New Megastigmane and Polyphenolic Components of Henna Leaves and Their Tumor-Specific Cytotoxicity on Human Oral Squamous Carcinoma Cell Lines

Polyphenols have a variety of phenolic hydroxyl and carbonyl functionalities that enable them to scavenge many oxidants, thereby preserving the human redox balance and preventing a number of oxidative stress-related chronic degenerative diseases. In our ongoing investigation of polyphenol-rich plants in search of novel molecules, we resumed the investigation of Lawsonia inermis L. (Lythraceae) or henna, a popular ancient plant with aesthetic and therapeutic benefits. The leaves’ 70% aq acetone extract was fractionated on a Diaion HP-20 column with different ratios of H2O/an organic solvent. Multistep gel chromatographic fractionation and HPLC purification of the Diaion 75% aq MeOH and MeOH fractions led to a new compound (1) along with tannin-related metabolites, benzoic acid (2), benzyl 6′-O-galloyl-β-D-glucopyranoside (3), and ellagic acid (4), which are first isolated from henna. Repeating the procedures on the Diaion 50% aq MeOH eluate led to the first-time isolation of two O-glucosidic ellagitannins, heterophylliin A (5), and gemin D (6), in addition to four known C-glycosidic ellagitannins, lythracin D (7), pedunculagin (8), flosin B (9), and lagerstroemin (10). The compound structures were determined through intensive spectroscopic investigations, including HRESIMS, 1D (1H and 13C) and 2D (1H–1H COSY, HSQC, HMBC, and NOESY) NMR, UV, [α]D, and CD experiments. The new structure of 1 was determined to be a megastigmane glucoside gallate; its biosynthesis from gallic acid and a β-ionone, a degradative product of the common metabolite β-carotin, was highlighted. Cytotoxicity investigations of the abundant ellagitannins revealed that lythracin D2 (7) and pedunculagin (8) are obviously more cytotoxic (tumor specificity = 2.3 and 2.8, respectively) toward oral squamous cell carcinoma cell lines (HSC-2, HSC-4, and Ca9-22) than normal human oral cells (HGF, HPC, and HPLF). In summary, Lawsonia inermis is a rich source of anti-oral cancer ellagitannins. Also, the several discovered polyphenolics highlighted here emphasize the numerous biological benefits of henna and encourage further clinical studies to profit from their antioxidant properties against oxidative stress-related disorders.


Introduction
The study of polyphenolic phytochemicals for drug development has grown significantly in recent decades.Polyphenols can participate in redox activities that are required for various metabolic events due to their many phenolic hydroxyl and carbonyl functionalities.As a result, they can prevent a variety of chronic degenerative illnesses and maintain human homeostasis [1].The plant polyphenols are widely varied in their chemical structures among different families of the plant kingdom or within the same family, and how they interact-whether specifically or not-with biomolecules relies heavily on both their own physicochemical properties and those of the biomolecule partners [2].
In our ongoing investigation of polyphenol-rich plants in search of novel molecules, we resumed the investigation of Lawsonia inermis L. (Syn.L. alba) (Lythraceae), or henna, as one with special healing qualities in ancient medicines.Henna, which refers to the dye prepared from the plant, is naturally grown from Northeast Africa to India and has been extensively used for centuries in the Middle East, Far East, and Northern Africa as a cosmetic dye for nails, hands, hair, and textiles.It has also been used to tackle skin issues, headaches, jaundice, amebiasis, and spleen enlargement [3][4][5].The plant extracts and purified constituents of henna account for a variety of activities, including anti-Alzheimer's, antioxidant, hepatoprotective, immunomodulatory, cytotoxic, antibacterial, antifungal, analgesic, anti-inflammatory and antipyretic, hypotensive, sedative, and anticancer effects (Figure 1) [3,[6][7][8][9][10].Oral administration of L. inermis leaf extracts significantly suppressed the growth of B16F10 tumors in mice with an increased tumor necrosis area and increased infiltration of mononuclear cells at the site of the tumor [11].The in vivo antitumor effect of L. inermis extracts was directly linked to the enhanced antioxidant activity, which is the main quality of plant polyphenols [11].In our preceding articles on henna leaf extracts, we demonstrated the occurrence of ellagitannins in abundance and reported on their anticholinesterase and cytotoxic activities [12,13].The structure and molecular weight of ellagitannins determine their inhibitory capacities against the initiation and spread of tumors, which are subsequently influenced by their antioxidant and binding qualities [12].

