2.1.1 Chemicals and reagents

Quercetin (Sigma-Aldrich, USA, Cas No: 117-39-5), gallic acid (Sigma-Aldrich, USA, Cas No: 5996-86-8), diosgenin (Toronto Research chemical, Canada, Cas No: orb1304570) and atropine (Sigma-Aldrich, USA, Cas No: 51-55-8) standards were used to estimate the total flavonoid, phenolic, saponins and alkaloids contents respectively. Ethanol (C ₂H ₅OH, 99.7%, EMSURE ^® ACS, ISO, Reagent, Merck Specialities Private Ltd, India, Cat no: 64-17-5) was used for extraction.

2.1.2 Instrumentation

Spectrophotometric measurements were performed using a Jenway ^® UV-visible spectrophotometer (Biochrom Ltd, Germany, model: 6715), Labconco FreeZone 4,5 Liters Freeze Dry System by Labconco Corporation, Labconco ^® (Kansas City USA, model: 7750020) was used for lyophilization. The fingerprinting of the Catha edulis leaf extract was performed using the Shimadzu Prominence UFLC High-performance liquid chromatography (HPLC) system manufactured by Shimadzu Corporation, Shimadzu ^® (Kyoto, Japan). A gas chromatography-mass spectrophotometer manufactured by Shimadzu Corporation, Shimadzu ^® (Shimadzu, Tokyo, Japan, model: GCMS-QP2020 NX) was used for characterization. All reagents were weighed using a RADWAG MYA.21.4Y. P microbalance produced by RADWAG Balances & Scales, RADWAG ^® (Radom, Poland).

Fresh Catha edulis leaves were collected from a farm in Mayindo village in Bulyantete parish, Kawolo subcounty, Buikwe district in Central Uganda (0.377471, 32.975878(Lat/Lon) 345 ^o north ( UBOS, 2020; Figure 2). The leaf was authenticated by a taxonomist before its voucher specimen was deposited at the Herbarium unit of Makerere University, Uganda where accession number: MHU51321 was allocated.

Catha edulis leaf was extracted according to method described by Limenie et al. (2020). Little modifications were made. Briefly, 500g of tender leaves were plucked and washed under running tap water with care. The leaves were then freeze-dried at −20 °C for 48h after which they chopped and crushed using a blender. Ethanol (70% v/v) was used in the extraction process to extract bioactive compounds such as alkaloids, flavonoids and phenolic compounds without significant degradation and thus maintaining stability ( Nn, 2015). All the crushed leaf materials were placed into a 1000 mL conical flask and 1000 mL 70% v/v (700 mL ethanol and 300 mL of distilled water (7:3v/v ratio)) was poured into the flask to completely cover the crushed leaf materials and wrapped with aluminum foil. The mixture was then placed on 72g rotary shaker in the dark for 48h at 25°C. After shaking, the mixture was filtered using a grade-I Whatman filter paper. The filtrate was placed in a rotary evaporator to remove the organic solvents at a regulated 36°C temperature, 3g rotation, and 240 Pascals of negative pressure.

The phytochemical content of khat plant materials can be altered by post-harvest operations and drying. Locally, after harvesting, growers wrap the harvested leaves in banana leaves to preserve their freshness and reduce the rapid decomposition of cathinone in the leaves preventing extraction because cathinone as the main alkaloid, undergoes decomposition during transportation ( Kelly, 2011; Feng et al., 2017). Consequently, it is essential to use suitable pre-extraction procedures to minimize the loss of active volatile chemicals. Freeze-drying (lyophilization) is a drying technique used to remove water from a sample by sublimation under vacuum ( Preethi Samyuktha et al., 2025). This process is suitable for drying unstable or heat-sensitive compounds that may be lost if allowed to dry for a long time ( Hazarika and Gosztola, 2020). Hence, lyophilization was used to dry the khat leaf extract used in this study. Lyophilized khat extract was obtained based on the method described by Ligor et al. (2022) using (Labconco FreeZone 4,5 Liters Freeze Dry System Model: 7750020). Briefly, the aqueous extract was placed in Falcon tubes and frozen for 12h at temperatures between 40 ^oC and 50 ^oC to prevent the formation of large ice crystals. A two-day freeze-drying method extracted approximately 95% of water from the solution using temperature control and vacuum pressure. After the desorption processing the sample was dried (desorption) to eliminate chemically bound water that remained in the dried substance. The dry (lyophilized) Catha edulis extract obtained from the process was kept at freezer at -20 ^oC until it was used for analysis.

