EPA: C20:5ω –3) and docosahexaenoic acid (DHA: C22:6ω –3), which can be synthesized in humans. Similarly, linoleic acid is an essential precursor to dihomo-γ-linoleic acid (DGLA: C20:3ω –6) and arachidonic acid (C20:4ω –6). As they are essential to life, linolenic and linoleic acids must be supplied to animals and humans through diet. In the JAPC of all JA clones, the highest contribution to the fatty acid profile was made by linolenic acid (38.6–42.7%) and linoleic acid (23.4–26.9%) as shown in Figures 1 and 2. The correct proportions of linoleic and linolenic acids are emphasized by anthropological

and epidemiological studies. The required ratio of omega-6 to omega-3 essential fatty acids is ~1:1, according to the evolutionary history of the human diet. In contrast, in the current Western diet, this ratio has shifted to 10–20:1, which is not beneficial to health and promotes the pathogenesis of many diseases [43]. We found a ratio of ~0.6:1 for omega-6 to omega-3 essential fatty acids in JAPC, which is very favorable and close to Paleolithic nutrition levels.

Concerning the harvest time, Alba and Kalevalas' JAPC exhibited higher oleic acid contents in the second harvest (when the nights were cooler). According to Barrero-Sicilia et al. [44], plants often respond to low temperature by increasing the levels of unsaturated fatty acids in the membrane and increasing membrane fluidity and stabilization. We found the opposing tendency in the saturated myristic acid (C14:0) contents of the JAPC, in which levels were higher in the first harvest (when the nights were warmer).

In summary, this study delivers deeper insights into JAPC originating from the fractionated green biomass of different JA clones focusing on its content of different phytochemicals that are potentially bioactive compounds and have several important uses. Future studies should investigate the anti-nutritional ingredients of JAPC, analyze the chemical composition of other fractions such as the brown juice and fiber, and calculate the economic viability of JA crops.

#### **5. Conclusions**

This paper discusses two important points related to JA. Firstly, we examined the potential production of leaf protein from its aerial parts. Secondly, we aimed to determine the quality of produced JAPC as a promising protein source that could be directed to human consumption and/or animal feeding. Biochemical analyses revealed that the JAPC is not only a good source of protein with a favorable amino acid composition but also a repository of essential fatty acids, flavonoid and non-flavonoid phytonutrients. The saturated palmitic acid (C16:0), stearic acid (C18:0), and the monosaturated form of stearic acid, oleic acid (C18:1ω–9), are often referred to as common fatty acids. They are biosynthesized in the plastids and partially incorporated into the cell and subcellular membranes [45]. The JAPC originates mainly from crushed cells of vegetative tissues containing membrane debris, which explains the relatively higher proportion of palmitic (16.4–17.9%) and oleic (6.6–11.6%) acids. Moreover, several important viable compounds were detected in JAPC. These compounds are known for their antibacterial, anti-inflammatory, and antipyretic activities, neuroprotection, anti-obesity, antiviral, and anti-hypertension activities, hepato- and cardio- protection, and central nervous system stimulation. However, the quantity and quality of the phytochemicals are specific to the species and can vary with the bioanalytical technology used. Hence, a quantitative analysis of identified phytocompounds needs to confirm the nutritional value of JAPC. Overall, the present results confirm that the green aerial parts of this underestimated plant can be a source of marketable products involving into green biorefinery concept.

**Author Contributions:** Conceptualization, L.K., M.F., and É.D.-S.; data curation, L.K. and É.D.-S.; formal analysis, É.D.-S.; funding acquisition, M.F.; methodology, Z.K.; project administration, É.D.-S.; software, É.N. and Z.C.; supervision, É.D.-S.; validation, J.K.; visualization, T.A.; writing—original draft, H.E.-R. and N.E.; and writing—review and editing, T.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The research was financed by the Higher Education Institutional Excellence Programme (NKFIH-1150-6/2019) of the Ministry of Innovation and Technology in Hungary, within the framework of the Biotechnology thematic programme of the University of Debrecen. This research was financed also by the "Complex Rural Economic and Sustainable Development, Elaboration of its Service Networks in the Carpathian Basin (Project ID: EFOP-3.6.2-16-2017-00001, Hungary)" research project. This paper was also supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. The authors thank Mohamed E. Ragab (Horticulture Department, Faculty of Agriculture, Ain Shams University, Egypt) for providing them with tubers of Fuseau.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Evaluation of Alpha-Amylase Inhibitory, Antioxidant, and Antimicrobial Potential and Phytochemical Contents of** *Polygonum hydropiper* **L.**

**Abdul Nasir 1,2 , Mushtaq Khan <sup>1</sup> , Zainab Rehman <sup>3</sup> , Atif Ali Khan Khalil <sup>4</sup> , Saira Farman <sup>1</sup> , Naeema Begum <sup>1</sup> , Muhammad Irfan <sup>5</sup> , Wasim Sajjad 4,\* and Zahida Parveen 1,\***


Received: 10 June 2020; Accepted: 3 July 2020; Published: 6 July 2020

**Abstract:** *Polygonum hydropiper* L. is a traditionally used medicinal plant. The present study was designed to explore the α-amylase inhibitory, antioxidant, and antimicrobial activities of *Polygonum hydropiper* L. Polarity-based solvent extracts (*n*-hexane, acetone, chloroform, methanol, ethanol, and water) of *Polygonum hydropiper* leaves and stem were used. Antioxidant activity was assessed by free radical scavenging assay (FRAP) and 2,2-diphenylpicrylhydrazyl (DPPH) free radical scavenging activity methods. Quantitative phytochemical analyses suggested that the stem of *Polygonum hydropiper* L. contains higher levels of bioactive compounds than its leaves (*p* < 0.05). The results suggested that stem-derived extracts of *Polygonum hydropiper* L. are more active against bacterial species, including two Gram-positive and three Gram-negative strains. Moreover, our results showed that the bioactive compounds of *Polygonum hydropiper* L. significantly inhibit α-amylase activity. Finally, we reported the polarity-based solvent extracts of *Polygonum hydropiper* L. and revealed that the stem, rather than leaves, has a high antioxidant potential as measured by FRAP and DPPH assay with IC<sup>50</sup> values of 1.38 and 1.59 mg/mL, respectively. It may also be deducted from the data that the *Polygonum hydropiper* L. could be a significant candidate, which should be subjected to further isolation and characterization, to be used as an antidiabetic, antimicrobial and antioxidant resource in many industries, like food, pharmaceuticals and cosmetics.

**Keywords:** water-pepper; FRAP; DPPH; polarity-based solvent extraction; α-amylase inhibition

#### **1. Introduction**

The Polygonaceae family is comprised of 48 genera and 1200 species [1,2]. Among the 60 species of the *Polygonum* distributed throughout the world, approximately 20 species are found in Pakistan [3]. They grow in moist places and shallow water. *Polygonum hydropiper* L. is an important medicinal plant of Polygonaceae family. It is commonly grown annually in the wet areas and is recognized as a common weed which is native to Southeast Asia [4]. Morphologically, the stem is round; its length usually varies from 40 to 70 cm [5].

This species has anti-inflammatory potential [6] and insecticidal properties [7], along with anticholinesterase, phytotoxic, anthelmintic, antiangiogenic, anticancer, antimicrobial, and antioxidant potential [8–11]. This plant is used in treating broad range of disorders, including gastrointestinal disturbances, neurological disorders, inflammation, and diarrhea [12]. Moreover, it is used to treat other diseases, like dyspepsia, itching skin, excessive menstrual bleeding, hemorrhoids, and cancer (particularly colon, breast, and prostate cancer) [13]. Furthermore, sap of the leaves is used to treat headache, pain, toothache, liver enlargement, gastric ulcer, dysentery, and loss of appetite. The juice of this plant is considered useful for treating wounds, skin diseases, and painful carbuncles [14]; it can also be used as an anthelminthic herb, in the treatment of snake-bites, and as a diuretic [15].

The phytochemicals of *P. hydropiper* L. include catechins, procyanidins, condensed tannins, and antitumor agents that are flavanols in nature [16]. Flavonoids with antioxidant activity and aldose reductase and tyrosinase inhibitory activities have also been found in *P*. *hydropiper* L. [4,17]. Due to the presence of drimane-type sesquiterpenes, *P*. *hydropiper* L. has some insecticidal and antifungal activities [18,19]. The antifungal activities of *P*. *hydropiper* L. are due to the presence of confertifolin, a natural antimicrobial compound [20]. Other bioactive compounds of *P*. *hydropiper* L. include gallic acid; ellagic acid; 3,3′ -di-O-methyl ether; anthraquinone; aromatic 6-lactone; and flavonoids such as viscosumic acid, oxymethyl-anthraquinones, rutin, hyperin, isoquercitrin, epicatechin, quercetin, kaempferol, and isorhamnetin [21]. It has been reported that this species contains pyrocatechol, 4-methyloxazole, caryophyllene, succinimide, vanillic acid, myristic acid, farnesol, arachidic acid, methyl ester, and capsaicin [10].

The excessive and long-term use of synthetic drugs causes several side effects [22]. In the human body, numerous cellular processes produce free radicals (FR) and reactive oxygen species (ROS). Such toxics may also be produced exogenously by pollutants, radiation, smoke, drugs, and xenobiotics [23]. Overproduction of different chemically reactive oxygen plagiaristic molecules such as hydrogen peroxide (H2O2), superoxide (O<sup>2</sup> −), and hydroxyl radicals (OH−) is highly toxic and leads to biomolecular damage, which in turn causes diabetes mellitus, cancer, atherosclerosis, and heart and neurodegenerative diseases [24]. In contrast, the overproduction of FR and ROS species can be averted by antioxidant substances. It is reported that secondary metabolites of plants like phenols, flavonoids, and alkaloids have antioxidant, antidiabetic, anthelminthic, lipid-lowering, anticoagulant, and antimicrobial properties [25].

