Pattern Recognition of Phytoconstituents and Bioactivities of Date Pit Extracts from Different Cultivars Grown in the Qassim Area

Abstract

A huge number of date varieties grow annually throughout the world. The genetic variation between different date varieties is reflected in their variable sensory characters and phytochemical contents. Date pits are considered a waste product of the date industry, despite their rich metabolic content. The present study attempts to generate visual clustering to clarify the diversity among fourteen date cultivars growing in the Qassim region, according to the phytochemical contents and biological potentials of their pits. The results indicated a wide variation in the total phenolic content (11.4–29.7 mg GAE/g), flavonoids content (21.9–37.1 mg RE/g), proanthocyanidine content (12.0–207.0 mg CE/g), and antioxidant potential (10.3–25.5 mg AEAC/g) among the tested cultivars. Screening the antimicrobial activity of extracts from the 14 tested cultivars indicated different activities against Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Salmonella enterica, and Candida albicans. Multivariate analysis of phytochemical content and biological activity, using different analytical models, allowed the classification of the 14 cultivars into four classes, Class-1: Barhi, Safawi, and Sukkari; Class-2: Khodry and Nabtat Ali; Class-3: Ruthana, Segae, Shaqra, and Sheishee; and Class-4: Hulwa Aljouf, Mabroom, Meneifi, Rushodia, and Wannana. These findings provide a scientific basis for the classification of date pits which facilitates the future selection of promising candidates for more phytochemical and biological exploration.

1. Introduction

As a matter of fact, Mother Nature supplies us with a remarkable inventory of plants as prospective therapeutic candidates. For centuries, date palm (Phoenix dactylifera L., Arecaceae) has been the iconic plant of desert regions in the Middle East and North Africa of which Arab countries constitute about 74.5% of the world’s output with around 800 different varieties of date [1]. The Kingdom of Saudi Arabia (KSA) is in the front of the leading date producers in the world along with Egypt and Iran [2,3]. The Qassim province, located in the center of Saudi Arabia, hosts beyond eight million palm trees, rendering it as one of the Middle East’s largest producers of dates, with an estimated 200 thousand tons of various kinds of valuable dates produced annually [4]. Date palm trees are cultivated mainly for their unique sweet fruits owing to their outstanding nutritional and economical value [5], in addition to a myriad of constituents that account for date palms’ diverse biological activities [6]. A number of studies have reported a wide range of biological effects of date fruits including antioxidant [7], cytotoxic [8], antimicrobial, anti-inflammatory [9], antidiabetic [10], cardio-protective, gastro-protective, and hepatoprotective activities [11].

Date pits, which constitute about 6–15% of the weight of ripe date fruit [12], are produced annually in huge amounts and used as animal food and for processing caffeine-free beverages besides a considerable portion that is discarded [3]. Interestingly, recent studies investigated the nutritional value and beneficial ingredients of date pits. Date pits have shown promising applications in purifying water [13], and it has been reported that they contain oleic acid, dietary fibers, and polyphenolic compounds that have been associated with a reduced incidence of cardiovascular diseases and improved overall well-being [3,14]. Nutrients and bioactive compounds in date pits are quite variable depending on the variety which may be attributed to genetic diversity, environmental factors, or variations in soil and harvest conditions [15]. The characterization and classification of date varieties are important from both nutritional and medicinal points of view.

The aim of the present study is twofold: (1) to screen phenolic, flavonoid, and proanthocyanidin contents as well as the antioxidant and antimicrobial potential of date pits. (2) to use the tested parameters for the classification of different varieties on a scientific basis to facilitate the future selection of date pits that seem similar to promising candidates for more phytochemical and biological exploration.

Accordingly, fourteen varieties of date pits from date fruits cultivated in the Qassim area in the KSA were investigated for their total phenolics, total flavonoids, and proanthocyanidins contents along with their antioxidant and antimicrobial potentials. Additionally, multivariate statistical analysis was employed to reveal the possible clustering pattern between the tested cultivars. Principal component analysis (PCA), principal coordinates analysis (PCO), and dendrogram plots were applied in a synergistic way to find out the possible clusters of the studied cultivars.