The fingerprinting of the Catha edulis leaf extract was performed using a UFLC HPLC system Shimadzu ^® (Kyoto, Japan). The system contains three main components; an online degasser DGU-20A5R, an ultraviolet (UV) detector and a Phenomenex Luna C18 column (250 x 4.6 mm, 5 μm) alongside temperature-controlled sample trays and an LC-20AD pump. The reversed-phase HPLC test utilized binary isocratic elution at 1.0 flow rate, 30 °C column temperature and a mobile phase of methanol/acetonitrile/0.01% trifluoroacetic acid (6:1:3). At a detection wavelength of 254nm, the injection volume was 10L. All the solvents used were of HPLC grade.

The presence of various bioactive compounds in the ‘ Kasenge’ khat variety was identified using GC-MS (Model: GCMS-QP2020 NX, Shimadzu, Tokyo, Japan). The machine used Helium gas (99.999%) as the carrier gas with viscosity compressor time of 0.2 seconds, total flow of 50.0 mL/min at a washing volume 8uL. The analytical conditions for the experiment were optimized as follows: the injection temperature was set at 250.00 °C, while the ion source temperature was maintained at 230.00 °C. The column flow rate was controlled at 1.69 mL/min, with a linear velocity of 47.2 cm/sec. Mass spectrometry was performed with a scan interval of 0.5 seconds, capturing fragments within the mass range of 50-500 m/z (Dalton). The total GC running time was 28.00 minutes and the compounds were identified based on their retention time, retention indices, and mass spectra. To interpret the names, molecular masses and structures of the identified bioactive compounds in khat samples, the spectrum of the unknown compounds was compared with that of the known compounds domiciled in the database of the National Institute of Standard and Technology (NIST) library. The relative percentage concentration of each compound was determined by comparing its average peak area with the total area ( Sharma et al., 2015).

2.7.1 Preparation of standard solutions

Atropine: A standard solution of atropine (1 mg/mL) of was prepared by mixing 10 mg of atropine with 10 mL of methanol. Quercetin: A standard solution of quercetin was prepared by mixing 10 mg quercetin with 10 mL methanol. Gallic acid: A standard solution of 1mg/mL was prepared by dissolving 10 mg gallic acid in 10 mL of distilled water. Diosgenin: A diosgenin standard solution was prepared by mixing 10 mg diosgenin in 10 mL of distilled water.

2.7.2 Determination of total alkaloid content (TAC)

The TAC of the Catha edulis extract was determined based on the methods of Shamsa et al. (2008) and Seifried et al. (2007). Briefly, 1 mg/mL of the Catha edulis extract was reconstituted with 2N HCl, filtered, and 1 mL of the mixture was added to a separating funnel along with 5 mL of bromocresol green (BCG) solution and phosphate buffer (pH 4.8). The complex formed was consecutively extracted into 1, 2, 3 and 4-mL chloroform with vigorous shaking in 10 mL volumetric flask and made to the volume. Chloroform-containing alkaloids were measured at 415 nm. The total alkaloid content was estimated as atropine (mg/g AE).

2.7.3 Atropine standard curve preparation

Atropine standard solutions (0.01, 0.2, 0.4, 1 and 2 mL) were transferred to five separating funnels that were carefully positioned on the retort stands. To each aliquot in the separating funnel, 4 mL chloroform, 5 mL bromocresol green (BCG) solution and phosphate buffer were added. The complex formed was extracted into 1-, 2-, 3- and 4-mL chloroform in a 10 mL volumetric flask and made to volume. The absorbance of the complex was measured at 415 nm against a blank solution containing all solutions other than atropine. The unknown TAC in the Catha edulis extract was estimated using the linear regression curve y = 0.0027x – 0.0149; r ² = 0.9823 from atropine following the measurement of absorbance.

2.7.4 Determination of total phenolic content (TPC)

The TPC of the Catha edulis extract was estimated using gallic acid as the standard as described by Ainsworth and Gillespie (2007). Aliquots of gallic acid standard solution were accurately measured and placed into 10 mL volumetric flasks at various concentrations (0.01, 0.2, 0.4, 1 and 2 mL). Sodium carbonate (7.5% w/v) and 10% Folin-Ciocalteu reagent were added to 1 ml of 0.5 mg/mL Catha edulis extract. Standard concentrations were prepared using similar procedures. All the tubes were incubated for 30 minutes at 40 ^oC. The absorbance was measured in triplicates at 760 nm. The line of regression, y = 0.0136x – 0.0585; r ² = 0.957 from the gallic acid standard curve was used to determine the TPC.