General Experimental Procedures
Electronic circular dichroism (ECD) and ultraviolet (UV) spectra were recorded on JASCO J-720W (JASCO, Tokyo, Japan) and JASCO V-530 (JASCO, Tokyo, Japan) spectro- The isolation-identification procedures of therapeutic metabolites is a potential research focus for the next steps of our studies.Further phytochemical investigations of different fractions of the L. inermis leaf extract may lead to the exploration of novel constituents that could explain the extract's various biological areas of significance.
In this study, a chromatographic investigation of phenolic-rich fractions of the henna leaf extract led to the isolation of a new megastigmane, lawsoiononoside (1), together with the first-time isolation of tannin-related phenolics, benzyl 6 -O-galloyl-β-D-glucopyranoside (2), benzoic acid (3), and ellagic acid (4), whereas an extensive investigation of the tanninrich fractions led to two glucopyranose-type ellagitannins (5 and 6) in addition to four known C-glycosidic ones (7)(8)(9)(10).The cytotoxicity of the abundant ellagitannins (8)(9)(10) to various human oral cancer and normal cell lines was examined in the present study.
The fractionation and purification procedures were monitored by normal-phase (NP) and reversed-phase (RP) high-performance liquid chromatography (HPLC).The NP-HPLC analyses were performed on a YMC-Pack SIL A-003 (YMC, Kyoto, Japan) column (4.6 × 250 mm) using a mobile phase composed of n-hexane/MeOH/tetrahydrofuran/ formic acid (55:33:11:1, v/v) + oxalic acid (450 mg/L).The flow rate was adjusted at 1.5 mL/min at room temperature, and eluates were monitored using a UV detector at 280 nm.The RPHPLC analyses were performed on a YMC-Pack ODS-A A-302 column (4.6 × 150 mm) (YMC, Japan) with a 0.01 M H 3 PO 4 /0.01 M KH 2 PO 4 /MeOH (1:1:0.5, v/v) mobile phase at a flow rate of 1.0 mL/min at 40 • C and UV detection at 280 nm.Preparative RP-HPLC was performed at 40 • C on a YMC-Pack ODS-A A-324 column (10 × 300 mm) at a flow rate of 2.0 mL/min and the same UV detector.The composition of the mobile phases used in the RPHPLC purifications are specified in the extraction and isolation procedures below.The gels used for column chromatography were Diaion HP-20 (Mitsubishi Chemical, Tokyo, Japan), MCI-gel CHP-20P, Toyopearl HW-40C (TOSOH, Tokyo, Japan), and Sephadex LH-20 (GE Healthcare Bio-Science AB, Sweden).

Plant Material
L. inermis leaves were obtained from mature trees around the court collections, Assiut city, Egypt.The plant was authenticated by Professor Salah M. I. El-Najjar, Department of Botany, Assiut University, Egypt.A specimen numbered Li-05013 was kept in the department of Pharmacognosy, Al-Azhar University, Assiut, Egypt.

Spectroscopic Data of Isolated Compounds
It is best to use human epithelial normal oral cells, if possible, for the comparison of drug-sensitivity with OSCC.However, when two human normal epithelial cells (human oral keratinocyte (HOK) and human gingival epithelium progenitors (HGEP)) were cultured in the regular culture medium (DMEM +10% heat-inactivated FBS), their growths were immediately stopped.Therefore, in order to maintain their growth, it was necessary to culture them in the commercially available special media supplemented with growth factors.However, we found that such stimulated epithelial (HOK/HGEP) cells began to grow like cancer cell lines, showing extremely high sensitivity against many anticancer drugs (camptothecin, SN-38 (active principal of irinotecan), doxorubicin, daunorubicin, etoposide, mitomycin C, 5-FU, docetaxel, melphalan and even molecular-targeted drug, gefitinib) [21].At present, normal human epithelial cells cannot be used as controls.Based on this background, we used three normal oral mesenchymal cells (HGF, HPLF, HPC) in the present study.Further studies are necessary to establish the optimization of the culture condition of normal epithelial cells for use as controls and the composition of HPC cells.