2. Materials and Methods

2.1. Chemicals

Ascorbic acid, catechin, gallic acid, rutin, Folin–Ciocalteu reagent, and DPPH (2,2 diphenyl-1-picrylhydrazyl) were bought from Sigma-Aldrich (St. Louis, MO, USA). All solvents were of high analytical grade.

2.2. Plant Material

Date fruits of fourteen cultivars under investigation were collected from Hadhim farm in the city of Buraidah, Qassim region, Saudi Arabia from 29 January to 10 February 2020. All the varieties were verified by the Ministry of Agriculture of Saudi Arabia.

2.3. Extraction

Date seeds were removed from fruits and properly washed with water to completely remove all impurities, and then they were dried for 2–3 days at room temperature (25 °C). Dry seeds were then completely ground to form fine powder using a grinder (PHR-04A HERB GRINDER). Powdered seeds were individually, exhaustively extracted by maceration in 80% methanol for 5 days and a stepwise extraction from residual seed material was performed until a clear supernatant liquid was obtained. The extracts were separately filtered and then evaporated under reduced pressure.

2.4. Total Phenolic Content (TPC)

The total phenolic content (TPC) of the date seed extracts was determined using Folin–Ciocalteu reagent [16]. Gallic acid was utilized as a standard at a concentration of 1 mg/mL in 80% methanol and serial dilutions of the standard were prepared (0.025–0.4 mg/mL). The standard’s concentration was graded in serial dilutions. A solution of each crude extract in methanol (1 mg/mL) prepared using Folin–Ciocalteu reagent (10% aqueous solution, 2.5 mL) was added to each of the extracts, and the standard dilutions (0.5 mL) in separate test tubes. Afterwards, anhydrous Na₂CO₃ (7.5% w/v, 2 mL) was added. After vortexing, the mixture was incubated at room temperature (25 °C) for 30 min. After incubation, absorbance was measured at 750 nm. The total phenolic content was evaluated as mg of gallic acid equivalent (GAE) per gram crude extract deduced according to the standard linear graph y = 4.7551x + 0.1645, R² = 0.9575 using the equation CV/M. C is the concentration extrapolated from the standard linear graph, V is the volume of extract in mL, and M is the mass of the extract used expressed in grams (g).

2.5. Total Flavonoids Content (TFC)

The total flavonoid content (TFC) was measured by the aluminum chloride spectrophotometric assay [16]. The assay depends on the quantification of the yellow-orange color of the complex between the flavonoids and the AlCl₃ that arises from their reaction. A stock solution (1 mg/mL) of the plant extracts was prepared in 80% methanol. Serial dilutions (0.025–0.4 mg/mL) of rutin were prepared, and then 0.5 mL of the extract and each standard concentration was separately pipetted into test tubes. Then, 2 mL of distilled water was added to each test tube, and after that, 0.15 mL of 5% NaNO₂ was. Each mixture was vortexed and left to stand for 6 min. Then, 10% AlCl₃ (0.15 mL) was added, and 5 min later, 1 M NaOH (1 mL) was added. Distilled water was added to make the solution up to 5 mL followed by incubation for 30 min. Afterwards, the absorbance was recorded at 430 nm. The total flavonoid content as mg/g of quercetin equivalent (RE/g) was calculated from the standard calibration curve y = 3.9439x + 0.1857, R² = 0.9716 following the abovementioned equation, CV/M.