Diabetes mellitus is characterized by hyperglycemia, lipedema, and oxidative stress and predisposes affected individuals to long-term complications afflicting the eyes, skin, kidneys, nerves, and blood vessels. Recently, it has been estimated that the prevalence of diabetes by 2025 will increase from 143 million to 300 million patients [26]. Various studies have indicated that dietary supplementation with combined antiglycation and antioxidant nutrients might be a safe and simple complement to traditional therapies targeting diabetic complications [27]. Hyperglycemia, through both enzymatic and non-enzymatic mechanisms, produces oxidative stress by producing free radicals. α-Amylase hydrolyzes (1,4)-α-D-glucosidic linkages in polysaccharides containing three or more (1,4)-α-linked D-glucose units, which ultimately increases the blood sugar level; α-amylase-inhibiting drugs such as acarbose, miglitol, and voglibose are frequently used in diabetes management [28,29]. However, α-amylase-inhibiting drugs cause severe side effects like bloating and abdominal uneasiness [30]. Therefore, the administration of natural resources to treat diabetes seems to be a promising strategy. To this end, targeting α-amylase inhibition by natural remedies may be an ideal platform in diabetes prevention [31,32]. In this context, the current study was designed to determine the bioactive compounds of *P. hydropiper*. Furthermore, the extracts of *P. hydropiper* were evaluated for their potential to inhibit α-amylase activity and reduce oxidative stress. Finally, the antimicrobial potential of *P. hydropiper* was demonstrated.

#### **2. Results**

#### *2.1. Determination of Bioactive Compounds*

In first series of experiments, class of secondary metabolites including alkaloids, tannins, flavonoids, β-carotene, and lycopene from the leaves (PHL) and stem (PHS) of *P. hydropiper* were extracted with their respective solvents and quantified. The comparative analyses found that concentrations of alkaloids and tannins were significantly higher in *P*. *hydropiper* stem than in leaves (t = −3.22, *p* = 0.032; t = −3.61, *p* = 0.023). However, flavonoids were found in comparable amounts in both stem and leaves (t = −0.427, *p* = 0.691) (Table 1). We also noticed that the leaves of *P. hydropiper* contained significantly higher concentrations of β-carotene and lycopene than those found in the stem of *P. hydropiper* (t = 2.90, *p* = 0.044; t = 4.31, *p* = 0.013).


**Table 1.** Phytochemical constituents of *P. hydropiper* leaves and stem.

<sup>a</sup> Values are means of triplicate determination (n <sup>=</sup> 3) <sup>±</sup> standard deviations; \* indicates significant difference (*p* < 0.05) between leaves and stem; PHL, *P. hydropiper* leaves; PHS, *P. hydropiper* stem.

#### *2.2. In Vitro Evaluation of* α*-Amylase Inhibition*

The different solvent extracts of *P. hydropiper* stem and leaves were investigated for their potential to inhibit α-amylase activity at six different concentrations (0.46, 0.94, 1.88, 3.75, 7.50, and 15 mg/mL). The dose–response calibration curves for n-hexane, acetone, chloroform, ethanol, methanol, and water extracts of *P. hydropiper* leaves and stem were constructed individually. Percent α-amylase inhibition and IC<sup>50</sup> values were determined from the dose–response calibration curves for each type of extract (Figures 1 and 2).

The α-amylase inhibitory activities of the leaf extracts were ranked in the following order: n-hexane (IC<sup>50</sup> 1.03 mg/mL; R<sup>2</sup> 0.566) > chloroform (IC<sup>50</sup> 1.53 mg/mL; R<sup>2</sup> 0.6492) > methanol (IC<sup>50</sup> 2.32 mg/mL; R <sup>2</sup> 0.7255) > acetone (IC<sup>50</sup> 4.70 mg/mL; R<sup>2</sup> 0.9919) > water (IC<sup>50</sup> 4.85 mg/mL; R<sup>2</sup> 0.7629) > ethanol (IC<sup>50</sup> 13.89 mg/mL; R<sup>2</sup> 0.3249). Differently from those of the leaf extracts, the α-amylase inhibitory activities of the stem extracts were ranked in the following order: chloroform (IC<sup>50</sup> 2.599 mg/mL; R <sup>2</sup> 0.8232) > methanol (IC<sup>50</sup> 3.517 mg/mL; R<sup>2</sup> 0.8375) > ethanol (IC<sup>50</sup> 5.672 mg/mL; R<sup>2</sup> 0.4736) > n-hexane (IC<sup>50</sup> 6.910 mg/mL; R<sup>2</sup> 0.4399) > acetone (IC<sup>50</sup> 11.86 mg/mL; R<sup>2</sup> 0.5608) > water (IC<sup>50</sup> 13.12 mg/mL; R <sup>2</sup> 0.6824).

#### *2.3. Antioxidant Capacity*

The antioxidant potential of the different extract types was analyzed at six concentrations (0.46, 0.94, 1.88, 3.75, 7.50, and 15 mg/mL) by using the free radical scavenging assay (FRAP) and the 2,2-diphenylpicrylhydrazyl (DPPH) assay. Table 2 shows the antioxidant potential measured by two assays.

Inhibition of α – **Figure 1.** Inhibition of α-amylase activity by different extracts (ethanol, (**A**); acetone, (**B**); methanol, (**C**); *n*-hexane, (**D**); chloroform, (**E**) and water, (**F**)) of *P. hydropiper* leaves (PHL) and stem (PHS). Solid lines represent hyperbolic dose–response curves, which were generated in GraphPad Prism. Values represent the means of triplicate measurements (n = 3).

α **Figure 2.** α-Amylase inhibitory activity of the different extracts tested: comparison of IC<sup>50</sup> values. The IC<sup>50</sup> values were calculated from dose-dependent percent inhibition. Values represent the means of triplicate measurements (n = 3). Bars represent the standard deviation.

**Table 2.** Antioxidant activity of *P*. *hydropiper* extracts in different solvents at different concentrations.


<sup>a</sup> Values are means of triplicate determination (n <sup>=</sup> 3) <sup>±</sup> standard deviations; *<sup>p</sup>* values less than 0.05 were considered to be statistically significant; \* *p* < 0.05, \*\* *p* < 0.01; PHL, *P. hydropiper* leaves; PHS, *P. hydropiper* stem.

The FRAP activities for different extract types of both stem (PHS) and leaves (PHL) were observed to be ranked in the following order: PHS ethanol (IC<sup>50</sup> 1.38 mg/mL; R <sup>2</sup> 0.7847) > PHS n-hexane (IC<sup>50</sup> 1.50 mg/mL; R <sup>2</sup> 0.8407) > PHS methanol (IC<sup>50</sup> 1.73 mg/mL; R <sup>2</sup> 0.8483) > PHS acetone (IC<sup>50</sup> 1.81 mg/mL; R <sup>2</sup> 0.8814) > PHL acetone (IC<sup>50</sup> 2.29 mg/mL; R <sup>2</sup> 0.977) > PHL methanol (IC<sup>50</sup> 2.30 mg/mL; R <sup>2</sup> 0.9121) > PHL ethanol (IC<sup>50</sup> 2.99 mg/mL; R <sup>2</sup> 0.8090) > PHL n-hexane (IC<sup>50</sup> 5.52 mg/mL; R <sup>2</sup> 0.7588). All types of extracts of the stem showed higher FRAP activity than those of the leaves of *P. hydropiper*.

The DPPH activities of both stem and leaves were also analyzed for different extract types at different concentrations. The acetonic extracts of both stem and leaves were more effective than ethanolic extracts. The activities was observed to be ranked in the following order: PHS acetone (IC<sup>50</sup> 1.59 mg/mL; R <sup>2</sup> 0.8330) > PHL acetone (IC<sup>50</sup> 2.94 mg/mL; R <sup>2</sup> 0.9244) > PHL ethanol (IC<sup>50</sup> 5.14 mg/mL; R <sup>2</sup> 0.9102) > PHS ethanol (IC<sup>50</sup> 6.88 mg/mL; R <sup>2</sup> 0.8126).

#### *2.4. Antimicrobial Activity*

Our results showed strong antibacterial activities for both acetonic and ethanolic extracts at all six concentrations (0.46, 0.94, 1.88, 3.75, 7.50, and 15 mg/mL) of *P. hydropiper* stem and leaves. Zones of inhibition of the tested extracts are depicted in Table 3. The acetonic stem extract showed antibacterial activity against five different microbial species (*Escherichia coli*, *Staphylococcus aurous*, *Klebsiella pneumoniae*, *Morganella morganii*, and *Haemophilus influenzae*); however, the leaves did not show antibacterial activity against *E. coli and S. aureus*. The ethanolic extracts of stem and leaves showed strong antiproliferative activities against all the microbial species tested (Table 3).


**Table 3.** Antimicrobial activity of different solvent extracts of *P. hydropiper* leaves and stem at different concentrations.

<sup>a</sup> Values are means of triplicate determination (n = 3); ND, not detected at this concentration; A, ampicillin (10 µg); DMSO, dimethyl sulfoxide (100%); Ant Ag, antimicrobial agent.

#### **3. Discussion**

The excessive use of synthetic drugs causes several side effects and may lead to conflict [22]. In contrast, traditional medicines are harmless, effective, and inexpensive drug candidates. In order to evaluate the biological activities of plant extracts with respect to traditional uses, *P. hydropiper* was previously screened for its antioxidant potency and antimicrobial and antipathogenic activities at low doses and with few solvent extractions [8,10]. We evaluated the antioxidant and antibacterial potential at high doses and with several different solvent extractions; additionally, we screened the α–amylase inhibitory potential of *P. hydropiper*.

Our results show that *P. hydropiper* leaves are rich in tannins and flavonoids, which is in strong agreement with the results of Nakao et al. (1999), who they reported condensed tannins, procyanidins, and catechins which are flavones in nature [16]. Yang et al. (2011) also reported the presence of flavonoid in *P. hydropiper* leaves [21]. Previously, it was reported that *P. hydropiper* species contained 4-methyloxazole (flavonoid in nature) and succinimide [8]. The therapeutic application of β-carotene showed a strong association with a lower risk of lung cancer [33]. β-Carotene along with phenytoin has great antiepileptic activity and can be used as a therapeutic agent in epilepsy management [34]. Tannins have been reported as anticancer and anti-inflammatory agents and can be used for ulcerated tissues [35–37]. Polyphenols are known not only to ease the oxidative stress status, but also to act on cellular signaling pathways, including vascular endothelial growth factor (VEGF)-mediated angiogenesis, endoplasmic reticulum (ER) stress, nitric oxide (NO· ) signaling, and nuclear factor E2-related factor 2 antioxidant pathways, thus preventing vascular complications in diabetes. Resveratrol, one of the most studied polyphenols, has been reported to restore the insulin receptor substrate 1 (IRS-1) and endothelial nitric oxide synthase (eNOSx) signaling pathway in endothelial cells under palmitate-induced insulin resistance [38,39]. As a source of tannins, *P. hydropiper*

may be used to treat cancer, inflammation, and ulcerated tissues. Furthermore, our findings revealed that the leaves of *P. hydropiper* contain a significantly high level of lycopene. The intake of lycopene reduces the risk of prostate cancer [40] and plays a protective role against nephrotoxicity [41]. Lycopene consumption can also regulate endothelial function, thereby reducing oxidative stress in healthy humans [42].