CC 50 values against three normal human oral mesenchymal cell lines (HGF, HPLF, and HPC) and four OSCC cell lines (Ca9-22, HSC-2, HSC-3, and HSC-4) were determined.The mean CC 50 values for the normal and tumor cell lines were calculated and the ratio of the means gives the tumor specificity (TS): TS = mean CC 50 normal cell lines/mean CC 50 OSCC cell lines 2.5.3.Statistical Analysis All analyses were carried out in triplicate to ensure robustness and reliability.The data are presented as mean ± standard deviation (SD).Graph Pad Prism 7 and Microsoft Excel 2010 were used for the statistical and graphical evaluations.
the S chirality of C-1.Among the four possible stereoisomers (1S3S, 1R3R, 1S3R, and 1R3S, Figure S41) of 1, these NOESY correlations were consistent with the 1S3R isomer.The H3-12 methyl signal, as well as the H3-13 methyl signal (δH 1.54, 3H, s), exhibited NOESY correlations with H2-7 (δH 2.14 and 2.20) and H2-8 (δH 2.48).The NOESY correlations between each pair of protons of H2-2 and H2-4 and H2-7 were also detected (Figures S15-S17).Importantly, the practically recoded ECD spectrum of 1 was compared with the computationally calculated ECD (see procedures in the Supplementary Materials) of the four possible stereoisomers.Despite the noisy Cotton effects of the practically measured ECD, the overall spectrum was consistent with that calculated for the 1S3R isomer (Figure S42).According to these findings, the new structure of 1 was concluded to be a gallate derivative of a megastigmane glucoside, as shown in Figure 2, and given the name lawsoiononoside (1).Except for the megastigmane aglycone β-ionone, this is the first report on the isolation of a megastigmane from henna.Megastigmanes are oxygenated isonorterpenoids with a C13 carbon skeleton and frequently referred to as oxidative by-products from β-carotenoids [28].The biosynthesis of compound 1 from β-ionone and gallic acid is assumed to  13 C NMR data (Table 1).The 1 H NMR spectrum of 1 (Figures S3-S5) displayed spectroscopic features typical of a galloyl moiety [22]; an aromatic 2H singlet (δ H 7.13, H-2 /H-6 ) that exhibits HSQC correlation with a non-oxygenated aromatic carbon (δ C 109.6, 2C, C-2 /C-6 ) and HMBC correlations with oxygenated aromatic carbon (δ C 146, 2C, C-3 /C-5 , and δ C 137.7, C-4 ), a quaternary aromatic carbon (δ C 120.5, C-1 ), and a carbonyl carbon (δ C 167.2, C-7 ).The 1 H NMR and 1 H-1 H COSY spectra of 1 (Table 1, Figures S6-S8) exhibited a resonating system of seven aliphatic proton spins at δ H 4.49-3.19(Table 1), with a large coupling pattern of the glucose H-2 -H-5 (J H-1 -H-2 = 7.8, J H-2 -H-3 = J H-3 -H-4 = J H-4 -H-5 = 9.6 Hz), highlighting the 4 C 1 conformation of the glucopyranose moiety [23].A large coupling constant of the glucose H-1 proton signal (δ H 4.49, J = 7.8 Hz) indicates the β-configuration of the glucose's anomeric center [22].The HSQC correlations of the glucose proton signals (Figure S10) enabled the assignments of the 13 C chemical shifts (δ C 102.2, 74.5, 77.5, 71.3, 74.8, 64.8) to the glucose carbons C-1 -C-6 , respectively (Table 1).The 13 C NMR spectrum of 1 (Figure S9) also exhibited a system of thirteen carbons (Table 1), characteristic of the megastigmane moiety [24].Aided by the 1 H-1 H COSY and HSQC spectroscopic data, the system is separated into two components.The first is composed of nine carbons, recognized by their 1D NMR and the HSQC data: one oxygenated methine carbon δ C 72. of the latter corresponds to the methyl group on an olefenic carbon, and the broadening of the signal is explained by the W-shaped 1 H-1 H coupling with the H-4 proton signal (δ H 1.94, 1H, br.dd) [25].The second component of the