INTRODUCTION:

Prosopis cineraria, commonly known as the inattentive plant, is a small to medium-sized, thorny, and irregularly branched tree that holds significant potential in traditional medicine. It has been identified as a potent bioresource for natural antioxidants¹ and anti-ulcer drugs. Consequently, there is a need for further research to elucidate the specific phytochemicals present in the plant and their underlying mechanisms of action.

Prosopis cineraria is an emerging medicinal plant known to contain various phytochemicals, including protein, fat, carbohydrates, fiber, alkaloids, fatty acids, glycosides, and polyphenols^2,3, which exhibit strong antioxidant properties.

Ghafeth plant extracts, derived from the Prosopis cineraria, are commonly used as flavorings, antioxidants, and in the production of food⁴ and cosmetics. Due to its natural antioxidant properties, it is preferred over synthetic alternatives like butyl hydroxyanizole (BHA) or butylid hydroxytoluene (BHT). In addition to its antioxidant activity, the Ghaf plant has been utilized in alternative medicine and complementary industries for its digestive properties, as a food preservative, stimulant, astringent, and diuretic⁵. Moreover, it has been associated with a wide range of beneficial health effects. The overuse and misuse of antibiotics have resulted in the emergence of antibiotic resistance among various pathogens. Consequently, scientists are actively exploring valuable plant resources to address the issue of antimicrobial resistance. Prosopis cineraria is extensively used in the treatment of conditions such as boils, skin disorders, as a blood purifier, and for dysentery treatment⁶. The plant shows promising potential for pharmaceutical and medicinal applications.

Prosopis cineraria is a rich source of phenolic compounds that can be widely applied in the food, beverage, feed, and pharmaceutical industries⁷. The objective of this study was to determine the polyphenol content and antioxidant activity of Prosopis cineraria through the analysis of liquid chromatography, mass spectrometry (LC-ESI-/MS), and high-performance liquid chromatography (HPLC-PDA). The identification of natural sources of bioactive substances and the development of extraction methodologies are of great interest in the fields of food and cosmetics^{8, 9}. Therefore, parameters such as total phenol content (TPC), total flavonoid content (TFC), antioxidant properties, and fatty acid content were assessed.

MATERIALS AND METHODS:

Materials:

All chemicals used in the extraction and analysis processes were of high-performance liquid chromatography (HPLC) and general reagent (GR) grade from Merck. Methanol, acetone, hexane, petroleum ether, dichloromethane, and distilled water were sourced from Merck. Specific compounds, such as gallic acid (>95%), p-hydroxybenzoic acid (>97%), vanillic acid (>97%), syringic acid (>97%), p-coumaric acid (>97%), ferulic acid (>97%), and rutin (>97%), were purchased from Sigma-Aldrich. Acetonitrile, ethanol, and acetic acid (85%) were of HPLC grade from Panreac.

Equipment:

The used equipment included: Agilent gas chromatography-mass spectrometry (GC-MS) device equipped with Chemstation analytical software and Willy-Nist research library, Agilent liquid chromatography-mass spectrometry (LC-MS)detector or photodiode array (PDA) detector equipped with Chemstation analytical software, Buchi rotary evaporator with vacuum capabilities, Jasco-2500 spectrophotometer, and Power-Sonic 405 ultrasonic wave model.

Extraction of phenols content in the Prosopis cineraria L. bark:

The bark of the Prosopis cineraria L. tree was thoroughly cleaned and dried in the shade until a stable weight was achieved^10-12. Subsequently, the bark was ground into a powder form. A total of 25g of the tree bark powder was accurately weighed and placed in a conical flask. Then, 70mL of a mixture containing 70% ethanol and 30% water was added to the flask, followed by thorough stirring. The pH of the mixture was adjusted to 3 using hydrochloric acid, and the flask was immersed in an ultrasonic bath operating at a frequency of 40 kHz and a power of 132 watts. The extraction process was conducted for two hours at a temperature of 37°C. After extraction, the resulting extract was filtered, and 2500 d/d was installed for 15 minutes. Subsequently, the extract was evaporated using a stream of nitrogen gas, and the volume was adjusted to 5ml. The resulting extract was stored in an opaque bottle in the refrigerator for further analysis using HPLC technology.