Enzymes that are primarilyinvolvedinincreasing blood glucose, such asα-amylase andα-glucosidase, have been targeted as a therapeutic approach in postprandial hyperglycemia [43] because their inhibition can cause reduction in postprandial hyperglycemia [44]. Our results indicate that α-amylase activity was inhibited by the extracts of *P. hydropiper* leaves only. Specifically, the *n*-hexane extract of the leaves showed the most potent antiamylase activity. Therefore, we suggest that leaf extracts of *P. hydropiper* could be used as a future therapeutic candidate in treating hyperglycemia.

The antioxidant activity of leaf and stem extracts was determined by two methods: the free radical scavenging assay (using ferric ion reducing agent) using and the DPPH scavenging assay. The results in our study indicate that this plant species was potently active, which suggests that all different extracts of *P. hydropiper* leaves and stem contained compounds that are capable of hydrogen donation to the free radical for the purpose of eliminating the odd electron, thus reducing the radicals' activity. This also implies that this plant species may be beneficial, especially at high concentrations, for treating the pathological damage caused by radicals' activities. All of the extracts of both stem and leaves at different concentrations were also active against the Gram-negative and Gram-positive bacteria. However, such activity was not shown by the acetonic extract of leaves against *E. coli* (which might be due to outer membrane in Gram-negative bacteria acting as a permeability barrier) and *S. aureus* [45]. Hence, we suggest that ethanolic extracts of *P. hydropiper* stem and leaves could be used as antioxidant and antibacterial agents and should be studied further.

#### **4. Materials and Methods**

#### *4.1. Chemical and Reagents*

Soluble starch, potassium ferrocyanide, trichloroacetic acid, ferric chloride, and solvents used for polarity-based extraction (n-hexane, acetone, chloroform, ethanol, and methanol) were purchased from Sigma-Aldrich (Lahore, Pakistan). Dimethylsulfoxide, quercetin, Folin's phenol, and tannic acid were obtained from Merck (Karachi, Pakistan). Porcine pancreatic α-amylase, ascorbic acid, and DPPH solution were purchased from Sigma-Aldrich (Lahore, Pakistan) for measurement of α-amylase, FRAP, and DPPH assay, respectively. All reagents were biochemical reagent grade.

#### *4.2. Sample Collection*

*Polygonum hydropiper*was collected fromMardan District, Khyber Pakhtunkhwa, Pakistan, in April 2016. The plant was identified as *P. hydropiper* and deposited in the Department of Botany, Abdul Wali Khan University, Mardan, Pakistan.

#### *4.3. Sample Preparation*

Fresh leaves and stem of *P. hydropiper* were separated, washed with tap water to remove dust, and then air-dried at 25 ◦C for 30 days in an air flux drying oven. The plant parts were crushed to fine powder (80 mashes) with the help of an electric blender. Powdered samples were placed in sterile sealed bags each containing a damp paper towel and kept at 4 ◦C for further analyses.

#### *4.4. Solvent–Solvent Extraction*

The extracts of powdered samples were isolated using the solvent–solvent extraction method. Several solvents (n-hexane, acetone, chloroform, ethanol, methanol, and water) were used to ensure the polarity-based extraction. Initially, each sample was extracted by shaking with n-hexane at a ratio of 1:10 (*w*/*v*) for 24 h at room temperature. The corresponding sample was then filtered by Whatman

filter paper. The pellet was separated, and the supernatant was transferred to a preweighed Falcon tube. The residual pellet was re-extracted with next solvent, which was slightly polar than n-hexane, using the same ratio, temperature, and time. The same procedure was repeated with all solvents, and all extracts were allowed to dry in oven at 37 ◦C. The dried sample was weighed and redissolved in dimethylsulfoxide (DMSO) at a final concentration of 15 mg/mL.

#### *4.5. Phytochemical Determination*

#### 4.5.1. Determination of Flavonoids

Flavonoid estimation was carried out by spectrophotometric assay as previously described [46]. Five grams of air-dried powdered sample was dissolved in 50 mL of 80% aqueous ethanol and incubated for 24 h in a shaker incubator. The extract was centrifuged at 10,000 rpm and 25 ◦C for 15 min. The pellet was discarded and the supernatant containing flavonoids was stored in a 50 mL Falcon tube at 4 ◦C. The flavonoid extract (250 µL) was mixed with 1.25 mL of sterile distilled water and 75 µL of 5% NaNO<sup>2</sup> solution. After 5 min, 150 µL of 10% AlCl3.H2O was added, and the mixture was incubated for 6 min. Thereafter, 500 µL of 1 M NaOH and 275 µL of sterile distilled water were added to the mixture. The solution was mixed, and absorbance was measured at λ of 415 nm. Different concentrations of quercetin (15–500 µg) were used as standards to calculate the standard curve, while 80% aqueous ethanol was used as blank.

#### 4.5.2. Determination of β-Carotene and Lycopene

β-Carotene and lycopene were extracted and quantified according to the method described by Lillian et al. [47]. Briefly, methanol extract was prepared by dissolving 10 g of air-dried powdered sample in 100 mL of methanol and incubating in a shaker incubator for 24 h. The extract was filtered by Whatman filter paper, the pellet was discarded, and supernatant was isolated. Thereafter, the methanol contents were evaporated by heating in a water bath. The dried sample was dissolved in acetone and n-hexane mixture (4:6). Finally, the reaction mixture containing β-carotene and lycopene was stored at 4 ◦C. The spectrophotometric analysis was carried out by measuring absorbance at λ's of 453, 505, 645, and 663 nm. β-Carotene and lycopene contents were calculated [48] by using the following equations:

$$\text{Lycepene } (\text{mg/50 mL}) = 0.0458 \text{A}\_{663} + 0.372 \text{A}\_{505} - 0.0806 \text{A}\_{453} \tag{1}$$

$$\beta-\text{carotene}\left(\text{mg/50 mL}\right) = 0.216\text{A}\_{663} + 0.304\text{A}\_{505} - 0.452\text{A}\_{453} \tag{2}$$

#### 4.5.3. Determination of Tannins

Tannin contents of *P. hydropiper* were extracted using the method of Makkar et al. [49]. Briefly, 0.5 g of each air-dried powdered sample was dissolved in 100 mL of 70% acetone and incubated while shaking for 6 h. The sample was filtered by Whatman filter paper, the pellet was discarded, and the supernatant was stored in a 50 mL Falcon tube at 4 ◦C. Different concentrations of tannic acid (3–50 mg) were prepared by serial dilution from stock solution (50 mg/100 mL of 70% acetone). The tannin extracts (50 µL) was mixed with 950 µL of sterile distilled water. Thereafter, 0.5 mL of Folin's phenol reagent (mixture of phosphomolybdate and phosphotungstate), used for phenolic and polyphenolic antioxidants detection, and 2.5 mL of 20% NaCO<sup>3</sup> solution were added and vortexed. The solution was incubated at room temperature for 40 min. Finally, the absorbance was measured at λ of 725 nm. During the experiment, 70% acetone was used as blank and treated as positive control.

#### 4.5.4. Determination of Alkaloids

Alkaloids were extracted by using the acid–base shifting method [46]. Briefly, dried powdered sample was dissolved in ethanol at a ratio of 1:10 (w/v) and left to shake for 24 h at room temperature. The extract was concentrated while drying in an oven. The dried sample was redissolved in ethanol

with 1% HCl. The mixture was then precipitated in a refrigerator for 3 days. The solution was filtered, and pH was maintained at 8–10 by the addition of ammonium hydroxide. This basic solution was extracted with chloroform by using a separating funnel. The chloroform layer containing alkaloids was recovered, and ethanol layer was discarded. The chloroform was evaporated by heating in a water bath. Finally, the sample was dried in an oven and alkaloid contents were measured.

#### *4.6.* α*-Amylase Inhibition Assay*

Screening of extracts for α-amylase inhibition was carried as previously described for the starch iodine assay [5]. In brief, the assay mixtures composed of 120 µL of 0.02 M sodium phosphate buffer (pH 6.9 containing 6 mM sodium chloride), 1.5 mL of porcine pancreatic α-amylase (PPA) solution (0.05 mg/2 mL H2O) and *P. hydropiper* extracts (sample from solvent–solvent extraction) at concentrations ranging from 0.46 to 15 mg/mL (w/v) were incubated at 37 ◦C for 10 min. Afterward, soluble starch (1%, 0.1 g/10 mL (w/v)) was added to each reaction test tube and incubated at 37 ◦C for 15 min. Then, 1 M HCl (60 µL) was added to stop the enzymatic reaction, followed by the addition of 300 µL of iodine reagent (5 mM I<sup>2</sup> and 5 mM KI). The color change was noted, and the absorbance was recorded at λ of 620 nm on a spectrophotometer (721 2C50811136 Shimadzu, Japan). The control reaction representing 100% enzyme activity did not contain any *P. hydropiper* extract. To eliminate the absorbance produced by extracts, appropriate extract controls without α-amylase were also included. A dark blue color indicates the presence of starch, a yellow color indicates the absence of starch, and a brownish color indicates partially degraded starch in the reaction mixture. In the presence of extracts, the starch will not degrade upon its addition to the enzyme assay mixture, hence giving a dark blue color complex. In contrast, the absence of color complex indicates the lack of inhibitor and means that starch is completely hydrolyzed by α-amylase. The percentage inhibition of α-amylase was calculated by the following formulas:

$$\mathbf{I}\_{\alpha-\text{amylase}}\% = \frac{\Delta \mathbf{A}\_{\text{control}} - \Delta \mathbf{A}\_{\text{sample}}}{\Delta \mathbf{A}\_{\text{control}}} \times 100\tag{3}$$

$$
\Delta \mathbf{A}\_{\text{control}} = \mathbf{A}\_{\text{test}} - \mathbf{A}\_{\text{blank}} \tag{4}
$$

$$
\Delta \mathbf{A}\_{\text{sample}} = \mathbf{A}\_{\text{test}} - \mathbf{A}\_{\text{blank}} \tag{5}
$$