megastigmane moiety was found to be composed of a chain of four carbons: the 1D together with the 2D ( 1 H-1 H COSY, Figures S6-S8) and HSQC (Figures S10-S12) spectra substantiated the presence of two methylene groups (δ H 2.14 and 2.2 (2H, dd, H 2 -7), correlated with a carbon signal at δ C 22.3 (C-7) and δ H 2.48 (2H, t, H 2 -8), correlated with a carbon signal at δ C 44.2 (C-8)).The other two carbons were identified as methyl and ketone carbons: the methyl proton signal at δ H 2.08 (3H, s, H 3 -10) exhibits an HSQC correlation with the carbon peak at δ C 29.8 (C-10) and an HMBC correlation with a ketonic carbonyl carbon peak at δ C 209.3.The same carbonyl carbon is also correlated with the H 2 -8 signal at δ H 2.48 in the HMBC spectrum.This four-carbon segment, proposed as -CH 2 -CH 2 -CO-CH 3 , was placed at the olefenic carbon C-6 (δ C 133.6), as deduced from the HMBC correlations of the H-7 signal at δ H 2.14 with C-6 (δ C 133.6) and the neighboring carbons C-1 (δ C 43.5) and C-5 (δ C 128.8).The connectivity of the structural components (galloyl, glucosyl, and megastigmane moieties) of 1 was then substantiated based on the HMBC correlations (Figures S13 and S14).The galloyl unit was positioned on the glucose C-6 ; this was based on the low-field shift of the glucose C-6 signal (δ C 64.8) in addition to a weak HMBC correlation of glucose-H-6 (δ H 4.16) with the galloyl carbonyl carbon peak (δ C 167.2).The down-field shift of the glucose anomeric carbon (δ C 102.2, C-1 ) indicated the presence of an O-glucosidic linkage between the megastigmane aglycone and glucose C-1 , which was confirmed by the HMBC correlation of the glucose H-1 (δ H 4.49) and the megastigmane moiety C-3 (δ C 72.8).
The 1 H NMR spectrum showed the H-3 signal at δ H 4.14 as a multiplet in the 1 H NMR spectrum, where the coupling constants of J 2ax-3 , J 2eq-3 , J 4ax-3 , and J 4eq-3 with the adjacent proton signals were assigned to be 12.6 Hz, 8.4 Hz, 9.6 Hz, and 5.4 Hz, respectively.These NMR data indicated the equatorial orientation of the substituted hydroxyl group at C-3.This was further evidenced by the 1 H-1 H nuclear Overhauser effect spectroscopy (NOESY) correlations between the axially oriented glucose H-1 proton (δ H 4.49) and both H-3 (δ H 4.14) and H-4eq (δ H 2.30) (Figures S15-S17) corresponding to the R chirality of C-3 in compound 1 [26].In the skeleton of megastigmanes, it has been documented, thus far, that the hydroxyl group at the C-3 position frequently has an equatorial orientation [27].The NOESY spectrum exhibited a key correlation of H-3 with H 2 -11 and NOESY correlations of both H-2ax (δ H 1.21, t) and H-4ax (δ H 1.94) with H 3 -12 (δ H 0.91, 3H, s, methyl), indicating the S chirality of C-1.Among the four possible stereoisomers (1S3S, 1R3R, 1S3R, and 1R3S, Figure S41) of 1, these NOESY correlations were consistent with the 1S3R isomer.The H 3 -12 methyl signal, as well as the H 3 -13 methyl signal (δ H 1.54, 3H, s), exhibited NOESY correlations with H 2 -7 (δ H 2.14 and 2.20) and H 2 -8 (δ H 2.48).The NOESY correlations between each pair of protons of H 2 -2 and H 2 -4 and H 2 -7 were also detected (Figures S15-S17).Importantly, the practically recoded ECD spectrum of 1 was compared with the computationally calculated ECD (see procedures in the Supplementary Materials) of the four possible stereoisomers.Despite the noisy Cotton effects of the practically measured ECD, the overall spectrum was consistent with that calculated for the 1S3R isomer (Figure S42).According to these findings, the new structure of 1 was concluded to be a gallate derivative of a megastigmane glucoside, as shown in Figure 2, and given the name lawsoiononoside (1).
