Phytochemical Screening and Cytotoxic Properties of Ethanolic Extract of Young and Mature Khat Leaves

Substance Abuse and Toxicology Research Center, Jazan University, Jazan, Saudi Arabia Faculty of Medicine, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia Department of Pharmaceutical Chemistry, College of Pharmacy, Jazan University, P.O. Box 114, Postal Code 45142, Jazan, Saudi Arabia Department of Pharmacognosy, College of Pharmacy, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia Department of Pharmacology and Toxicology, Faculty of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi Arabia Medicinal and Aromatic Plants Research Institute, National Center for Research, P.O. Box: 2424, Khartoum-11111, Sudan Faculty of Medicine, University of Tabuk, Tabuk, Saudi Arabia Pharmacy Practice Research Unit, Department of Clinical Pharmacy, College of Pharmacy, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia Poison Control and Medical Forensic Chemistry Center, Ministry of Health, Jazan, Saudi Arabia

biological plausibility that chronic khat use may induce memory deficits and impair cognitive flexibility [10]. e differential patterns of memory deficits may reflect the differences in dose effect as well as time-dependent impairment [11]. Cathinone, a major constituent of khat, is structurally and functionally similar to amphetamine and cocaine. Cathinone caused the proliferation of gonadotrophs but decreased the lactotrophs and corticotrophs in anterior pituitary sections of animals in high dose and long-term exposure, while an effect of low dose on these cells was insignificant [12]. Few articles contribute to death among khat chewers to khat-induced heart failure, but several other studies have demonstrated that khat chewing has unfavorable cardiovascular effects [13]. Like amphetamine, the reflection of undesired actions of cathinone on the myocardium is observed through variations in heart rate, blood pressure, and vascular actions [14]. However, further studies are needed to address the risk factors in khat chewers that may explain khat-induced cardiotoxicity, cardiomyopathy, and heart failure.
Khat is reported to induce toxic hepatitis together with high-titer anti-nuclear antibody mimicking serologic patterns of autoimmune hepatitis and potentially associated with malignant and malignant oral disorders [15]. ere are several pieces of evidence connecting khat chewing to genetic damage of the oral mucosa and cancer [16,17] Esophageal and gastric carcinoma have been observed in khat chewers in both men and women in Yemen [18]. A similar review of oral cancers presenting over two years in the Asir region of Saudi Arabia showed strong circumstantial evidence linking the long-term use of khat with an increased rate of oral malignancies [19].
Khat young and mature leaves have a different degree of psycho-stimulating and variable biological properties. Khat chewers, however, prefer young leaves over mature for the more desired outcomes and fewer side effects. e young leaves are believed to possess higher central nervous system-(CNS-) stimulating activities than the mature leaves. is difference in CNS-related activities could be due to the presence of the same constituents in both young and mature leaves; however, in different quantities or due to the presence of different constituents should be investigated. No assessment and comparison of the complete phytochemical profiles of young and mature leaves of Catha edulis has been conducted. erefore, we have undertaken this study to investigate the phytochemical constituents of both young and mature khat leaves and to find out the degree of cytotoxicity of their selected extracts.

Collection of Plant Material.
Young leaves (locally known as Nwaif ) and mature leaves (locally known as Gafra) of three cultivars of khat (Catha edulis) locally known as Gaifi, Kofat, and Gahasha were collected from the Jazan region of Saudi Arabia and divided into two groups each: young Gaifi (N1), young Kofat (N2), and young Gahasha (N3) for young leaves and mature Gaifi (G1), mature Kofat (G2), and mature Gahasha (G3) for mature leaves. All khat varieties were identified by Dr. Yahiya Masrahi, Department of Botany, Faculty of Science, Jazan University. e collected leaves were washed, dried in the shade at room temperature, and powdered.

Preparation of Leaves
Extract. All dried leaves powder (200 g each) was exhaustively extracted with 80% ethanol in the Soxhlet apparatus for 6 to 8 hours. Colored extracts were evaporated under reduced pressure to get brown viscous masses. Sample N1, N2, N3, G1, G2, and G3 were labeled and stored at 4°C in the dark for GC-MS analysis and cytotoxic activity.