#### *4.7. Antioxidant Activity Determination*

#### 4.7.1. Free Radical Scavenging Assay (FRAP)

Herein, the ability of extracts to reduce ferric ions was determined by using a previously described method [50], with modifications. The extract (750 µL) of each sample was mixed with an equal amount of phosphate buffer (0.2M, pH 6.6) and 1% potassium ferricyanide (a source of ferric ions). The mixture was incubated at 50 ◦C for 20 min. After incubation, an equal amount of trichloroacetic acid (10%) was added to stop the reaction. The sample was then centrifuged at 3000 rpm for 10 min. The upper layer (1.5 mL) was separated and mixed with an equal amount of distilled water and 0.1 mL of FeCl<sup>3</sup> solution (0.1%). A blank was also prepared by using same procedure, and the absorbance was measured at λ of 700 nm as the reducing power. In parallel, ascorbic acid (Vitamin C) was used as standard positive control. The assay was repeated in triplicate, and percentage inhibition was calculated using the following formula:

$$\% \text{Scavenging effect} = \frac{\text{Control absorbance} - \text{Sample absorbance}}{\text{Control absorbance}} \times 100\tag{6}$$

#### 4.7.2. 2,2-Dipheny-1-Picrylhydrazyl (DPPH) Scavenging Assay

The DPPH (2,2-dipheny-1-picrylhydrazyl) free radical scavenging capacity of the extract was determined by using a previously described method [51], with modifications. The solution was prepared by dissolving the 0.006 g of DPPH in 100 mL dimethylsulfoxide (DMSO). The extract (1 mL) of each sample was mixed with an equal amount of DMSO and used to prepare desired concentrations of sample (i.e., six concentrations from 0.468 to 15 mg/mL). The solutions of the required concentrations were then transferred into a test tube by taking 1 mL of sample and 2 mL of DPPH solution. The blank was prepared by mixing 1 mL of DMSO with 2 mL of DPPH. All solutions were then incubated for 30 min at 37 ◦C. The absorbance of each concentration was taken at λ of 517 nm. The percent scavenging activity was calculated as:

$$\% \text{Scavenging activity} = \frac{\text{A}\_0 - \text{A}\_1}{\text{A}\_0} \times 100\tag{7}$$

where A<sup>0</sup> represents absorbance of the control and A<sup>1</sup> is the absorbance of the sample. Each experiment was performed in triplicate.

#### *4.8. Antimicrobial Activity Determination*

#### 4.8.1. Selection of Microorganisms

Antimicrobial activity was evaluated against*Escherichia coli*,*Staphylococcus aurous*,*Klebsiella pneumoniae*, *Morganella morganii*, and *Haemophilus influenzae*. Microorganisms were obtained from the National Institute of Health (NIH) in Islamabad, Pakistan. The stock inoculums were subcultured using the streaking method. Inoculums of all microbes were prepared in sterilized LB-broth medium (Miller's LB Broth, Sigma-Aldrich, St. Louis, MO, USA). Twenty grams of LB-broth powder was added to 1 L of distilled water and autoclaved for 15 min at 121 ◦C. The autoclaved liquid medium (5 mL) was then poured into separate test tubes and settled to cool at 50 ◦C. Bacterial inoculums were transferred to the medium-filled test tube and incubated while shaking at 37 ◦C for 24 h. Later, optical density of each culture was taken at λ of 660 nm and an absorbance level of 0.5–1.0 was considered for the optimal determination of antimicrobial activity.

#### 4.8.2. Preparation of the Culture Medium

The culture medium was prepared by dissolving 20 g of LB-broth medium in 1000 mL of distilled water. A turbid solution was obtained, which was heated until it became a clear transparent solution, using continuous shaking to dissolve the agar completely. The medium was sterilized at 121 ◦C for 15 min at 15 pounds of pressure. Sterilized medium (35 mL) was poured into petri dishes under the laminar flow hood and left to solidify at room temperature.

#### 4.8.3. Antimicrobial assay

Antimicrobial activity was evaluated by agar well diffusion method. Seventy-five microliters of each microbial culture was spread individually on separate plates. A sterile cork-borer was used to bore wells (9 mm) in each inoculum-spread plate. Acetone and ethanol extracts (100 µL of each) were pipetted into individual wells. In each plate, a negative control (DMSO 100%) and a positive control (ampicillin 10 µg) were treated as standard. The plates were incubated at 37 ◦C for 24 h. For each plate, zones of inhibition were measured in millimeters.

#### *4.9. Statistics*

All the statistical analyses were performed using IBM SPSS Statistics 20.0 software (Armonk, NY, USA). The graphs were created in GraphPad Prism 5.0 software (La Jolla, CA, USA). One-way ANOVA test was performed for the comparison of groups, and an independent samples *t*-test was performed for differences between the two groups. All the data are presented as mean and standard error of

triplicate measurements. The IC<sup>50</sup> values were calculated from dose-dependent percent inhibition using GraphPad Prism. Statistically significant difference was considered for *p* < 0.05.

#### **5. Conclusions**

The present study highlights that *P. hydropiper* possesses a strong α-amylase inhibitory potential and reveals its potency to be used as a strong source of future therapeutic agents in diabetes. Our study also indicates that this plant species may be beneficial for treating the pathological damage caused by radicals' activities and bacterial infections. Future studies are required to unveil the novel bioactive compounds of *P. hydropiper*, which might be helpful in studying the precise mechanisms of α-amylase inhibition, antioxidant potential, and antimicrobial activity.

**Author Contributions:** Conceptualization, A.N. and M.K.; methodology, A.N., M.K., and Z.R.; software, A.N., M.K., and Z.R.; validation, A.N. and M.I.; formal analysis, A.N., A.A.K.K., S.F., N.B., M.I., and W.S.; investigation, Z.P., W.S., and A.A.K.K.; resources, Z.P.; writing—original draft preparation, A.N. and Z.P.; writing—review and editing, A.A.K.K., S.F., N.B., M.I., W.S., and Z.P.; visualization, A.N., A.A.K.K., S.F., N.B., M.I., W.S., and Z.P.; supervision, Z.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by Higher Education Commission Pakistan (Grant No: 20-3589) and Directorate of Science and Technology.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Phytochemicals and Biological Activity of Desert Date (***Balanites aegyptiaca* **(L.) Delile)**

**Hosakatte Niranjana Murthy <sup>1</sup> , Guggalada Govardhana Yadav <sup>1</sup> , Yaser Hassan Dewir 2,3,\* and Abdullah Ibrahim <sup>2</sup>**


**Abstract:** Many underutilized tree species are good sources of food, fodder and possible therapeutic agents. *Balanites aegyptiaca* (L.) Delile belongs to the Zygophyllaceae family and is popularly known as "desert date", reflecting its edible fruits. This tree grows naturally in Africa, the Middle East and the Indian subcontinent. Local inhabitants use fruits, leaves, roots, stem and root bark of the species for the treatment of various ailments. Several research studies demonstrate that extracts and phytochemicals isolated from desert date display antioxidant, anticancer, antidiabetic, antiinflammatory, antimicrobial, hepatoprotective and molluscicidal activities. Mesocarp of fruits, seeds, leaves, stem and root bark are rich sources of saponins. These tissues are also rich in phenolic acids, flavonoids, coumarins, alkaloids and polysterols. Some constituents show antioxidant, anticancer and antidiabetic properties. The objective of this review is to summarize studies on diverse bioactive compounds and the beneficial properties of *B. aegyptiaca*.


plants10010032

Dewir, Y.H.; Ibrahim, A. Phytochemicals and Biological Activity of Desert Date (*Balanites aegyptiaca* (L.) Delile). *Plants* **2021**, *10*, 32. https://dx.doi.org/10.3390/

Received: 28 November 2020 Accepted: 22 December 2020 Published: 25 December 2020

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional

**Citation:** Murthy, H.N.; Yadav, G.G.;

**Keywords:** bioactive compounds; polysterols; polyphenols; saponins; therapeutic properties

#### **1. Introduction**

*Balanites aegyptiaca* (L.) Delile (Family: Zygophyllaceae) is an underutilized fruityielding tree (Figure 1A) native to Africa and distributed in tropical and subtropical regions of Africa, from Senegal in the west (16 ◦W) to Somali in the East (49 ◦E) and Jordan in the north (35 ◦N) to Zimbabwe in the south (19 ◦S). *B. aegyptiaca* is also distributed in India, Myanmar, Iran, Jordan, Oman, Palestine, Saudi Arabia, Syria and Yemen [1]. Young leaves (Figure 1B) and tender shoots are used as vegetables. Leaves and fruits are used as fodder for livestock [1]. Fiber obtained from tender bark and older dried bark is used for the preparation of medicines (Figure 1C). Unripe and ripe fruits (Figure 1D,E) are edible and popularly known as "desert date". The fruits are processed into beverages and liquor. Timber is suitable for the construction of furniture, domestic items and musical instruments. The wood produces high-quality charcoal fuel and industrial activated charcoal. Gum or resin produced from stems are used as glue. Seeds contain about 49% edible oil (Figure 1F,G),which is also used in the production of biodiesel fuel [1].

*B. aegyptiaca* is used in African and Indian traditional medicine. Roots and bark are purgative and anthelmintic. A decoction of roots is used to treat malaria. The bark is used to deworm cattle, and the roots are boiled into a soup and used to treat edema and stomach pains. Roots are also used as an emetic [1]. The fruit is usedto treat jaundice in Sudan [2]. Seed oil is used as a laxative and for the treatment of hemorrhoids, stomach aches, jaundice, yellow fever, syphilis and epilepsy [3]. Bark extracts are used to kill freshwater snails and copepods. A decoction of bark is also used as an abortifacient and antidote in West African traditional medicine [4].

affiliations.

**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/).

**Figure 1.** Morphology of *Balanitesaegyptiaca* (L.) Delile: (**A**) habit, (**B**) leaves, (**C**) stem bark, (**D**) ripened fruits, (**E**) rind (left) and pulp (right), (**F**) seed kernels and (**G**) seed oil.

