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Article

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

by
Mohamed A. A. Orabi
1,*,
Esam A. Orabi
2,*,
Ahmed Abdullah Al Awadh
3,
Mohammed Merae Alshahrani
3,
Basel A. Abdel-Wahab
4,
Hiroshi Sakagami
5 and
Tsutomu Hatano
6
1
Department of Pharmacognosy, College of Pharmacy, Najran University, Najran 66454, Saudi Arabia
2
Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke Street West, Montréal, QC H4B 1R6, Canada
3
Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Najran University, Najran 66454, Saudi Arabia
4
Department of Pharmacology, College of Pharmacy, Najran University, Najran 64462, Saudi Arabia
5
Meikai University Research Institute of Odontology (M-RIO), 1-1 Keyakidai, Saitama 350-0283, Japan
6
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700-8530, Japan
*
Authors to whom correspondence should be addressed.
Antioxidants 2023, 12(11), 1951; https://doi.org/10.3390/antiox12111951
Submission received: 25 September 2023 / Revised: 29 October 2023 / Accepted: 30 October 2023 / Published: 1 November 2023
(This article belongs to the Special Issue Extraction and Characterization of Natural Bioactive Compounds)
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Versions Notes

Abstract

:
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.

1. 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].
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 tannin-rich fractions led to two glucopyranose-type ellagitannins (5 and 6) in addition to four known C-glycosidic ones (7–10). The cytotoxicity of the abundant ellagitannins (8–10) to various human oral cancer and normal cell lines was examined in the present study.

2. Materials and Methods

2.1. 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) spectrophotometers, respectively. The optical rotation was measured on a JASCO DIP-1000 (JASCO, Tokyo, Japan) digital polarimeter. High-resolution electrospray ionization mass (HRESIMS) spectra were acquired on a Micromass AutoSpec OA-TOF (Manchester, UK) spectrometer. Samples in H2O/MeOH (1:1, v/v) + 0.1% NH4OAc were infused into the ESI source at a flow rate of 20 μL/min. The NMR spectra were acquired on a Varian INOVA AS600 (Varian, Palo Alto, CA, USA) instrument (600 MHz for 1H and 151 MHz for 13C). Chemical shifts are given in δ (ppm) relative to that of the solvent signal [(CH3)2CO-d6 (δH 2.04; δC 29.8)] on the tetramethylsilane (TMS) scale.
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 H3PO4/0.01 M KH2PO4/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).

2.2. 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.

2.3. Extraction and Isolation

Dry L. inermis powdered leaves (200 g) were defatted by repeated steeping in n-hexane for 1.5 L × 3 consecutive days. The defatted marc was squeezed well and left to dry from the n-hexane, and then it was homogenized in 70% aq acetone (4 × 1.5 L). The solvent was dried off under vacuum, and the solute was coarsely fractionated on a Diaion HP-20 column (10 × 81 cm, i.d.) with H2O (3 L), 50% aq MeOH (4.5 L), 75% aq MeOH (3 L), MeOH (3 L), and 70% aq (CH3)2CO (2 L), successively. Each eluate was dried at 40 °C under vacuum to afford the dry fraction weights of 37.7 g, 17.24 g, 1.6 g, 0.43 g, and 0.066 g, respectively. The different eluates, except for the 50% aq MeOH eluate, revealed plant metabolites with tR < 4 min in the NP-HPLC chromatograms [14].
The MeOH fraction (0.43 g) from the Diaion column was dissolved in MeOH. The MeOH-soluble part was vacuum-dried (115 mg) and then dissolved in a 15% aq MeOH. The soluble part was chromatographed on an ODS column (2.2 × 40 cm, i.d.) with aq MeOH (15%, 25%, and 35%) and MeOH. The 25% and 35% aq MeOH eluates afforded benzoic acid (3, 10 mg) and ellagic acid (4, 14 mg), respectively.
The Diaion 75% aq MeOH fraction (1.6 g) was dissolved in H2O, and the H2O-soluble part was applied to an MCI-gel CHP-20P column (1.1 × 37 cm, i.d.) and eluted with H2O and H2O/MeOH (9:1, 8:2, 7:3, 6:4, 5:5, 0:10, v/v). The H2O/MeOH (6:4, v/v) eluate (210 mg) was further worked on an ODS column (2.2 × 40 cm, i.d.), and eluted with H2O and H2O/MeOH (9:1, 8:2, 7.5:2.5, and 5:5, v/v), collecting 700 drops/fraction. Fractions 58–65 (7.3 mg) from the H2O/MeOH (8:2, v/v) eluate were further purified using preparative RP-HPLC with [H2O/CH3CN (8:2, v/v) + 1% CH3COOH)] and yielded pure lawsoiononoside (1, 1.3 mg) and benzyl 6′-O-galloyl-β-D-glucopyranoside (2, 1.8 mg).
A part (10 g) of the diaion 50% aq MeOH fraction was further fractionated on a Toyopearl HW-40C column (2.2 × 72 cm) eluted with aq EtOH (50%, 60%, and 70%), 70% aq EtOH/70% aq CH3)2CO gradients (9:1, 8:2, 7:3, 6:4, and 5:5, v/v), and finally 70% aq CH3)2CO, successively. The Toyopearl factions (1000 drops/fraction) were analyzed via NP-HPLC and/or RPHPLC, and the fractions with similar chromatographic profiles were combined.
Toyopearl fractions T90–T105 (35 mg), eluted with 50% aq EtOH, were chromatographed on an MCI-gel CHP-20P column (1.1 × 37 cm) using H2O and then H2O/MeOH (9:1, 8.5:1.5, 8:2, 7.5:2.5, 7:3, 6.5:3.5, 5:5, and 0:10) as mobile phases. The early eluate with H2O/MeOH (8:2, v/v) afforded crude (40 mg) and pure gemin D (6, 3.5 mg). Toyopearl fractions T105–T124 (87 mg), eluted with 50% aq EtOH, were purified on an MCI-gel CHP-20P column (1.1 × 37 cm) with the same elution mode. The H2O/MeOH (8.5:1.5, v/v) eluate afforded pedunculagin (8, 22.9 mg), while the H2O/MeOH (6.5:3.5, v/v) eluate afforded heterophylliin A (5, 2.6 mg). Using the same MCI-gel CHP-20P column and eluants, the Toyopearl fractions T156–T183 (259 mg) afforded flosin B (9, 8.1 mg) in the early eluate with H2O/MeOH (7.5:2.5, v/v). The H2O-insoluble part of the Toyopearl fractions T240–T347 (347 mg) afforded lagerstroemin (10, 46.8 mg). The H2O-soluble part was then chromatographed using the same MCI-gel CHP-20P column (1.1 × 37 cm) with the same elution profile. The early eluate with H2O/MeOH (8.5:1.5, v/v) afforded lythracin D (8, 13.4 mg).