2.7.5 Determination of total saponins content (TSC)

TSC was determined using the method described by Senguttuvan et al. (2014) with modifications. Each tube contained a normal aliquot (0.01, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mL) of diosgenin and a 0.25 mL aliquot of 1 mg/mL Catha edulis extract. Additionally, each tube received 0.25 mL of 8% vanillin reagent and 2.5 mL of 72% sulfuric acid. The mixtures were incubated in a water bath for 15 min at 60 °C and transferred to ice-cold water for 5 min. Absorbance was measured at 560 nm after cooling. The line of regression, y = 0.0015x + 0.0766; r ² = 0.9746 from the diosgenin standard curve was used to determine the TSC.

2.7.6 Determination of total flavonoid content (TFC)

TFC of Catha edulis was determined using a modified aluminium chloride colorimetric method described by Baba and Malik (2015). A 1 mL aliquot of Catha edulis extract (1 mg/mL) was mixed with 3 mL of methanol and vortexed. Subsequently, 0.2 mL of a 10% aluminium chloride (AlCl ₃) solution and 0.2 mL of 1 M sodium acetate solution were added to the mixture. The solution was incubated for 30 min. Absorbance was measured at 420nm (in triplicate). A calibration curve, y = 0.006x – 0.0267; r ² = 0.9975 of standard quercetin at various concentrations (0.01, 0.02, 0.04, 0.1, and 0.2 mg/mL) was used to determine the total amount of flavonoids in the Catha edulis extract.

Spectrophotometric determination of the total contents of alkaloids, phenols, saponins and flavonoids using the corresponding regression equation of the calibration curves indicated different concentrations in the “ Kasenge” khat variety. The total alkaloid content in the khat extract determined from the regression equation of the calibration curve (y = 0.0027x – 0.0149; R ² = 0.9823) was 0.0063 mg/g AE. The total phenolic content determined using the regression equation of the calibration curve (y = 0.0136x-0.0585; R ² = 0.957) was 0.099 mg/g GAE. Total saponins content was revealed to be 0.69mg/g DE using the regression equation of calibration curve (y = 0.0015x + 0.0766; R ² = 0.9746). The total flavonoid content in the sample was 0.047 mg/g, and expressed as quercetin equivalents (QE). This value was calculated using the regression equation (y = 0.006x - 0.0267) derived from the calibration curve, which showed a strong correlation coefficient (R ² = 0.9975). The results are presented in Table 1 and are illustrated in Figures 3- 6.

HPLC analysis of the lyophilized Catha edulis extract showed a diverse mixture of phytochemical components in its chromatogram featuring 34 individual peaks ( Figure 7). The different phytochemicals in the extract correspond to specific peaks showing their relative abundance based on their retention times measured in minutes. This study used fingerprinting as a chemical characterization method essential for quality control, standardization and comparative analysis between different Catha edulis varieties. The examined compounds exhibited retention times ranging from 3.797 to 40.558 min demonstrating the presence of polar and non-polar phytochemicals. Multiple high-intensity peaks emerged during the analysis at retention times of 4.401, 5.685, 17.777, 26.806 and 31.624 min. This peak at 31.624 minutes demonstrated the highest area percentage (33.048%) indicating its dominant presence in the extract. The peak table showing the retention time of the identified phytochemicals in the khat extract is presented in Table 2 ( Mbina et al., 2025).

GC-MS analysis of the lyophilized khat extract revealed a diverse profile of the bioactive compounds. The GC-MS analysis identified 21 peaks with corresponding area percentages. Among the major compounds identified in the Kasenge khat variety was 9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester (Z, Z, Z), a linolenic acid glycerol ester constituting 35.01% of the total peak area, hexadecanoic acid, ethyl ester and methyl ester, derivatives of palmitic acid collectively accounted for over 21% of the composition, phytol, a diterpene alcohol, constituting 10.38% area, butyl 9,12-octadecadienoate (7.58%) and 2,4-Di-tert-butylphenol with an area percentage of 3.22% ( Figure 8 and Table 3).