2.6. Proanthocyanidins Content (PAC)

The total proanthocyanidins content (condensed tannins) was evaluated as described in [16]. To 0.5 mL of each of the stock solutions (extract and serial dilutions of catechin as a standard), 3 mL of vanillin (4% w/v) was added, followed by HCl (1.5 mL). The mixture was then vortexed followed by 15 min of incubation at room temperature (25 °C), and then absorbance was read at 500 nm. The estimation of the total proanthocyanidin content as mg of catechin equivalent (CE)/g of the crude extract was performed using the calibration curve y = 0.3723x + 0.0277, R2 = 0.9984 from the above formula, CV/M.

2.7. Evaluation of Antioxidant Activity

The antioxidant activities of date seed extracts were estimated using DPPH free radical scavenging [16]. Estimations were made against the standard antioxidant compound, ascorbic acid. A solution of 2, 2-diphenyl-1-picrylhydrazyl (500 μM) in absolute ethanol was kept in a dark bottle. In total, 300 μL of extract or standard (in serial dilutions) was added into separate test tubes, and then 3 mL of DPPH solution was added. The mixture was vortexed and incubated at room temperature (25 °C) for 30 min. Thereafter, absorbance was recorded at 517 nm. Extrapolation of the scavenging activity of the tested samples in the inhibitory percentage of DPPH was performed using the equation:

Scavenging effect (%) = [1 − (absorbance of sample/absorbance of control)] × 100

The standard curve was designed by plotting the percentage of the free radical scavenging activity of ascorbic acid against its concentration. The final assay result was stated as mg of ascorbic acid-equivalent antioxidant capacity in 1 g of extract (mg AEAC/g). The final results were obtained from the calibration curve y = 298.74x + 11.988, R² = 0.9879.

2.8. Antimicrobial Activity Screening

The antimicrobial activity of all date pits extracts was evaluated using the agar well diffusion method as previously described [17] with some modifications [18,19]. The fourteen extracts were examined for antibacterial activity against Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 43300), Escherichia coli (ATCC 25922), and Salmonella enterica (ATCC 14028), and for their antifungal activity against Candida albicans (ATCC 60193). Briefly, different indicator strains were suspended in sterile saline until turbidity of 0.5 McFarland was achieved. Then, the strains were streaked on the surface of Mueller–Hinton agar plates (Himedia, Mumbai, India) by a cotton swab, followed by the formation of 10 mm cups in each pre-inoculated plate using a sterile borer. Next, 100 µL from each extract at a concentration of 10 mg/mL in 60% DMSO in water (v/v) was applied to the wells of separate indicator strains. The plates were then kept for 1 h at 4 °C before being incubated at an appropriate temperature for microbial growth. After 24 h of incubation, the plates were evaluated for the antimicrobial activity of the date seeds’ extracts by measuring the inhibitory zones surrounding each well. The standard antibacterial agent was rifampicin solution (SANDOZ, Egypt). Additionally, a solution of 60% DMSO in water (v/v) was used as a negative control.

2.9. Chemometric Analysis

2.9.1. Principal Component Analysis (PCA)

Principal component analysis is a traditional and very common way of cluster analysis that works through the decomposition of the main data matrix intro scores and loadings matrices, where the useful information is concentrated in early principal components. Scores are related to the samples (different cultivars in our case), and loadings are related to the variables.

X = T·P

X stands for the original raw data matrix that consists of 14 rows (cultivars) and 9 columns (variables), T is the scores, and has an equal number of rows as the original data matrix; the dimensions of T will be 14 × A. P denotes the loadings and has the same number of columns as the original data matrix; the dimensions of P will be A × 9. A is the total number of principal components (PCs), and in our study, we will use only two PCs that span most of the useful information and discard as much noise in the data. By drawing the scores of the first two PCs, one can have an idea about the clusters available in the data, whereas by plotting the loadings of the first two PCs, the effect of variables on the clustering pattern can be deduced to some extent [20].

2.9.2. Principal Coordinates Analysis (PCO)

Principal coordinates analysis (PCO) is a general approach performed for the visualization of the dissimilarity matrices (i.e., show how (dis)similar each sample is from one another).