#### **2. Nutritional Composition of Fruits, Seeds and Leaves**

Ripe fruits display a thin brownishepicarp (Figure 1E), dark brown and fleshy mesocarp (Figure 1E) and thick endocarp nut (Figure 1F). The edible partsof the pulp and kernel yield oil. The pulp is rich in carbohydrates (62.63%) and protein (9.19%; Table 1) [5]. Fruit pulp shows lesser amounts of fat (2.58%) and dietary fiber (2.93%). The overall energy value is 346.82 kcal/100 g. Fruits are also rich in minerals, including calcium, magnesium, phosphorus, potassium and sodium (Table 1) [6]. Iron, copper, manganese, lead, chromium, cobalt, cadmium and selenium are reported in lower concentrations (Table 1). Major fatty acids in fruit pulp are oleic (37.17%), linoleic (27.73%) and palmitic (22.02%; Table 2) [7]. The fruit pulp also exhibits amino acids (Table 3) [8] and vitamins (Table 3). Antinutritional factors are comparatively less (Table 4) [5].

Seeds are rich in fixed oil content (49.00%) with a significant content of proteins (32.40%) and carbohydrates (8.70%; Table 1) [9,10]. Seed oil is used for edible purposes; major fatty acids arelinoleic (47.84%), oleic (22.80%), palmitic (16.68%) and stearic (11.67%) (Table 2) [11]. It has been demonstrated that biodiesel from seed oil meets all international biodiesel standards [11]. Seeds contain minerals, such as potassium, phosphorus and calcium in higher concentrations (Table 1) and amino acids (Table 3) [9]; seed cake isused for animal feed. However, seeds also contain oxalate (8.51 mg/g DW), antinutrient and possibly toxic constituents (Table 4).

Young leaves and shoots are used as vegetables in African countries. Leaves and shoots are also popular livestock fodder [1]. Leaves are a good source of carbohydrates (28.12%) and proteins (15.86%) and contain ash (9.26%) and dietary fiber (30.75%; Table 1) [12]. Leaves also provide minerals (Table 1), fatty acids (Table 2), amino acids (Table 3) and vitamins (Table 3) [12,13]. Leaves contain antinutrients in meager concentrations (Table 4).


**Table 1.** Nutritional and mineral composition of desert date pulp, seeds and leaves.

\* NR = not reported.




**Table 2.** *Cont.*

\* ND = not detected.

**Table 3.** Amino acid and vitamin composition of desert date pulp, seeds and leaves.



**Table 3.** *Cont.*

\* NR = not reported.

**Table 4.** Antinutritional components of desert date pulp, seeds and leaves.


\* ND = not detected.

#### **3. Phytochemicals Isolated from Desert Date**

*B. aegyptiaca* produces a variety of secondary metabolites, such as polyphenols (phenolic acids, flavonoids and coumarins), alkaloids, steroids, saponins (spirostanolsaponins, furostanolsaponins and open-chain steroidal saponins) and pregnane glycosides, isolated from plant tissues, such as fruit, seeds, leaves, stem bark, roots and galls (Table 5).

#### *3.1. Polyphenols*

Polyphenols exhibit phenolic structural features with one or more aromatic rings, each with one or more hydroxyl groups [15]. Polyphenols are grouped into phenolic acids, flavonoids, stilbenes, lignans and tannins. These compounds are important as natural therapeutic agents involved in the prevention of degenerative diseases, particularly cancers, cardiovascular diseases and neurodegenerative diseases [16].

Phenolic acids are nonflavonoid polyphenolic compounds of benzoic acid and cinnamic acid. Major phenolic acids, which are isolated from tissues of *B. aegyptiaca*, include caffeic acid (**1**), ferulic acid (**2**), gentisic acid (**3**), p-coumaric acid (**4**), sinapic acid (**5**), syringic acid (**6**), vanillicacid (**7**), 2-methoxy-4-vinylphenol (**8**), 2,6-dimethoxyphenol (**9**), 2-methoxy-3(-2 propenyl)-phenol (**10**), 2-methoxy-4-(1-propenyl)-phenol (**11**), 2,4-di-tert-butyl-phenol (**12**), 2,6-di-tert-butyl-phenol (**13**) and 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-1-propanone (**14**) (Table 5; Figure 2) [2,17–19].

**Figure 2.** Structures of phenolic compounds isolated from desert date.

#### *3.2. Flavonoids*

α Flavonoids exhibit a diphenyl propane–flavone skeleton with a three-carbon bridge between phenyl groups and commonly cyclized with oxygen. Epicatechin O-glucoside (**28**), hyperoside (**19**), isorhamnetin (**18**), isorhamnetin-3-O-glucoside (**23**), isorhamnetin 3,7 diglucoside (**25**), isorhamnetin 3-O-galactoside (**27**), isorhamnetin 3-O-robinobioside (**26**), isorhamnetin 3-rutinoside (**24**), kaempferol (**15**), myricetin (**16**), quercetin (**17**), quercetin 3 glucoside (**21**), quercetin 3-rutinoside (**22**) and quercitrin (**20**) are isolated from different tissues of *B. aegyptiaca* (Table 5; Figure 3) [19–22].

.

**Figure 3.** Structures of flavonoids and coumarins isolated from desert date.


**Table 5.** Phytochemicals isolated from various parts of desert date.




**Table 5.** *Cont.*

#### *3.3. Coumarins*

Coumarins are phenolic compounds displaying fused benzene and α-pyrone rings and are known for anti-inflammatory, anticoagulant, antimicrobial, anticancer, antioxidant and neuroprotective properties [43]. Bergapten (**29**) and marmesin (**30**) are coumarins extracted from stem bark (Table 5; Figure 3) [23].

#### *3.4. Alkaloids*

Alkaloids are compounds that contain basic nitrogen atoms [44] and show varied biological activities. They are especially useful for cancer treatment. N-cis-feruloyltyramine (**32**), N-trans-feruloyltyramine (**31**) and trigonelline (**33**) are some of the alkaloids isolated from stem bark and fruit (Table 5; Figure 4) [2,20].

#### *3.5. Phytosterols*

Phytosterols are bioactive compounds found naturally in food with chemical structures similar to cholesterol. Various clinical studies consistently show that intake of phytosterols, such as beta-sitosterol, campesterol and stigmasterol, is associated with a significant reduction in levels oflow-density lipoprotein in humans. *B. aegyptiaca* produces several steroids, such as campesterol (**40**), cholesterol (**39**), diosgenin (**34**), 6-methyldiosgenin (**36**), rotenone (**37**), β-sitosterol (**38**), stigmasterol (**41**) and yamogenin (**35**) (Table 5; Figure 4) [24–27].

**Figure 4.** Structures of alkaloids, steroids and pregnane glycosides isolated from desert date.

Pregnane glycosides are naturally occurring sugar conjugates of C<sup>21</sup> steroidal compounds, isolated from various plants and many show anticarcinogenic properties [45]. Pregn-5-ene-3β,16β,20(R)-triol 3-O-(2,6-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (**42**) andpregn-5-ene-3β,16β,20(R)-triol 3-O-β-D-glucopyranoside (**43**) were extracted from the fruits of desert date (Table 5; Figure 4) [28].

#### *3.6. Saponins*

β β β α β Saponins are bioorganic compounds that exhibit triterpenoid or steroidal skeletons that are glycosylated by varying numbers of sugar moieties attached at different positions. Steroidal saponins are further classified intospirostanol, furostanol and open-chain steroidal saponins [46]. Saponins exhibit a widerange of biological properties, including hemolytic factorsand anti-inflammatory, antimicrobial, insecticidal, anticancer and molluscicidal activities [47]. Various spirostanol, furostanol and open-chain steroidal saponins, which are isolated from fruits, seeds, roots and stem bark are presented in Table 5 and Figures 5 and 6 [24,29–42].

β β β

**Figure 5.** Structures of spirostanolsaponins isolated from desert date.

**Figure 6.** Structures of furostanoland open-chain steroidal saponins of desert date.

#### **4. Biological Activity**

Extracts and compounds from extractionsof *B. aegyptiaca* todate exhibita wide range of biological activity (Table 6).

#### *4.1. Antioxidant Properties*

Various kinds of physical and physiological stresses lead to the overproduction of oxidants in the human body, which can cause oxidative damage of DNA, proteins and lipids. Furthermore, this damage is responsible for several disorders in the human body such as cardiovascular diseases, cancer and aging. It was reported that minor fruits and nuts possess abundant antioxidant phytochemicals, and the consumption of minor fruits and nuts is beneficial to the human body [48]. The antioxidant effects ofmethanol extracts ofstembark on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) scavenging is demonstrated and accounted for in the total soluble phenolic and flavonoid contents [49]. Furthermore, Hassan et al.'s [49] results show that methanol extracts display the highest phenolic content (35.17 mg gallic acid equivalents/g) and considerable flavonoid content (112.83 mg quercetin equivalents/g). Methanol extract showed the highest free radical scavenging activity atIC<sup>50</sup> = 40 µg/mL and IC<sup>50</sup> = 125.85 µg/mL in DPPH and ABTS assays, respectively. The antioxidant properties of aqueous fruit extracts were assessed in streptozotocin-induced diabetic rats [50]. Oral administration produced a significant (*p* < 0.01) increase in mean plasma total antioxidant levels and a significant (*p* < 0.01) decrease in malondialdehyde levels. The antioxidant properties of leaf and root extracts were also demonstrated [51]. Balanitin 1 and balanitin 2 (saponins) were isolated from bark extracts and demonstrated antioxidant propertiesin vitro, using a method based on the Briggs–Rauscher oscillating reaction [39]. Polyphenols such as quercetin and kaempferol are the major components responsible for antioxidant activities [52]. In addition, phytosterols including ß-sitoterol, stigmasterol and campesterolhave been reported to exert antioxidant activity [53]. Polyphenols, phytosterols and saponins together might be responsible for the antioxidant activity of desert date.

#### *4.2. Antimicrobial Properties*

Plants synthesize several antimicrobial compounds, including phenolics such as simple phenols, phenolic acids, quinones, flavonoids, flavones, flavonols, tannins, coumarins, terpenoids, essential oils and alkaloids [54]. The mechanism of action of these compounds ranges from membrane disruption, substrate deprivation, intercalation into the cell wall/or DNA and enzyme inhibition. Desert date is rich in all these phytochemicals and demonstrates potent antimicrobial activity. The bark of *B. aegyptiaca* is widely used in African folk medicine for the treatment of wounds and skin diseases. The effects of aqueous ethanolic extracts of bark on bacteria isolated from wounds have been reported [55]. These extracts inhibited the growth of *Pseudomonas aeruginosa* and *Staphylococcus aureus* in vitro. The in vitro antifungal activity of saponin-rich extracts of fruit mesocarpwas explored against phytopathogenic fungi [56]. These extracts were moderately active (34.7%) against *Alternariasolani* and highly active (89.01%) against *Pythium ultimum*, and activity was significantly higher compared to the fungicide, metalaxyl (15 µg/mL). The antifungal activity of ethanolic and methanolic extracts of root bark and fruit have been demonstratedagainst *Aspergillus niger*, *Candida albicans*, *Penicilliumcrustosum* and *Saccharomyces cerevisiae* [57].