Characterization of phenolic compounds in the extract of the Prosopis cineraria L. bark using HPLC-DAD:

The characterization and quantification of phenolic compounds in the extract of the Prosopis cineraria L. bark were carried out using the Agilent 1200 HPLC device under the following analytical conditions: analytical column (RP-C-18, 250×4.6mm, 5μm), injection volume (20μL), temperature (30°C). PDA (Photodiode Array) detector was employed for phenolic compound analysis, utilizing wavelengths of 280nm and 325nm, which are known to be effective for the determination of most phenols¹³. The mobile phase was composed of mobile phase A (water/acetic acid, 2:98, v/v), and mobile phase B (acetonitrile/acetic acid/water, 50:0.5:49.5, v/v/v). Gradient elution was used (Figure 1).

Figure 1. Gradient elution program used in the characterization and quantification of phenolic compounds.

Characterization of phenolic compounds in the extract of the Prosopis cineraria L. bark using LC ESI/MS technology:

Approximately 25g of dried bark powder from the Prosopis cineraria tree was accurately weighed and transferred to a 50ml flask. 15mL of methanol was added to the flask, and the flask was placed in an ultrasonic bath for 30 minutes to facilitate extraction. The resulting extract was then transferred to tubes and centrifuged at 5000rpm, allowing the floating material to be collected in a separate tube^13-17. The same procedure was repeated for the remaining residue, and the filtrates were combined. This process ensured the complete extraction of the sample. The sample solution was subsequently filtered using a 0.22μ nylon filter. It was then concentrated to a volume of 3mL using a rotary evaporator before being subjected to analysis using the LC-MS method with electrospray ionization. Analytical Conditions of LC-MS: analytical Column (Synergi Hydro-RP C18, 250×4.6mm, 4μm) with a C18 ODS pre-column (4.0 × 2.0mm), flow rate (0.8mL/min), voltage settings (3.5kV and 500V). Additionally, the following conditions were maintained: nitrogen gas temperature at 300°C, nitrogen gas flow at 11L/min at 250°C, and nitrogen gas mist at 45 psi.

Extraction of active substances from leg peel powder of Prosopis cineraria L.:

A total of 150g of leg peel powder from Prosopis cineraria was subjected to comprehensive extraction using Soxhlet extraction with hexane, followed by ethyl acetate and methanol. The extraction process was carried out for 4hours, and the resulting extracts were collected and evaporated using a rotary evaporator until dryness. The dried extracts were dissolved in 1 mL of hexane solvent^18-20 and subsequently analyzed using GC-MS technology.

Analysis of active compounds in the extract of leg peel of Prosopis cineraria L. using GC-MS technology:

Before analysis, appropriate analytical conditions were determined, including the optimal thermal program for efficient compound separation, carrier gas flow rate, suitable analytical column, constant flow conditions, spectral scanning range, and fragmentation energy compatible with the comparison library. The active ingredients were then separated using gas chromatography (GC), and the separated and unknown compounds were identified using MS technology.

The analysis was performed using the GC Agilent Technologies 6890 N network GC system coupled with organic mass spectroscopy. The following analysis conditions were employed: column (HP-5ms, 30m× 0.25mm ID, 0.25μm), carrier Gas (Helium with a constant flow), oven Temperature (initial temperature: 55°C for 2 minutes, temperature ramp: 55-90°C at a rate of 3°C/min, hold at 90°C for 3 minutes, temperature ramp: 90-120°C at a rate of 5°C/min, hold at 120°C for 5minutes, temperature ramp: 120-170°C at a rate of 3°C/min, hold at 170°C for 3 minutes, temperature ramp: 170-265°C at a rate of 5°C/min, hold at 265°C for 25minutes), detector (MSD (Mass Selective Detector, with a transfer line temperature of 230°C and full scan in the range of 35-500 amu), injection Volume (1μL). The components of the plant extract were identified by comparing the retention times obtained from gas chromatography with the mass spectra of the compounds to the data available in the library (Willy & Nist)^21-23.