PCO has a major advantage over PCA: it can deal with any (dis)similarity mathematical measure and hence is more flexible [N.B. PCA uses the Euclidean distances between samples only].

PCO’s disadvantage is that it is designed only to investigate similarities between samples, and it is difficult to manifest the influence or significance of the variables [unlike the PCA loadings plot]. The input to PCO is an I × I dissimilarity matrix D whose elements contain dissimilarity indices. Euclidean distance is the most commonly used with PCO and gives affirmative results to PCA with similar clusters and similar to dendrograms, hence it was used in our study [21].

2.9.3. Dendrogram Plot

Hierarchical clustering is presented in the form of a dendrogram, where the samples are arranged in a row, based on their similarities. It works by using nearest-neighbor linkage and correlation coefficients for similarities [20].

For dendrograms, the principal input is a similarity measure, whereas it is a dissimilarity measure for principal coordinates analysis. [20].

2.10. Software

All applied chemometric methods were performed using Matlab^® 7.1.0.246 (R14) (Mathworks, Natick, MA, USA). The codes for the PCA and PCO analysis were kindly provided by Gavin R. Lloyd. Built in Matlab, the function for dendrogram plots was used. All calculations were performed using an Intel(R) Core (TM) i7-10750H CPU, 2.60 GHz, and 8.00 GB of RAM in Microsoft Windows 10.

3. Results and Discussion

3.1. Phytochemical Content

Date pits seem to be a potential source of phytoconstituents, although they are usually considered as waste by-products of the date industry. The current results confirmed the richness of pits from some of the studied date cultivars with valuable metabolites. This greatly recommends these cultivars as potential inexpensive sources of antioxidant compounds. Herein, the total phenolics, flavonoids, and proanthocyanidins contents were determined for the fourteen studied cultivars.

3.1.1. Total Phenolic Content (TPC)

Previous investigations of date pits reported the presence of variable phenolic compounds including pyrogallol, benzoic acid, cinnamic acid, ellagic acid, catechol, syringic acid, and chlorogenic acid [22]. In this study, the TPCs of the fourteen cultivars were determined using the method of Jimoh et al. [16]. The results shown in (Figure 1) demonstrated notable differences in the phenolic contents between the studied date pit cultivars, where Khodry, Shaqra, Ruthana, Segae, and Sheishee contained the highest amounts of total phenolics (30.2, 29.7, 29.6, 29.6 and 29.5 mg GAE/g, respectively), whereas Barhi, Safawi, and Sukkari showed the lowest TPC levels (11.4, 15.4 and 20.0 mg GAE/g, respectively). Other cultivars, Mabroom, Meneifi, Nabtat Ali, Wannana, Hulwa Aljouf, and Rushodia, exhibited moderate TPCs (27.9, 27.6, 27.3, 26.3, 26.2, and 25.3 mg GAE/g, respectively). Several previous studies investigated the TPC of other date pit cultivars from Algeria, Oman, Pakistan, and Tunisia [23,24,25,26,27] and reported variable phenolic contents in each cultivar. Additionally, a similar study investigated ten cultivars from Saudi Arabia where the TPC content of the Barhi, Sukkari, and Sheishee cultivars was reported to have 42.6, 77.7, and 66.8 mg GAE/g dry weight, respectively [28]. These results exceeded our data, a situation that could be attributed to different factors including growing circumstances, soil, fertilizers, storage conditions, or different analysis techniques [29]. Furthermore, low phenolic contents in some plants might be attributable to the actions of certain enzymes such as phenolase and polyphenol oxidase; the activation of these enzymes is dependent on temperature, pH, and other factors [30].