#### *4.3. Hepatoprotective Properties*

A methanolic extract of leaves was evaluated for hepatoprotective activity against carbon tetrachloride (CCl4)-induced hepatic damage in rats [58]. Administration of the extract (200 and 400 mg/kg per os) markedly reduced the CCl4-induced elevation of serum marker enzymes, such as glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, alkaline phosphatase and bilirubin. Similarly, fruit mesocarp and stem bark aqueous extracts amelioratedCCl4-induced hepatotoxicity in rats, as measured by liver enzyme activity, blood parameters and histopathology [59]. Ethanolic extracts of bark protected hepatocytes against paracetamol-and CCl4-induced hepatotoxicity in rats, analogous to silymarin [60]. Bioactive compounds, primarily obtained from dietary sources, contain a wide range of free radical scavenging constituents, including polyphenols, alkaloids and phytosterols, which are responsible for hepatoprotective effects [61]. Desert date is rich in

polyphenols, phytosterols and saponins. It has depicted very good antioxidant potential and thus increased the cellular antioxidant defense system, which may be responsible for the hepatoprotective effects of desert date.

#### *4.4. Anticancer Properties*

Cancer is a major health problem. Radiotherapy, chemotherapy and surgical removal are the current treatment methods. However, these methods have varied disadvantages such as drug resistance and toxic effects on nontargeted tissues. Therefore, researchers are searching for naturally available plant-based bioactive compounds for cancer therapy [62]. Among the plant-based bioactive compounds, saponins and phytosterols have significant importance in reducing the risk of cancer [63,64]. Varioussteroidal saponins isolated from various tissues of *B. aegyptiaca* are reported to display anticancer activities. For example, a mixture of balanitin-6 and balanitin-7 (28:72) isolated from kernels showgrowth inhibition in human cancer cell lines in vitro [31]. Balanitin-6/balanitin-7 exhibited higher antiproliferative activity than well-known natural cancer therapeutic agents, such as etoposide and oxaliplatin. Balanitin-6/balanitin-7 displayed its highest activity against A549 nonsmall cell lung cancer (IC50, 0.3 µM) and U373 glioblastoma (IC50, 0.5 µM) cell lines. Balanitoside extracted from the fruit also showed anticancer activity against Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice [36]. Mice injected intraperitoneally with balanitoside (10 mg/kg body weight) displayed decreases in liver and serum enzyme levels. Issa et al. [65] studied an aqueous extract of pulp on the development and growth of EAC and metastasis to the liver and spleen. Treatment with the extract (400 mg/kg) inhibited tumor growth and proliferation in ascetic fluid, inducing a significant decrease in tumor volume, total cell volume and viable cell count and prolongedmouse survival. The authors also recorded significant decreases in levels of lipid peroxidation and increased superoxide dismutase and catalase activity and P53 (a tumor suppressor protein) expression. The saponin, balanitin-7 isolated from seed kernels, showed antiproliferative activity [32]. These agents showed potent antiproliferative activity against MCF-7 human breast cancer cells and HT-29 human colon cancer cells, with IC<sup>50</sup> values of 2.4 and 3.3 µM, respectively.

#### *4.5. Anti-Inflammatory Properties*

Inflammation is a pattern of response to injury, which involves the accumulation of cells, exudates in irritated tissue, which allows protection from further damage. A variety of in vitro and in vivo experiments has shown that certain flavonoids and saponins possess anti-inflammatory activity [66]. The mechanism by which flavonoids and saponins exert their anti-inflammatory effects involves the inhibition of cyclooxygenase and lipoxygenase activities [67]. Desert date exhibited potent anti-inflammatory activity; for example, Speroni et al. [39] studied the in vivoanti-inflammatory activity of methanol and butanol extracts and two saponins, viz. balanin-B1 and balanin-B2, isolated from *B. aegyptiaca* bark in rats with edema induced bycarrageenin. Both extracts exhibited a significant reduction of rat paw edema. The inhibition produced by methanolextract, butanol extract, balanin-B1 and balanin-B2 were 32%, 68%, 62% and 59%, respectively. Likewise, the influence of seed oil on liver and kidney fractions in rat serum was evaluated [68]. Seed oil (100 mg/kg) in the rat dietdecreased nitrogen oxide and lipid peroxidation. Further, mRNA and protein expression of tumor necrosis factor-α and interleukin-6 were downregulated, leading to a reduction of cyclooxygenase-2, reflecting anti-inflammatory activity.

#### *4.6. Antidiabetic Activity*

Diabetes is a chronic disease that occurs either when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces. Several medicinal plants have demonstrated hypoglycemic and hyperglycemic activities; these activities seem to be mediated through increased insulin secretion via stimulation of pancreatic cells, interfering with dietary glucose absorption or through insulin-sensitizing action [69]. Kamel et al. [38] demonstrated the antidiabetic effect of an aqueous extract of

fruit in streptozotocin (STZ)-induced diabetic mice after oral administration. They also identified steroidal saponins, 26-O-ß-D-glucopyranosyl-(25R)-furost-5-ene-3ß,22,26-triol-3-O-[α-L-rhamnophyranosyl-(1→1)]-[ß-D-xylopyranosyl-(1→3)]-[α-L-rhamnopyronosyl-(1→4)] ß-D-glucopyranoside and its 22-methyl ether in the extract and recognized two additional saponins, 26-O-ß-D-glycopyranosyl-(25R)-furost-5-ene-3ß,22,26,-triol-3-O-[2,4-di-O-α-Lrhamnopyranosyl)-ß-D-glucopyranoside and its methyl ether. A combination of saponinsexhibited greater antidiabetic activity than individual saponins. Gad et al. [70] administered fruit extracts (1.5 g/kg body weight) to STZ-induced diabetic rats and studied the glycogencontent of liver and kidney and on some key enzymes of liver involved in carbohydrate metabolism. STZ (50 mg/kg body weight) caused a five-fold increase in blood glucose level, an 80% reduction in serum insulin level, a 58% decrease in liver glycogen and a seven-fold increase in kidney glycogen content. A marked increment in the activity of glucose-6-phosphatase activity and decreasedactivity of glucose-6-phosphate dehydrogenase and phosphofructokinase were recorded. Treatment of rats with fruit extract reduced blood glucose levels by 24% and significantly decreased liver glucose-6-phosphatase activity. The authors also demonstrated that the extract inhibited α-amylase activity in vitro. The major component in the extract was diosgenin, based on high-performance thin-layer chromatography. Additionally, Al-Malki et al. [71] showed that ethyl acetate extract containing β-sitosterol modulatedoxidative stress induced by streptozotocin.


**Table 6.** Biological activities of compounds isolated from various parts of desert date.


**Table 6.** *Cont.*

Hassanin et al. [74] tested a crudeethanolic fruit extract and itsbutanolic and dichloromethane fractions on stress-activated protein kinase/c-Jun N-terminal kinase (SAPK-JNK) signaling in experimental diabetic rats. Six groups of male Wistar rats were used: normal control, diabetic, diabetic rats treated with crude, butanol or dichloromethane factions (50 mg/kg body weight), and diabetic rats were treated with gliclazide as a reference drug. Treatments continued for one month. Extract treatments produced a reduction in plasma glucose, hemoglobin A1c, lactic acid, lipid profile and malondialdehyde levels, which induced an increase in insulin and reduced glutathione (GSH) levels and catalase and superoxide dismutase activities. Moreover, the authors observed the downregulation of apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase 1 and protein 53 and the upregulation of insulin receptor substrate 1 in rat pancreas. Glucose transporter 4 was upregulated in rat muscle. Liquid chromatography and high-resolution mass spectrometry (LC-HRMS) analysis identified balanitin-2, hexadecenoic acid, methyl protodioscin and 26-(O-β-D-glucopyranosyl)-3-β-[4-O-(β-D-glucopyranosyl)-2-O-(α-Lrhamnopyranosyl)-β-D-glucopyranosyloxy]-22,26-dihydroxyfurost-5-ene in crude extract and balanitin-1 and trigonelloside C in butanoland dichloromethane fractions of crude

extract. Ezzat et al. [40] isolated several compounds from pericarp, including stigmasterol-3-O-β-D-glucopyranoside (**a**), a pregnane glucoside: pregn-5-ene-3β,16β,20(R)-trio1-3-O-β-D-glucopyranoside (**b**); a furostanolsaponin: 26-(O-β-D-glucopyranosyl)-22-O-methylfurost-5-ene-3β,26-diol-3-O-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-Dglucopyranoside (**c**). The latter component possessed significant α-glucodidase (AG) and aldose reductase inhibitory activities in streptozotocin-induced diabetic Wistar rats. Compound (**c**) also caused a significant increment in insulin and C-peptide levels.

#### *4.7. Molluscicidal Activity*

Regarding the effects of fruit extracts on juvenile and adult *Bulinusglobosus* and *B.truncatus,* two Planorbid (ramshorn) freshwater snails have been reported [75]. LC<sup>95</sup> values were 16.9 and 19.7 µg/mLand 14.2 and 12.0µg/mLforjuvenile and adults of *B. globosus* and *B. truncatus*, respectively. Seed, endocarp, mesocarp and whole fruit extracts were assessed against adult *Biomphalariapfeifferi,* another Planorbid snail, and *Lymnaeanatalensis,* a Lymnaeid pond snail [76]. LC<sup>90</sup> values were 77.70, 120.04, 89.50 and 97.55 mg/L against *Biomphalariapfeifferi* for seed, endocarp, mesocarp and whole fruit extracts, respectively, and 102.30, 138.21, 115.42 and 127.69 mg/L against *Lymnaeanatalensis.* Furthermore, the molluscicidal activity of seed oil on *Monachacartusiana*, aHygromiid land snail, has been demonstrated [77]. Bioactive compounds were identified assaponins, such as diosgenin, yamogenin and 3,5-spirostadiene.