Extraction of active substances from Prosopis cineraria L. seed powder and their determination by GC-MS technology:

Prosopis cineraria seed powder (150g) is comprehensively extracted by Soxhlet hexane. For 4 hours, the extract was collected, evaporated with a rotary evaporator until dry, dissolved with 1ml of hexane systems, and analyzed with GC-MS technology to determine the content of the extract of fatty acids.

Analytical procedure for Prosopis cineraria L.seed oil analysis:

A volume of 100μL of seed oil was accurately weighed and transferred to a volumetric flask. To this, 9.80mL of hexane was added, and the mixture was stirred using a vortex mixer for 5 minutes. Subsequently, 100μL of N₂ KOH solution, dissolved in methanol, was added. The flask was covered and stirred for one minute. The mixture was then placed in a methylator and centrifuged at 4000rpm for 10minutes^23-39. The floating layer was carefully removed for injection into the GC-MS system. For analysis, the EI (Electron Ionization) system was employed with an ionization power of 70 volts. A volume of 1μL of the prepared sample was injected into the GC-MS system using the fractionation mode with a split ratio of 50:1. The mass range for detection was set from m/z 50 to 650amu. The library search for compound identification was performed using the NIST and Wiley 2008 GC/MS libraries. The analysis was conducted under the following analytical conditions: column: DB-Wax (30m × 0.25mm ID, 0.25μm), carrier Gas: Helium with constant flow, oven Temperature (initial temperature: 85°C for 2 minutes, temperature ramp: 85-150°C at a rate of 2°C/min, hold at 150°C for 3 minutes, temperature ramp: 150-250°C at a rate of 5°C/min, hold at 250°C for 5 minutes), Detector ((Mass Selective Detector MSD, with a transfer line temperature of 230°C and full scan in the range of 35-500 amu), injection Volume: 1 μL, split: 1:50. The components of the seed oil extract were identified by comparing the retention times obtained from gas chromatography with the mass spectra of the compounds to the data available in the library (Wiley & NIST).

Determination of total phenolic content (TPC) in leg peel extract and seed powder using Folin-Ciocalteu reagent:

The determination of Total Phenolic Content (TPC) relies on the formation of a blue complex between the Folin-Ciocalteu reagent (consisting of phosphomolybdate and phosphotungstate) and phenolic compounds in an alkaline medium. The intensity of the resulting color is directly proportional to the concentration of phenolic compounds present in the solution. This enables a colorimetric titration, where the absorption is measured at a wavelength of 755 nm using a spectrophotometer. The concentration of polyphenols can be determined by comparing the results to a calibration curve based on gallic acid standards⁷.

Sodium carbonate solution (Na₂CO₃) 20% was prepared by weighting 20 grams of sodium carbonate and then dissolving in 100mL of distilled water. The solution is heated in a water bath to ensure complete dissolution of the carbonate. The gallic acid standard solution is prepared by dissolving 0.4g of gallic acid in 100mL of distilled water with gentle heating on a water bath.

A series of concentrations (20, 40, 60, 80, and 100 mg/100 mL) was prepared from the mother solution of gallic acid standard (400mg/100mL). For each concentration, 0.1mL of the solution is taken and mixed with 5mL of distilled water, 0.5 mL of Folin-Ciocalteu reagent, and 2 mL of 20% sodium carbonate solution. The final volume is adjusted to 10mL with distilled water, and the mixture is thoroughly mixed to ensure homogeneity. The reaction is allowed to proceed for 40 minutes to develop a stable color. The absorbance was determined at 755nm. The determination coefficient was 0.9987 indicating a very good linearity (Figure 2). The quantification of total phenolic content involved a quantitative analysis of phenol compounds. This was achieved by determining the linearity equation derived from the standard curve of gallic acid, utilizing the absorbance values obtained and expressed as gallic acid equivalents (GAE). The results were reported as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g dw) for the sample.