3.1.2. Total Flavonoids Content (TFC)

Various flavonoids have been detected in date pits including quercetin, luteolin, naringenin, kaempferol, and isorhamnetin [28]. Abu-Reidah et al. revealed that date pits are rich in flavonols that occur mainly as O-glycosides [31]. Herein, the flavonoid content in each of the studied cultivars was determined using the aluminum chloride method. The results (Figure 1) highlighted the highest TFC in Shaqra, Segae, and Sheishee (37.1, 35.6, and 35.2 mg RE/g, respectively). Additionally, considerable flavonoids contents were recorded in Sukkari, Nabtat Ali, Wannana, Ruthana, Khodary, Hulwa Aljouf, Mabroom, and Rushodia (29.2, 29.0, 27.2, 26.8, 25.2, 24.4, 24.1, and 23.5 mg RE/g, respectively), whereas the lowest contents were detected in Meneifi, Barhi, and Safawi (21.4, 21.9, and 22.4 RE/g, respectively). The present findings recorded the TFC of cultivars: Barhi, Sukkari, and Sheishee as 21.9, 29.2, and 35.2 RE/g, respectively. These results were less than those reported by El-Mergawi et al. (2016) [28] for the same cultivars (30.8, 60.8, and 56.4 mg QE/g dry weight, respectively). This may be due to the different analytical methods and standards used or due to other factors as discussed in the previous section. The presence of appreciable amounts of flavonoids in different cultivars of date pits and the association of flavonoids with potential bioactivities suggests further investigation to identify the flavonoid contents and bioactivity of different date pit cultivars.

3.1.3. Proanthocyanidins Content (PAC)

PACs of the fourteen date pits extracts were estimated as mg of catechin equivalent per gram of the crude extract, and the results in Figure 1 clearly demonstrated a remarkable variation among the tested cultivars. The highest levels were observed in the Segae, Sheishee, and Shaqra cultivars (207, 197.5, 193.3 mg CE/g), that was followed by the Ruthana, Khodary, Nabtat Ali, Mabroom, Meneifi, Wannana, Hulwa Aljouf, Rushodia, Sukkari, and Safawi cultivars (165.5, 134.5, 134.0, 93.4, 90.6, 89.8, 73.3, 61.8, 48.0, and 34.3 mg CE/g, respectively), while the lowest PAC was obviously exhibited by the Barhi cultivar (12 mg CE/g). These results greatly exceeded the results of eight Algerian cultivars reported by Djaoudene et al. (2019) [24] that ranged from 49.17 to 85.13 mg CE/g. Several previous reports have investigated the proanthocyanidine content in date varieties. Messaoudi et al. detected three proanthocyanidins in some variety of dates [23], whereas Habib et al. revealed by HPLC analysis the presence of proanthocyanidin dimers, trimers, and tetramers in date pits of the Khalas variety [32]. Other studies have stated the protective activity of proanthocyanidin-rich date pit extract against hepatorenal toxicity [33], as well as its vasoactive potential [34].

Based on the current results, the three cultivars, Segae, Sheishee, and Shaqra, were strongly recommended for further in-depth phytochemical and biological exploration.

3.2. Evaluation of Antioxidant Activity

The stable free radical DPPH can be scavenged by transferring an electron or hydrogen from other compounds [35]. In the present study, an investigation of the antioxidant capacity of the different date pits extracts was performed by measuring their ability to scavenge the DPPH radical. The results (Figure 1) noted the Sheishee and Segae cultivars as having the most characteristic scavenging effect (26.6 and 26.5 mg AEAC/g, respectively). Furthermore, other cultivars displayed characteristic antioxidant efficacy such as the three cultivars, Khodary, Mabroom, and Nabtat Ali (25.5 mgAEAC/g), as well as the two cultivars, Menefi and Shaqraa (25.4 AEAC/g). Rushodia, Ruthana, Hulwa Aljouf, and Wannana also exhibited good activity estimated as 25.2, 25.2, 25.0, and 22.6 mg AEAC/g, respectively. On the other hand, the three cultivars, Barhi, Safawi, and Sukkari, exhibited the lowest antioxidant activity expressed as 10.3, 16.5, and 19.9 mg AEAC/g, respectively. Several previous studies investigated the antioxidant activity of other cultivars and reported variable antioxidant potentials [23,24,25,26,27]. The current results clearly indicated the relationship between the observed antioxidant activity and the content of phytoconstituents in each cultivar. The antioxidant activity of the two cultivars, Sheishee and Segae, was outstanding; that clearly reflected the impact of their high contents of PAC, TPC, and TFC. The results of the three cultivars, Barhi, Safawi, and Sukkari, added more confirmation that the contents of TPC, PAC, and TFC were the lowest among the tested cultivars, and also the same was true for their antioxidant activities. The relationship between the phytochemical content and the antioxidant potential of an extract was previously stated [27]. These data support the claim that some date pit cultivars that are considered waste products could serve as sources of antioxidant compounds, which may help in the treatment of different diseases.