#### *4.8. Other Activities*

Several studies demonstrate the antinematode andantiplasmodial activities of *B. aegyptiaca* extracts. Shalaby et al. [78] showed the effects of methanolicfruit extracts on enteral and parenteral stages of *Trichinellaspiralis* (pork worm). The authors also evaluated the effectiveness of methanolic extract against preadult migrating larvae and encysted larvae of *Trichinellaspiralis* in rats and compared them with the commonly used anthelmintic chemical, albendazole. Methanolic extract (1000 mg/kg body weight) for five successive days throughout the parasite lifecycle led to a marked reduction in migrating and encysted larvae by 81.7% and 61.7%, respectively. In another study, the efficacy of a methanolicextract on *Toxocaravitulorum* (roundworm), a major parasite in cattle and buffalo, was assessed [79]. They incubated parasites in a ringer solution containing 10, 30, 60, 120 and 240 µg/mL of ethanolic extract for 24 h. The most prominent activity at 240 µg/mLcausedthe disorganization of body cuticle musculature. Kusch et al. [17] evaluated a crude extract of seeds for antiplasmodial activity. An IC<sup>50</sup> value for chloroquine-susceptible *Plasmodium falciparum* NF54 was 68.26 µg/µL. The compound responsible for this activity was6-phenyl-2(H)-1,2,4-triazin-5-one oxime. The authorsalso showed that two phenolic compounds, 2,6-di-*tert*-butyl-phenol and 2,4-di-*tert*-butylphenol, displayedantiplasmodial activity at IC<sup>50</sup> values of 50.29 and 47.82 µM, respectively.

#### **5. Conclusions**

*B. aegyptiaca* or desert date is an underutilized tree species. The nutritional status of the fruits, leaves and seeds indicate that this species could be exploited as a food source. Seed oil might also be a good source of biodiesel. Leaves and young shoots are nutritionally rich and could be exploited as cattle feed. Furthermore, fruits, leaves, roots and the bark of stem and roots are substantial sources of bioactive phytochemicals that display a host of possibly useful biological properties. *B. aegyptiaca* might prove to be a valuable source of bioactive agents for use in human and veterinary medicine.

**Author Contributions:** Conceptualization and methodology, H.N.M. and G.G.Y.; validation, H.N.M. and G.G.Y.; investigation, H.N.M., G.G.Y., Y.H.D. and A.I.; formal analysis H.N.M. and G.G.Y.; resources, H.N.M. and Y.H.D.; data curation, H.N.M., G.G.Y. and Y.H.D.; writing—original draft preparation, H.N.M. and G.G.Y.; writing—review and editing, H.N.M., Y.H.D. and A.I.; visualization, H.N.M., G.G.Y., Y.H.D. and A.I. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors extend their appreciation to the Deputyship for Research and Innovation, "Ministry of Education" Saudi Arabia for funding this research work through the project number IFKSURP-59. H.N.M. is thankful to the University Grants Commission-Basic Scientific Research (UGC-BSR) mid-career award (Grant No. F.19-223/2018/ (BSR)).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable

**Data Availability Statement:** Please refer to suggested Data Availability Statements in section "MDPI Research Data Policies" at https://www.mdpi.com/ethics.

**Acknowledgments:** The authors thankful to the Researchers Support and Services Unit (RSSU) for their technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Review Euphorbia antisyphilitica* **Zucc: A Source of Phytochemicals with Potential Applications in Industry**

**Romeo Rojas <sup>1</sup> , Julio César Tafolla-Arellano <sup>2</sup> and Guillermo C. G. Martínez-Ávila 1,\***


**\*** Correspondence: guillermo.martinezavl@uanl.edu.mx; Tel.: +52-81-8329-4000 (ext. 3511)

**Abstract:** *Euphorbia antisyphilitica* Zucc, better known as the candelilla plant, is one of the 10 nontimber forest products of greatest economic importance in the desert and semi-desert regions of Mexico. Moreover, it is a potential source of some functional phytochemicals such as polyphenolic compounds, wax and fiber, with potential applications in food, cosmetic and pharmaceutical industries. Thus, this review aims to describe these phytochemicals and their functional properties as antimicrobial, antioxidant, reinforcing and barrier agents. In addition, a suitable valorization of the candelilla plant and its byproducts is mandatory in order to avoid negative effects on the environment. This review provides, for the first time, an overview of the alternative methodologies for improving candelilla plant production, pointing out some of the agricultural aspects of the cultivation of this plant.

**Keywords:** candelilla plant; antioxidant properties; antimicrobial activity; polyphenols; candelilla cultivation; candelilla wax

**Citation:** Rojas, R.; Tafolla-Arellano, J.C.; Martínez-Ávila, G.C.G. *Euphorbia antisyphilitica* Zucc: A Source of Phytochemicals with Potential Applications in Industry. *Plants* **2021**, *10*, 8. https://doi.org/10.3390/ plants10010008

Received: 25 November 2020 Accepted: 21 December 2020 Published: 23 December 2020

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

The genus *Euphorbia* belongs to the *Euphorbiaceae* family, and it is one of the largest genera of higher plants, with more than 2000 recognized species, such as *Euphorbia thymifolia*, *Euphorbia neriifolia*, *Euphorbia antiquorim*, *Euphorbia chamaesyce*, *Euphorbia helioscopia* and *Euphorbia antisyphilitica* [1,2]. One of the most important species of this family is *E. antisyphilitica* Zucc, better known as the candelilla plant, which is used traditionally as an herbal remedy in countries such as India and other arid and semiarid countries [1]. It naturally grows in the desert and semi-desert regions of northern Mexico, and this nontimber plant is a very important economic resource for the people living in these areas, due to the extreme climatic conditions which restrict agricultural activities [3,4]. The candelilla plant grows in clusters, with thin wax-covered stems that protect them as thick layers giving tolerance against environmental conditions (i.e., temperature variations) and biotic agents (i.e., insects) [5,6]. Therefore, this plant is mainly used to obtain wax, which can be considered as a multipurpose agent for several industries due to its unique properties and multiple applications in the formulation of food, cosmetic and pharmaceutical products. In addition, the candelilla plant has proven to be a good source of other useful phytochemicals such as fiber, wax and polyphenolic compounds (i.e., catechin and ellagic acid), as shown in Figure 1. In this sense, this review provides interesting information generated from around the world about some constituents and bioactive compounds from the candelilla plant and their functional properties.

**Figure 1.** Phytochemicals and functional properties of E. antisyphilitica Zucc.

#### **2. Phytochemicals Present in Candelilla Plant and Byproducts**

Usually, a proximal chemical analysis is carried out to determine the chemical constituents of a plant material. In this sense, the chemical analysis of candelilla plants has been reported by Rojas-Molina et al. [5] and Ventura-Sobrevilla et al. [7], as shown in Table 1. Although the chemical composition of plants is strongly influenced by factors such as weather conditions, and season and genetic variability, it can be seen from the reported proximal analysis that lipids and ashes are the major constituents of candelilla plants, which are related to the presence of candelilla wax as mentioned above. Moreover, it has been reported that the candelilla plant possesses a large number of high-quality bioactive phytomolecules with potential applications in different industries acting as antimicrobial and antioxidant agents, and providing other technological advantages [8–13]. Figure 2 shows the structure of the main phytochemicals identified in the candelilla plant and its byproducts.

Table 2 shows the phytochemicals obtained from the candelilla plant and its byproducts, and the functional activity of these compounds. Since candelilla wax is one of the most important constituents of E. antisyphilitica, it is necessary to know its chemical composition, which has been reported as follows: n-alkanes (hentriacontane as the main component) > high molecular weight esters > alcohols and sterols > free acids (7–9%) [4,5,14–17]. Another important structural component of the candelilla plant is fiber, which can be used as a support in the production of hydrolytic enzymes and as a reinforcing agent, as explained below [11,18]. It has been established that candelilla bagasse fiber (CBF) comprises cellulose (45%), hemicellulose (16%), lignin (37%), pectin (1.8%), wax (0.5%) and water-soluble

extract (8–12%), a composition that is similar to other natural fibers (i.e., sisal and jute fiber) with industrial applications [11]. In addition, the presence of some polyphenolic compounds has also been reported in the candelilla plant. Rojas-Molina et al. [5] and Ventura-Sobrevilla et al. [7] detected the presence of hydrolysable and condensed tannins, as well as the presence of catechin, ellagic and gallic acid. The first study about the extraction of ellagic acid from E. antisyphilitica was reported by Aguilera-Carbo et al. [19]; the authors reported that the candelilla plant has at least double the quantity of this phenolic compound than other plant materials such as Turnera diffusa and Jatropha dioica. Recently, the putative structure of a high molecular weight (860.7 g mol−<sup>1</sup> ) ellagitannin called candelitannin has been reported in candelilla residues [9]. Finally, flavonoids and other molecules (samonins and quinons) can be found in the methanolic extracts of the candelilla plant [8]; however, no further information has been provided about these components. Regardless of these reports, there is currently limited information on the chemical characterization of this plant, which provides an opportunity for innovative studies in this field due to its economic importance.

**Table 1.** Proximal chemical analysis carried out in candelilla plants.


**Figure 2.** Main phytochemicals identified in E. antisyphilitica and byproducts: (**a**) ellagic acid; (**b**) gallic acid (candelilla whole plant); (**c**) candelitannin (candelilla byproducts); (**d**) hentriacontane (wax); (**e**) cellulose (candelilla bagasse fiber).

#### **3. Agricultural Aspects of Cultivation**

According to a very recent study published by Vargas-Pineda et al. [20], candelilla plants have a distribution area of more than 19.1 million hectares in North America under the current climatic conditions. Nevertheless, although the collection of candelilla plants is an important economic activity for communities from northern Mexico, any overharvesting should be avoided. Villa-Castorena et al. [21] conducted a comprehensive study related to

the production of candelilla seedlings by cuttings. In this study, four eco-types of candelilla plants called Cuencamé, Cuatrociénegas, Tlahualilo and Viesca, four growing substrates (sandy soil, mixture of river sand and coconut fiber (1:1), mixture of river sand and peat moss (1:1) and mixture of peat moss, perlite and vermiculite (1:1:1)), and four chemical treatments (ProRoot, magic root, phenoxyiacetic acid and a treatment without chemical application) for root promotion were evaluated. According to the authors, untreated Cuatrociénegas eco-type had the highest ability to promote superior rooting of the cuttings due to its special genetic characteristics which allow this eco-type to produce more endogenous auxins for emitting more roots without the presence of other chemicals. For the other eco-types, the cuttings treated with growth media of peat moss with perlite and vermiculite and the mixture of river sand and peat most improved the percentage of rooted cuttings and the shoot growth of this plant. In this sense, the production of candelilla plants can be explored in controlled environments such as greenhouses in order to provide seedlings of good quality for the reforestation of affected areas. In addition, it has been reported that candelilla plants exhibited greater relative growth rate (0.15 g·g −1 ·d −1 ) and relative water content (88%), which was associated with the physiological status of this plant, allowing its consideration for suitable growth and production as green roofs in arid regions [22].