Figure 2. Plot of absorbance values versus concentration of galic acid (mg/100mL).

Determination of total flavonoid content (TFC):

The total flavonoid content is determined using the following method: 250μL of leg peel powder extract and seed powder extract are separately mixed with 75μL of 5% sodium nitrite (NaNO₂) and shaken using a vortex mixer for 5minutes. Then, 150μL of 10% aluminum chloride (AlCl₃) and 500μL of NaOH solution (M1) are added. The volume is then extended to a final volume of 3mL with distilled water.

The absorbance is measured at 430nm, and the amount of flavonoids is determined using a standard curve of quercetin within the range of 0.2-0.12mg/mL. The results are expressed as milligrams of quercetin equivalents per gram of sample (mg QE/g).

In this measurement, the sum of standard flavonoids in the form of quercetin is utilized. The total determination of flavonoids is expressed using the linear equation obtained from the standard quercetin titration curve, represented as follows: y = 0.0019x – 0.0035, with an R2 value of 0.9997. Here, "y" represents the sample's absorption, while "x" represents the amount of quercetin in micrograms per milliliter (μg/mL).

Determination of antioxidant activity of polyphenols in Ghafeth bark and seed powder:

The antioxidant activity of polyphenols in Ghafeth bark powder extract and seed powder is determined using the DPPH (1,1-diphenyl-2-picrylhydrazyl) test, which measures the ability to inhibit free radicals. 0.0189g of DPPH is weighed and dissolved in 100mL of methanol.

0.5 mL of Ghafeth bark powder extract and seed powder extract are taken separately and mixed with 5mL of DPPH solution (0.48mmol/L). The mixture is then left at room temperature for 55 minutes. Afterward, the solution is filtered, and the absorbance is recorded at a wavelength of 517nm. The ability to inhibit free radicals is calculated using the following relationship:

Scavenging Activity = 1 - (A sample / ADPPH) * 100

Where A sample is the absorbance of the sample and ADPPH is the absorbance of the DPPH solution without the sample.

RESULTS AND DISCUSSION:

The phenolic compounds in the extract of Prosopis cineraria L. bark were characterized and quantified using HPLC-DAD and LC- ESI/MS (Table 1, 2).

Table 1. Quantification of Phenols in the extract of Prosopis cineraria L. bark using HPLC. The experimental condition was previously mentioned in the methods.

Phenols	Retention time (T_R)	Concentration μg/mL
Galic acid	8.3	15.78
P-hydroxybenzoyic acid	11.9	9.21
Vanillic acid	14.7	8.74
Syringic acid	19.8	9.21
P-coumaric acid	32.4	8.56
Ferrulic acid	40.2	4.91
Rutin	48.9	2.11

Table 2. the analysis of using LC ESI/MS. The experimental condition was previously mentioned in the methods.

Proposed compounds	Molecular Formula	T_R (min)	Mode of ionization	Molecular Weight
Coumarin	C₉H₆O₂	9.554	[M+H]+	146.0374
2-Hydroxybenzoic acid	C₇H₆O₃	11.294	M-H-	138.0304
Caffeic acid	C₉H₈O₄	15.871	M-H-	180.0417
(+)-Catechin	C₁₅H₁₄O₆	19.19	M-H-	290.0771
Sinapic acid	C₁₁H₁₂O₅	22.639	M-H-	224.068
m-Coumaric acid	C₉H₈O₃	29.25	M-H-	164.0459

The leg peel extract of the Prosopis cineraria plant was analyzed by GC-MS method and found to contain the following compounds (Table 3). These are bioactive compounds that may act as good antimicrobial, antiviral, antioxidant and anti-inflammatory agents.