3.3. Antimicrobial Activity Screening

Several earlier investigations on the antimicrobial activity of date seeds demonstrated that date pits could be considered as a promising natural source of bioactive antimicrobial compounds [36,37,38,39,40]. Notably, previous research has shown that the crude extract of date seeds has a wide range of antimicrobial bioactivity against a variety of indicator microorganisms, including S. aureus, E. coli, and C. albicans [36,37,38]. Moreover, a recent study found that date palm seeds from India displayed broad-spectrum antibacterial activity against a wide range of pathogens including S. aureus, E. coli, and Salmonella enteritidis [39]. Furthermore, in another study, date palm seeds were evaluated for their antibacterial activity against the Gram-negative bacteria, E. coli and Klebsiella pneumonia, and were reported to exhibit positive bioactivity against both strains [37]. Herein, Table 1 demonstrated that the three date pit extracts, Barhi, Safawi, and Wannana, did not exhibit activity against any of the tested microbes. As previously mentioned, the two cultivars, Barhi and Safawi, exhibited the lowest TPC and PAC among all of the tested cultivars, whereas the TPC, PAC, and TFC in the Wannana cultivar were intermediate. On the other hand, all other extracts showed positive inhibition against, at least, one indicator strain. Both Hulwa Aljouf and Meneifi showed only antibacterial activity against S. aureus, whereas Sukkari showed activity against L. monocytogenes. The five extracts; Mabroom, Rushodia, Segae, Shaqra, and Sheishee revealed selective antibacterial activity against the examined Gram-positive bacteria, L. monocytogenes and S. aureus. Interestingly, the three cultivars, Segae, Shaqra, and Sheishee, exhibited the highest content of all measured constituent TPC, PAC, and TFC. The Khodry cultivar displayed antibacterial activity against the tested Gram-positive bacteria, as well as one Gram-negative bacterium: E. coli. Furthermore, the extracts of the Nabtat Ali and Ruthana cultivars exhibited the broadest antimicrobial activity against the tested Gram-positive bacteria and one of the Gram-negative bacteria, in addition to the fungus, C. albicans. The observed antimicrobial activity could be linked to the rich phytochemical content of these cultivars. Accordingly, the present study suggests an extensive chromatographic investigation of the most interesting species in order to address the phytoconstituent(s) responsible for the corresponding antimicrobial activity.

3.4. Variation between the Fourteen Studied Date Pits Cultivars

The pits from fourteen date cultivars, growing in the Qassim region, Saudi Arabia, were used in the study. All cultivars displayed different values for phenolics, proanthocyanidins, and flavonoid contents. Moreover, they displayed different free radicle scavenging potentials and antimicrobial activities against five microorganisms (L. monocytogenes, S. aureus, E. coli, Salmonella enterica, and C. albicans). The nine traits, DPPH scavenging potential, TPC, PAC, TFC, and antimicrobial activity against the five microorganisms were used to classify the fourteen cultivars. Different cluster analysis tools were applied in order to confirm the results of the diversity and similarity among the tested cultivars.