Identification of Constituents.
GC-MS identified the most constituents by comparing their retention indices with those of authentic standards available in the laboratory or with the retention indices, which were in close agreement with the reference. GC-MS achieved further identification. e fragmentation patterns of mass spectra were compared with those stored in the spectrometer database using the NIST08 and Wiley 9 built libraries.

Hierarchical Cluster Analysis (HCA).
Due to significant variations of the contents of various types of khat leaves, the most abundant components from all samples were subjected to multivariate chemometric analysis. Hierarchical cluster analysis (HCA) was performed, and agglomeration and dendrograms were developed to assess the relationship between the components of the different types of extracted khat and to find out the proper classification of these studied samples and to detect the proper classification of khat types using SPPS software version 22.0.

Cell Culture.
ree cancer cell lines, MCF7 (Michigan Cancer Foundation-7; human breast adenocarcinoma), A2780 (human ovary adenocarcinoma), and HT29 (human colon adenocarcinoma), were used in this study to assess the cytotoxicity of different ethanolic extracts. Besides, we also used MRC5, normal human fetal lung fibroblast. All cells were obtained from the ATCC (American Type Culture Collection). e three cancer cells were subcultured in the RPMI (Roswell Park Memorial Institute)-1640 media (10% FBS, fetal bovine serum), while MRC5 was maintained in Eagles Minimum Essential Medium (EMEM, 10% FBS), all at 37°C, 5% CO 2 and 100% relative humidity).

Cytotoxicity Assay.
As previously reported by Bkhaitan et al., the cytotoxic activity of the six extracts was evaluated by the MTT assay (MTT is the abbreviation for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). e three cell lines and one normal fibroblast were separately cultured in a 96-well (3 × 10 3 /well) and incubated at 37°C overnight. Final extract concentrations were 0, 6.25, 12.5, 25, 50, and 100 μg/mL of DMSO (dimethyl sulfoxide) 0.1%; n � 3). Plates were incubated for 72 h, followed by addition of MTT to each well. en, the plates were incubated for 3 hr, the supernatant was aspirated, and DMSO was added to each well. Absorbance was read on the multiplate reader. e optical density of the purple formazan A 550 is proportional to the number of viable cells. Extract concentration causing 50% inhibition (IC 50 ) compared to the control cell growth (100%) was determined. GraphPad Software (GraphPad Software, San Diego California USA) was used for analysis.

GC-MS Analysis.
Separation and identification of different phytoconstituents present in the closely related genus in mature and young leaves was analyzed by GC-MS. Proportional chromatograms of unlike varieties of khat extracts are shown in Figure 1.
e phytochemical composition of the young and mature extracts, including psychoactive cathine and cathinone, is shown in Table 1. e total area percentage for identified components was 63.56%, 53.98%, 62.17%, 36.67%, 52.57%, and 67.12% for N1, G1, N2, G2, N3, and G3, respectively. Cathine, a chief psychostimulant constituent, was found in all three varieties of young leaves in different proportions, viz., 0.55%, 0.77%, and 0.67%, for N1, N2, and N3, respectively, whereas, only 0.35% and 0.16% found in G1 and G3 samples, respectively. e lowest amount was found in G3 and the highest amount, 0.77%, in the extract of N2. However, no trace of either cathine or cathinone was found in the extracts of mature G2. Cathinone was found in a lesser amount than cathine, 0.05% to 0.26%, in all extracts which were studied.
Based on GC-MS results and the hierarchical cluster analysis in the table of the agglomeration schedule (Table 3) for cluster solution, there is a sudden jump/gap in the distance coefficient. e solution before the gap indicated a good solution. Accordingly, we could determine two major clusters of samples based on the furthest neighbor clustering method. e first cluster consists of N1, N3, N2, and G2, and the second cluster consists of G1 and G3. In the first cluster, N1 and N3 combined and showed similar characteristics and therefore can be included in the same subcluster leaving N2 and G2 forming the second subcluster in this first group. e hierarchical cluster analysis results are shown in Table 3 and Figure 2.
In previous cytotoxicity studies [20,21], the methanolic extract of Catha edulis showed cytotoxicity on MCF 7 and HL60 cells with IC 50 33-200 μg/ml, respectively. However, the effect of those extracts was not tested in normal cells to evaluate the possible adverse effect of chewed khat on the health of normal living cells. us, khat extracts in this study showed comparable cytotoxicity on the cancer cells, but more interestingly, they were more cytotoxic against the normal cells, which could be associated with the different mouth cavity lesions.