Table 3 . Characterization of active substances in the extract of leg peel powder of Prosopis cineraria L. using GC-MS.

RT	Compound	formula	Area %
5.12	Umbellulone	C₁₀H₁₄O	3.2
6.45	Alloaromadedrene	C₁₅H₂₄	1.5
7.54	cis-calamenene	C₁₅H₂₂	5.1
7.92	Cedrol	C₁₅H₂₆O	9.9
8.47	1-Cubenol	C₁₅ H₂₆ O	2.5
9.21	Acorenol	C₁₅H₂₆O	2.7
9.75	Cadinol	C₁₅H₂₆O	3.8
10.93	1.4 Eudname	C₁₅H₂₆O	1.4
11.47	Eicosane	C₂₀H₄₂	5.8
12.45	Pentatriacontene	C₃₅H₇₀	5.1
13.57	Cubenol	C₁₅H₂₆O	1.7
14.11	Muurolol	C₁₅H₂₆O	2.8
14.87	3-Cyanobenzaldehyde	C₈H₅NO	3.7
15.43	1,4-Benzenedicarbonitrile	C₈H₄N₂	1.9
16.21	2-Methoxy-4-vinylphenol	C₉H₁₀O₂	8.7
17.56	Ethyl 2-hydroxybenzyl sulfone	C₉H₁₂O₃S	2.6
18.41	9-Tricosene	C₂₃H₄₆	2.9
19.82	Methyl 13-phenyl-tridecanoate	C₂₀H₃₂O₂	3.2
20.79	Hexadecanoic acid	C₁₇H₃₂O₃	0.5
21.55	1,3-Benzenedicarboxylic acid	C₂₆H₄₂O₄	1.3
21.93	Bis (2-ethylhexyl) ester	C₂₄H₃₈O₄	7.4
22.72	Squalene	C₃₆H₅0	3.8
23.57	Dotriacontane	C₃₂H₆₆	3.4
24.88	Vitamin E	C₂₉H₅₀O₂	5.8

The components of the seed oil extract were identified by comparing the retention times obtained from gas chromatography with the mass spectra of the compounds to the data available in the library (Wiley and NIST) (Table 4).

Table 4. Determination of Fatty acid content in Prosopis cineraria Seeds.

Fatty acids		Area%
Palmitic acid	(C 16:0)	9.95
Stearic acid	(C 18:0)	11.6
Arachidic acid	(C20:0)	2.63
Behenic acid	(C 22:0)	3.64
Pamitolic acid	(C16:1)	0.66
Oleic acid	(C 18:1)	63.41
cis-11-Eicosenoic acid	(C 20:1)	4.39
Linoleic acid	(C 2:18)	2.05
Linolenic acid	(C18:3)	1.65

The total phenolic content was determined from the linearity equation derived of gallic acid, utilizing the absorbance values obtained and expressed in terms of gallic acid equivalents (GAE). The results were reported as milligrams of gallic acid equivalents per gram of dry weight (mg GAE/g dw) of the sample. (table 5).

Table 5. Total phenols in the extract of the bark powder of the Ghafet plant and the seed powder.

Sample Number	Absorbance	Concentration mg GAE/g dw
P1 - Ghafeth Bark Powder	0.798	86.84
P2 -Thickened Seeds	0.899	92.58

Quantitative analysis of total flavonoids is performed from the quercetin standard curve, using the measured absorbance expressed in quercetin equivalents (QE). The determination of total flavonoid content in the leg peel powder extract is 55.74 mg QE/g, and in the seed powder extract is 63.98 mgQE/g (Figure 3).

Figure 3. Total Flavonoid Content (TFC) in the extract of leg peel powder and seed powder of Prosopis cineraria L..