3.4.1. Principle Component Analysis (PCA)

Principal component analysis (PCA) is a statistical technique usually adopted to reveal the similarities between varieties as well as the influence of each variable for the class. All measured phytoconstituents and biological activities were subjected to PCA in order to identify diversity and similarities among the studied date cultivars. The data were mean-centered prior to PCA. The first two principal components accounted for 99.73% of the total variance within the data set. PC1 explained 97.22%, and PC2 explained 2.51% of the data variation. The PCA scores plot showed that the 14 studied cultivars were separated into four different groups. Figure 2a (scores plot of the first two PCs) demonstrated the distribution of the 14 cultivars into four classes, whereas in Figure 2b (loadings plot) the relationship between the samples (cultivars) and the variables was indicated. The PCA loadings plot reflected the lower impact of the five antimicrobial variables compared to the first four variables (DPPH scavenging activity, TPC, PAC, and TFC). Hence, another plot known as the biplot was introduced to overlay the more significant first four variables and the 14 cultivars in colors. Figure 2c is the PCA biplot, which indicated the clustering of the 14 cultivars into four groups influenced extremely by PAC, whereas the other three variables, DPPH, TPC, and TFC, stay in the middle of the plot. It can be seen that PAC has a large influence on class 3 (green). The distribution of the 14 cultivars among the four classes is as follows: Class-1 included Barhi, Safawi, and Sukkari; Class-2: Khodry and Nabtat Ali; Class-3: Ruthana, Segae, Shaqra, and Sheishee; and Class-4: Hulwa Aljouf, Mabroom, Meneifi, Rushodia, and Wannana.

It is noteworthy that this is the first report on the multivariate analysis of the pits (seeds) of the aforementioned 14 date cultivars. A previous study was conducted on the fruit flesh of 12 Saudi Arabian cultivars. It used PCA to classify the 12 fruit cultivars based on their metabolic profiling and bioactivity analysis results [25]. One more study was also conducted on the fruit flesh of 20 cultivars collected from the United Emirates. It discussed the suitability of 29 physical–chemical traits for PCA and concluded that only three traits were decisive in fruit classification [25].

3.4.2. Principal Co-Ordinates Analysis (PCO)

Although PCO is based on a different mathematical principle compared to PCA, it provides similar clustering when the Euclidean distance measure is used in the calculation of similarity indices. Figure 3 shows the clustering of the 14 cultivars similarly to the PCA scores plot, which affirms the clustering pattern.

3.4.3. Dendrogram Plot

Furthermore, a dendrogram plot (Figure 4) was generated, which inclines a different mathematical approach, where it is based on the calculation of the correlation coefficient among samples (cultivars) and then tries to present their similarity in a hierarchical order. Again, the same clustering pattern of the PCA scores plot could be affirmed, where the 14 cultivars can be grouped into four groups. In the illustrated dendrogram, the first arm included the four cultivars: Ruthana, Segae, Shaqra, and Sheishee (Cluster-3). The three cultivars, Segae, Shaqra, and Sheishee, exhibited the same antimicrobial activity, whereas Ruthana had the broadest activity against S. enterica and C. albicans. In the second arm the two cultivars, Khodari and Nabtat Ali (Cluster-2), displayed similar antimicrobial activity, with the Nabtat Ali cultivar showing extra activity against C. albicans. Considering the phytochemical content (TPC and PAC), the cultivars included under the two clusters, 2 and 3, exhibited the highest content among all of the tested cultivars. This is also clear in the DPPH scavenging activity results of these cultivars. These findings could provide a basis for the selection of these cultivars for more phytochemical and biological investigation. The third arm included the Barhi, Safawi, Sukkari, (Cluster-1), and Wannana cultivars, where the three cultivars, Barhi, Safawi, and Wannana, were inactive and Sukkari displayed activity only against the sensitive strain L. monocytogenes. The TPC, PAC, and DPPH scavenging activity of the four cultivars recorded the lowest values among all tested cultivars. Accordingly, the observed antimicrobial activity could be due to the limited phenolic content in these cultivars. Finally, the fourth arm pointed out the remaining four cultivars: Hulwa Aljouf, Mabroom, Meneifi, and Rushodia (Cluster-4). In this cluster, all cultivars displayed selective activity against Gram-positive bacteria. Hulwa Aljouf and Meneifi were active against S. aureus only, whereas Mabroom and Rushodia were active against S. aureus and L. monocytogenes. All four cultivars exhibited appreciable antioxidant activity and content of phenolic compounds as indicated by TPC, TFC, and PAC.