Except for the megastigmane aglycone β-ionone, this is the first report on the isolation of a megastigmane from henna.Megastigmanes are oxygenated isonorterpenoids with a C13 carbon skeleton and frequently referred to as oxidative by-products from βcarotenoids [28].The biosynthesis of compound 1 from β-ionone and gallic acid is assumed to be as shown in Numerous megastigmane glucosides have been shown to have antibacterial, anti-inflammatory, anticancer, and hepatoprotective effects, which are strongly correlated with their antioxidant capacity [29][30][31].The antioxidant activity of megastigmane glycosides has been demonstrated through their capacity to scavenge DPPH free radicals and suppress the process of lipid peroxidation [31].The bioactivities of this class of metabolites, coupled with their antioxidant power, suggests the need for further studies in the future on these intriguing bioactive small molecules as a scaffold of drug discovery.Numerous megastigmane glucosides have been shown to have antibacterial, antiinflammatory, anticancer, and hepatoprotective effects, which are strongly correlated with their antioxidant capacity [29][30][31].The antioxidant activity of megastigmane glycosides has been demonstrated through their capacity to scavenge DPPH free radicals and suppress the process of lipid peroxidation [31].The bioactivities of this class of metabolites, coupled with their antioxidant power, suggests the need for further studies in the future on these intriguing bioactive small molecules as a scaffold of drug discovery.).These are characteristics for an ellagic acid.Based on the molecular ion peak at m/z 301 [M − H] − in the ESIMS spectrum, we confirmed the structure of 4 to be ellagic acid [17].
It is worth noting that Ye et al., 2007, have reported two resonances (δ H 7.14 and 7.47) for the equivalent ellagic acid protons (H-5 and H-5 , respectively), which are incorrect and misleading NMR data [32].
Structure of the O-Glycosidic Ellagitannins 5 and 6 Heterophylliin A (5) and gemin D (6) were isolated first from the plant, and hence, their structures were determined from the following spectroscopic data and comparison with the literature values: • Compound 5 was isolated as an off-white amorphous powder.Its 1 H NMR spectrum (Figure S29) exhibited aromatic proton signals at δ H 7.23, 7.03 (each 2H, s, galloyl H-2/H-6) of two galloyl units and two 1H singlets (δ H 6.61 and 6.48), indicative of the presence of a hexahydroxydiphenoyl (HHDP) unit [22].A spin system of seven aliphatic proton sets, as evident from the 1 H-1 H COSY correlations (Figure S30), were assigned for the 4 C 1 D-glucopyranose core as follows: δ  [14].These data, together with the comparison with the literature values [18], led to the identification of 5 as heterophylliin A (5, Figure 5).together with the comparison with the literature values [18], led to the identification of 5 as heterophylliin A (5, Figure 5).These spectroscopic data, which are reasonably identical with those previously published, confirmed the identity of 6 as gemin D (Figure 5) [19].

Cytotoxicity of Ellagitannins against Oral Cancer Cell Lines
The anticancer properties of L. inermis leaf, root, flower, and bark extracts have been thoroughly studied using animal models and cancer cell lines [33].Water extracts of L inermis leaves inhibited the growth of various cancer cell lines to varying degrees [34,35].
In the current study, four C-type glycosidic ellagitannins, which are typical of lythraceae plants, were among the isolated groups of phytochemicals that were detected in high concentrations in the aqueous acetone extract of henna leaves.Lately, it has become more common to investigate the applicability of ellagitannins to treat illnesses brought on

Cytotoxicity of Ellagitannins against Oral Cancer Cell Lines
The anticancer properties of L. inermis leaf, root, flower, and bark extracts have been thoroughly studied using animal models and cancer cell lines [33].Water extracts of L. inermis leaves inhibited the growth of various cancer cell lines to varying degrees [34,35].