On the other hand, some stress conditions in the cultivation of candelilla plants, such as the use of lime on the cultivation soil, can improve the production of secondary metabolites (i.e., epicular wax) due to an increase in the pH of the plant tissues due to the alkaline conditions in the soil [23]. According to the authors, candelilla plants cultivated on a liming soil (10 g per pot) reach, on average, more than 54% extractable wax when compared to the control and other abiotic stress conditions (plastic cover and solar reflection). However, no additional studies related to the effect of the stress conditions on additional secondary metabolite expression of this plant were found, which enables the development of new research in this regard.

#### **4. Functional Properties of the Phytochemicals**

Due to their functional characteristics, phytochemicals of candelilla plant have the potential to be used in several processes in food, cosmetic and biotechnological industries, as they have demonstrated antimicrobial and antioxidant properties (phenolics and wax), good barrier properties (wax) and reinforcing and biotechnological properties (fiber).

#### *4.1. Antimicrobial Properties*

In the last decade, interest in the study of the phytochemical constituents of E. antisyphilitica has grown (Table 2). In addition, it has been reported that plants from northern Mexico have several secondary metabolites to which some biological activities are attributed [8,12]. Thus, the studied molecules have proven to be highly effective at acting as antifungal and antimicrobial bacterial agents [8,9,12]. In their study, Serrano-Gallardo et al. [12] evaluated non-toxic methanolic extracts of four different plants used as a traditional remedy from a semi-desert region in Mexico, including E. antisyphilitica. In their study, the authors evaluated different concentrations of the plant extracts (500, 100 and 2000 µg mL−<sup>1</sup> ) against two reference bacterial strains, Staphylococcus aureus BAA44 and Klebsiella pneumoniae 9180, and four bacteria isolated from clinical samples (S. aureus, K. pneumoniae, Pseudomonas aeruginosa and E. coli). From the obtained results, the authors found that the candelilla extracts presented antimicrobial activity against all the tested bacteria at 500 µg mL−<sup>1</sup> (as a minimum inhibitory concentration), which can be related to the phytochemical profile detected in this plant (saponins and quinones). This is according to the previous results reported by Vega-Menchaca et al. [8], who determined that methanolic extracts from candelilla leaves showed antimicrobial activity against clinically isolated bacteria strains such as S. aureus, E. coli O157 and Enterobacter aerogenes 9183. In comparison, in the study of Vega-Menchaca et al. [8], the minimum inhibitory concentration needed for the inhibition of S. aureus was lower (26.8 µg mL−<sup>1</sup> ) than that reported by Serrano-Gallardo et al. [12], which could be due to the presence of

flavonoids in candelilla extracts evaluated by Vega-Menchaca et al. [8], as explained by the authors. However, it is important to consider other factors which can affect phytochemical composition of candelilla plants such as weather conditions and season.

Candelilla byproducts also contain other bioactive phenolic compounds that have been related to the antimicrobial activities of candelilla extracts against Erwinia amylovora, Xanthomonas axonopodis and Clavibacter michiganensis [10]. According to the authors, polyphenolic compounds from the hydro-alcoholic extracts obtained from the candelilla byproducts are responsible for the inhibitory effect against the pathogenic bacteria. This antimicrobial potential could be related to the presence of ellagitannins such as candelitannin in these plant materials which has shown effective antifungal properties against four phytopathogenic fungal strains: Alternaria alternata, Fusarium oxysporum, Colletotrichum gloeosporioides and Rhizoctonia solani [9]. The antimicrobial activities exhibited by the phenolic-rich extracts from candelilla plants may be attributed to the interaction of these compounds with the cell membrane causing several modifications to it and changes in various intracellular functions, such as interspecific permeability causing microbial death [24]. Thus, polyphenolic compounds from the candelilla plant can be an important alternative to traditional antimicrobial agents, and a complement to antibiotic therapy. On the other hand, it has been proven and hypothesized that, by itself, candelilla wax has antimicrobial effects on E. coli ATCC 10536 and some fungal strains such as Botrytis cinerea, Colletotrichum gloeosporioides and Fusarim oxysporum, when it is used in the formulation of edible coatings and films [25,26]. However, due to scarce information in the literature, the specific role of candelilla wax in this effect is still unknown.

#### *4.2. Antioxidant Properties*

Free-radical scavenging of extracts from candelilla byproducts was evaluated by Burboa et al. [10]. The authors reported that ethanolic extracts can inhibit more than 88% of the DPPH• radicals according to the methodology used for this analysis, which was related to the presence of phenolic compounds in these extracts. This is in line with the findings reported for other species of the Euphorbiaceae family, in which polyphenolic compounds have been detected, such as tannins and flavonoids, which are well known for their antioxidant capacity [1]. Nevertheless, regardless of the high content of phenolic compounds, the evaluation and exploitation of antioxidant properties of candelilla plant extracts are still scarcely explored. In this sense, more research should be conducted with the purpose of generating new and innovative knowledge about the antioxidant properties of the polyphenolic compounds and other phytochemicals from this plant material, as they have great potential to be applied in food, pharmaceutical and cosmetic industries.

#### *4.3. Barrier Properties of Candelilla Wax*

Barrier properties of candelilla wax against water vapor and gas transfer are two of the most important features for researchers, and they are usually measured by the formulation of edible coatings and films. The moisture barrier property is the most recognized characteristic of candelilla wax, which is based on its hydrophobic nature and capacity to form a compact network in synergy with other structural compounds such as proteins and carbohydrates [6,14,27,28]. According to Kowalczyk et al. [27], candelilla wax-based films exhibit low water and oxygen permeability compared to other lipid sources, which decreases when the concentration increases (from 0.5 to 2.0%). This can be attributed to its ability to increase film surface hydrophobicity. In the same way, some authors have recorded a decrease in the weight loss of food products such as avocado, "Golden Delicious" apples, Fuji apples and Persian limes. This could be due to the morphology of the coating surface which has a homogenous particle size and less roughness, reducing the open area of the emulsified solids network and therefore avoiding loss of moisture from the fruit [6,28–30]. However, more studies must be conducted regarding the low oxygen permeability of candelilla wax-based coating and films, as it can affect some quality and sensory attributes of coated food products due to the anaerobic respiration and changes in pH [31,32].


**Table 2.** Phytochemicals from candelilla plant, methods for characterization, registered yields and potential functional activity.

SEM = scanning electron microscope; FT-IR = Fourier transform infrared spectroscopy; HPLC = high-performance liquid chromatography; GC = gas chromatography.

#### *4.4. Other Functional Applications*

Due to the candelilla wax extraction process generating large amounts of lignocellulosic waste, they can be used for different technological purposes. Regarding the biotechnological aspects, candelilla fiber has been used as a support for fungal growth to the production of ellagitannase due to its ellagitannin content [18]. In this study, the authors evaluated four agroindustrial byproducts, and, although it was concluded that candelilla stalks have great potential for use as support for solid-state fermentation (SSF), the lowest values of ellagitannase were obtained using this plant material. Thus, future studies must focus on the standardization of this biotechnological process in order to increase enzymeactivity titles. On the other hand, due to their physico-chemical characteristics, candelilla fiber has also been used as a reinforcing agent in the formulation of new composites based on polypropylene (PP) and CBF [11]. In this study, the authors found that this byproduct is stable at around 200 ºC, which improves the thermal stability and tensile properties of the CBF–PP composites due to the crystallinity index of the cellulose and the disruption of the free moment of the polymeric chains in the matrix, respectively. In a more recent study, it was demonstrated that candelilla fiber contains intracuticular wax and resins, which has the benefit of being a compatibilizer between the fiber and the polypropylene [35]. This is according to a recent study by Pulido-Barragán et al. [34], who reported that CBF is a good plant material for obtaining cellulose nanocrystals, which can be used as a reinforcing, structural or thermal agent, as well as for 3D printing and as a constructive nanocellulosic paper agent, among other technical applications due its the specific physico-chemical properties. Although these studies were well conducted, limited information on the application of candelilla fiber is available, providing an opportunity for the investigation of other functional properties of this plant material.

#### **5. Final Remarks and Perspectives**

Since the candelilla plant is an endemic of the semiarid regions of northern Mexico, this country has been recognized for its potential to be the main producer of candelilla wax. However, this review provides interesting and innovative information associated with the promising applications and sustainable valorization of other phytochemicals from the candelilla plant not published elsewhere. In addition, it provides an opportunity for developing more investigations into the physicochemical characterization of polyphenolic compounds (different to ellagic acid), and the fiber present in the candelilla plant and its byproducts. Furthermore, considering its great potential to be used as a source of components with several functional applications, researchers should focus more on the evaluation, stability and application of the phytochemicals of this plant, as they could help to replace synthetic molecules used at the industrial level. In addition, the waste disposal for animal feed (after the SSF process) and the evaluation of CBF as a reinforcing agent (after the extraction of wax and phenolic compounds) could be of great interest for creating a "zero waste" process as an integral use of this plant resource.

**Author Contributions:** Conceptualization, writing—original draft preparation (G.C.G.M.-Á); investigation, writing—review and editing, project administration, and funding acquisition (R.R. and G.C.G.M.-Á); investigation, writing—original draft preparation, writing—review and editing (J.C.-T.-A.). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Forest Commission and the Mexican Council for Science and Technology (CONAFOR-CONACYT), through the Sectoral Fund for Forestry Research, Development and Technological Innovation with the project Diseño y construcción de equipo semiautomático para la extracción de cera de candelilla orgánica (number B-S-131466).

**Acknowledgments:** Authors thank the staff of the National Forest Commission for the facilities given to develop this project.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