4. Conclusions

Although date pits are considered a waste product in the date industry, they could be a valuable source for medicinal or nutritional agents. The provided research demonstrated variable metabolite contents that were reflected in the different biological potentials of the 14 tested cultivars. Chemometric analysis of the variable traits of the studied cultivars concluded the successful classification of these cultivars. All applied models, PCA, PCO, and dendrogram plots, drive synergistic conclusions about the clustering patterns of the 14 cultivars. These findings highlighted the possible medicinal value of date pits as well as the importance of modern analytical techniques as a tool to help in the selection of the most promising cultivars for subsequent research.

Author Contributions

E.A. initiated the project, hypothesis, and design and wrote the main manuscript. E.I.A.M., M.A.Z. and N.A. performed the phytochemical assays, analyzed their results, prepared related figures and tables, and took part in writing the manuscript. A.S.A. and M.A.A. collected and extracted the date pit samples. M.S. performed the antimicrobial activity assay. I.A.N. performed the chemometric study and analyzed its results with the preparation of related figures. S.I.B. and K.B. took part in analyzing data and writing the manuscript text. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Acknowledgments

The authors would like to extend their sincere appreciation to the Researchers Supporting Project number (RSP2023R497) at King Saud University, Riyadh, Saudi Arabia.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures and Table

Figure 1. Total phenolics, total flavonoids, and proanthocyanidins contents and antioxidant activity of fourteen varieties of date pits extracts [Values are represented in mean ± S.E.M (n = 3); total phenolics content (TPC) was expressed as mg gallic acid equivalents in 1 g of dried extract (mg GAE/g); total proanthocyanidins content (PAC) was estimated as mg of catechin equivalent (mg CE)/g of the crude extract; total flavonoids content (TFC) was estimated as mg rutin equivalents in 1 g of dried extract (mg RE/g). DPPH free radical scavenging activity was expressed as mg ascorbic acid equivalent antioxidant capacity (AEAC) in 1 g of dry extract].

Figure 2. Principal component analysis showing: (a) score plot of the fourteen date pit cultivars; (b) loading plot of the 9 variables; (c) biplot of overlaid scores and loadings.

Figure 3. Principal coordinates analysis (PCO) plot of the fourteen cultivars.

Figure 4. Dendrogram plot showing similarities and correlations among different fourteen studied cultivars.

Table 1

Antimicrobial activity of fourteen date pit extracts against different indicator strains.

Cultivar Number	Cultivar Name	L. monocytogenes	S. aureus	E. coli	S. enterica	C. albicans
1	Barhi	-	-	-	-	-
2	Hulwa Aljouf	-	+	-	-	-
3	Khodry	+	+	+	-	-
4	Mabroom	+	+	-	-	-
5	Meneifi	-	+	-	-	-
6	Nabtat Ali	+	+	+	-	+
7	Rushodia	+	+	-	-	-
8	Ruthana	+	+	-	+	+
9	Safawi	-	-	-	-	-
10	Segae	+	+	-	-	-
11	Shaqra	+	+	-	-	-
12	Sheishee	+	+	-	-	-
13	Sukkari	+	-	-	-	-
14	Wannana	-	-	-	-	-

Positive antimicrobial activity was only recorded for inhibition zones with a diameter equal to or more than 12 mm.

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