In the current study, four C-type glycosidic ellagitannins, which are typical of lythraceae plants, were among the isolated groups of phytochemicals that were detected in high con-centrations in the aqueous acetone extract of henna leaves.Lately, it has become more common to investigate the applicability of ellagitannins to treat illnesses brought on by oxidative stress, such as cancer and neurodegenerative diseases [36].Ellagitannins from various plants have demonstrated prominent multi-mechanistic antitumor properties [37].The selective cytotoxicity against human oral squamous cell carcinoma (OSCC) vs. normal oral cells was examined for four ellagitannins: lythracin D (7), flosin B (9), lagerstroemin (10), of unknown cytotoxicity, and pedunculagin (8), a known antitumor agent [38][39][40].All four ellagitannins, as well as doxorubicin (DXR), were cytotoxic toward the OSCC cell lines, whereas 5-FU was cytostatic.Pedunculagin and lythracin D demonstrated significant tumor-specific cytotoxicity (TS = 2.8 and 2.3, respectively), while flosin B and lagerstroemin exhibited low specificity (TS = 1.7 and 1.5, respectively) (Table 2).The primary mechanism by which ellagitannins and their derivatives, including ellagic acid, exhibit anticancer activities is through their antioxidant capacity, which is dependent on both iron chelation activity and direct radical scavenging and varies with the degree of hydroxylation [41].According to the literature, ellagitannins' antitumor properties are primarily influenced by their antioxidant activity and their ability to reduce inflammation; research-based evidence suggested that they have the ability to regulate secretory growth factors and proinflammatory molecules like IL-6, TGF-β, TNF-α, IL-1β, and IFN-γ [42].Research has demonstrated that a number of oligomeric ellagitannins have in vivo anticancer effects against mouse models of sarcoma 180 and MM2, which was linked to a strengthened host immune response.In vitro research using tumor cell lines has shown that a number of ellagitannins, as well as their constituent acids gallic and ellagic, showed a strong cytotoxicity against carcinoma cell lines and a low cytotoxicity toward normal cells [20].Our findings in the present study are consistent with the previous reports on ellagitannin's cytotoxic activity [20,42] and highlight the potential of the development of L. inermis ellagitannins as anti-oral cancer drugs.Furthermore, the occurrence of C-type glycosidic ellagitannins in abundance is significant from a chemotaxonomic perspective as well as for explaining the multiple biological benefits of henna, such as its anti-inflammatory, antioxidant, and anticancer properties.
Lagerstroemin (10) Increased glucose uptake of rat adipocytes and could be responsible for lowering of blood glucose level, as shown by Lagerstroemia speciosa extract [54].

Conclusions
To further our search for the exploration of new phytomolecules from polyphenolicrich plants, we isolated ten compounds with various characteristics from L. inermis leaves.The novel megastigmane structure lawsoiononoside (1) is also reported here based on intensive spectroscopic data.Given the interesting bioactivities of megastigmanes [29][30][31], our discovery of 1 will spur an additional exploration of the unique megastigmane structures from L. inermis.The cytotoxicity of the ellagitannins lythracin D (7) and pedunculagin (8) against the OSCC cell lines indicates the potential development of anti-oral cancer therapeutics based on L. inermis.According to recent studies, ellagitannins' antioxidant, and anti-inflammatory properties, which include the ability to modulate proinflammatory mediators including IL-6, TGF-β, TNF-α, IL-1, and IFN-γ, are mostly involved in their anticancer effects [42].The tannins 7-10, albeit having a relatively moderate TS, may be an appropriate radiosensitizer to diminish tumor resistance in cancer radiotherapy, as recently shown to be the case for pentagalloyl glucose and a gallotannin-rich extract from Bouea macrophylla seed [55].The different identified chemicals discussed here also draw attention to the numerous biological benefits of henna and encourage additional clinical research in order to benefit from the antibacterial, anti-inflammatory, antioxidant, hepatoprotective, and anticancer properties of this promising ancient plant.

Figure 3 .
This biotransformation involves the oxidation of C-3 and C-11 of the megastigmane basic skeleton (Step A), followed by the glucosylation (Step B) and esterification of the glucose OH-6 with gallic acid (Step C).Direct glycosylation of the megastigmane with a 6-O-galloyl glucose is also possible.Antioxidants 2023, 12, x FOR PEER REVIEW 10 of 19 be as shown in Figure 3.This biotransformation involves the oxidation of C-3 and C-11 of the megastigmane basic skeleton (Step A), followed by the glucosylation (Step B) and esterification of the glucose OH-6 with gallic acid (Step C).Direct glycosylation of the megastigmane with a 6-O-galloyl glucose is also possible.

Table 2 .
Cytotoxicity of ellagitannins 7-10 against human OSCC cell lines and normal oral cells a .