The antioxidant activity of polyphenols in Ghafeth bark powder extract and seed powder is determined using the DPPH (Figure 4).

Figure 4. Determination of antioxidant activity of polyphenols in the extract of leg peel powder and seed powder of Prosopis cineraria L. using DPPH method.

The total phenolic content and antioxidant properties of the bark extract from the wild Ghafeth plant were examined in this study, revealing significant positive linear correlations between phenols and antioxidants. This research represents the first comprehensive analysis of the wild Ghafeth leaf extract. The HPLC-DAD method employed in this study demonstrated superior characteristics such as sensitivity, selectivity, and speed of analysis. The obtained results shed light on the traditional and current uses of the wild Ghafeth plant in folk medicine, while also supporting its potential applications as a functional food and as a source of health-promoting ingredients. The high total phenolic content, confirmed by HPLC technology using the Folin-Denis reagent, indicates a substantial presence of antioxidants. The results further indicate that the Syrian wild Ghafeth plant extract possesses a significant total antioxidant capacity, estimated in terms of ascorbic acid equivalents. These findings provide evidence for the potential utilization of Ghafeth plant as a natural antioxidant in various industrial applications, considering its pharmaceutical and medical significance. Additionally, the use of GC-MS technology allowed the identification of several components in the volatile essential oil of the plant. Notably, Cedrol was present at a level of 9.9, cis-calamenene at 5.1, 2-Methoxy-4-vinylphenol at 8.7, Vitamin E at 5.8, and Cadinol at 3.8. These compounds exhibit various beneficial properties such as anti-inflammatory, antimicrobial, antifungal, and antioxidant effects. The Ghafeth plant serves as a valuable source of these phytochemicals, particularly phenolic compounds, which possess significant medicinal applications including antibacterial, antifungal, anti-inflammatory, and antioxidant properties. Previous laboratory and biological studies have confirmed the biological efficacy of P. cineraria, particularly its antioxidant, antimicrobial, analgesic, anticancer, and heart-protective effects. However, further research is needed to substantiate its health benefits, which have predominantly been established through laboratory investigations, particularly in terms of antioxidant and antimicrobial effects.

CONCLUSION:

This study focused on the separation and determination of active compounds, phenols, and antioxidants in the Ghafeth plant (Prosopis cineraria) using liquid chromatography and gas chromatography technology with mass spectrometry. The objective was to assess the composition of bioactive substances present in the plant and evaluate its potential in traditional medicine and various industries.

The results of the analysis revealed the presence of several important compounds in the Ghafeth plant extracts. Phenolic compounds such as gallic acid, p-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, ferulic acid, and rutin were identified and quantified. These compounds are known for their antioxidant properties and can be utilized in the food, beverage, feed, and pharmaceutical industries. The research also highlighted the significance of the Ghafeth plant in traditional medicine. It has been traditionally used in the treatment of various conditions, including skin disorders, boils, dysentery, and as a blood purifier. The plant showed promising potential for pharmaceutical and medicinal applications, particularly in addressing the issue of antimicrobial resistance. Furthermore, this study demonstrated the equivalency of plant products to commercially available drugs, emphasizing the importance of natural sources of bioactive substances. The extraction methodologies employed in this research provide valuable insights for the fields of food and cosmetics. The Ghafeth plant (Prosopis cineraria) is a rich source of active compounds, phenols, and antioxidants. Its extracts possess potential applications in various industries and traditional medicine. The findings of this study contribute to the understanding of the phytochemical composition and medicinal properties of the Ghafeth plant, paving the way for further research and development in the field of natural bioactive substances.

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Received on 08.04.2024 Revised on 16.08.2024

Accepted on 23.10.2024 Published on 10.04.2025

Available online from April 12, 2025

Research J. Pharmacy and Technology. 2025;18(4):1604-1610.

DOI: 10.52711/0974-360X.2025.00230

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