Discussion
Out of total 26, 26, and 20 chemical constituents present in ethanolic extracts of young leaves, 6, 4, and 2 different chemical entities were found in N1, N2, and N3, respectively. ese chemical compounds were not found in other extracts and were unique for them. N1 contains six unique compounds: 1-phenyl propanol, cathine, 1-hydroxy-1-phenyl-2-(acetylamino) propane, methyl α-Dglucopyranoside, isobutyl tetradec-3-enyl fumarate, and phytol. N1 has almost equipotent cytotoxic activity on both normal MRCS (30.63) and human breast adenocarcinoma, MCF7 (29.91), and observed the least activity on HT29. It has no advantage over cancerous cells. N2 has four unique compounds: 2,11,11-tetramethyldodecane, benzene α-hydroxyethyl acetate, tert-nonyl mercaptan, and 3-ethyl-6-trimethylsilyloxyoctane. It is more cytotoxic to normal cells than MCF7. N3 has α-hydroxy ethyl benzene acetate and pentatriacontane. It was cytotoxic compared to all other extracts, and was found that its activity is more on normal cells. N1 was the least cytotoxic among the three, and N3 is the most toxic among all extracts.
No compounds were common between N1 and G1 or N3 and G3 but two common chemical constituents in N2 and G2 extracts were observed: 1-hydroxy-1-phenyl-2-(acetylamino) propane and 9-octadecenamide. Similarly, sixteen unique compounds were present in N1, and none were found in G1; thirteen individual compounds were found in G1, but none observed in N1. N2 has six unique compounds, but the same was not found in G2; G2 has nine other distinct compounds that were absent in N2. N3 has eight unique compounds, but they were not found in G3; however, G3 has ten other compounds that were absent in N3.
e extracts of mature and young leaves have an almost different set of chemical constituents. It could be the reason why there is a difference in their cytotoxicity. Further study is required to identify the exact role of each of these chemical constituents found in these extracts.
In terms of the presence of cathinone and cathine, we found that the maximum content of cathinone in N1 (0.22) and N2 (0.26) and N3 is only 0.16. Cathinone was not found in mature leaves G1 but was found in G2 and G3 (0.09 and 0.16, respectively). Similarly, the presence of cathine was less in mature leaves G2 (0.35) and G3 (0.05) but was not found in mature G1 compared to young leaves N1. e maximum cathine was found in N1 (0.77). Methoxyamphetamine (0.29) was found only in the N1 extract. It could be the reason for the variation in psychostimulant activities in young and mature leaves.
Isolating and characterizing the different chemical constituents in the ethanolic extract of both mature and young leaves of Catha edulis and matching its cytotoxic activity is the strength of this study; however, identifying the biological activities of each constituent found in these extracts requires further study. Journal of Chemistry 7

Conclusions
GC-MS investigations revealed and identified several remarkable phytochemicals with significant variations among them in the young and mature ethanolic extracts of the three khat cultivars. e study confirmed the presence of psychoactive cathine and cathinone in high quantities in the young leaves compared to matured leaves. Ethanolic extracts of khat showed significant cytotoxicity, IC 50 ranging from 22-59 μg/mL on the cancer cells, compared to previous claims (IC 50: ranging from 33-200 μg/mL); however, these extracts were also exhibiting cytotoxicity against the normal cells (MRC5 IC 50 : 6-41 μg/mL). Hence, the substantial cytotoxic effect on normal cells may pose many hazards to the health of khat consumers. erefore, awareness campaigns on stopping the usage of khat should be implemented and facilitated in affected societies.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
All the authors declare that there are no conflicts of interest.