2.4. Spectroscopic Data of Isolated Compounds

  • Lawsoiononoside (1): Colorless gummy solid, [α]27D: −125.6 (c 1.0, MeOH); UV λmax (MeOH) nm (log ε): 221 (4.2), 240 (4.0), 277 (3.6); ECD (MeOH) [θ] (nm): +2.0 × 103 (222), −2.1 × 103 (332), +0.3 × 103 (376); NMR data; HRESIMS m/z 563.2072 [M + Na]+ (calcd for C26H36O12Na, 563.2099) and m/z 539.2128 [M − H]− (calcd for C26H35O12, 539.2134).
  • Benzoic acid (2): Colorless crystalline solid, 1H NMR (600 MHz, DMSO-d6) δH 12.9 (br.s, 1H, COOH), 7.95 (dd, J = 1.8, 7.2 Hz, 2H, H-2/H-6), 7.62 (tt, J = 1.8, 7.2 Hz, 1H, H-4), and 7.50 (dd, J = 7.2, 7.2 Hz, 2H, H-3/H-5); 13C NMR (151 MHz, DMSO-d6) δC 167.23 (C-7), 132.89 (C-4), 130.7 (C-1), 129.2 (C-2/C-6), 128.5 (C-3/C-5) [15].
  • Benzyl 6′-O-galloyl-β-D-glucopyranoside (4): White amorphous powder, 1H NMR (600 MHz, Me2CO-d6: D2O; 9:1): δH 7.33 (2H, dd, J = 1.2, 7.8 Hz, H-2/H-6), 7.26 (2H, dt, J = 1.2, 7.8 Hz, H-3/H-5), 7.20 (1H, dt, J = 1.2, 7.8 Hz, H-4), 7.13 (2H, s, gal H-2″/H-6″), 4.79, 4.59 (each 1H, d, J = 12 Hz, H-7), 4.55 [1H, dd, J = 1.8, 12 Hz, glc H-6′], 4.39 (1H, d, J = 8.4 Hz, glc H-1′), 4.34 (1H, dd, J = 6.6, 12.6 Hz, glc H-6′), 3.29 (1H, dd, J = 8, 9 Hz, glc H-2′), 3.44 (1H, t, J = 9 Hz, glc H-3′), 3.44 (1H, t, J = 9 Hz, glc H-4′), 3.57 (1H, ddd, J = 1.8, 6, 9 Hz, glc H-5′); 13C NMR (151 MHz, Me2CO-d6: D2O; 9:1) δC: 167.2 (gal C-7″), 146.0 (2C, gal C-3″/C-5″), 138.6 (gal C-4″), 137.7 (C-1), 128.9 (2C, C-3/C-5), 128.8 (2C, C-2/C-6), 128.2 (C-4), 121.0 (gal C-1″), 109.7 (2C, gal C-2″/C-6″), 102.7 (glc C-1″), 77.5 (glc C-3′), 74.8 (glc C-5′), 74.5 (glc C-2′), 71.2 (glc C-4′), 70.9 (C-7′), 64.5 (glc C-6′); ESIMS m/z 421 [M − H]− [16].
  • Ellagic acid (4): Pale-yellow amorphous powder, 1H NMR (600 MHz, DMSO-d6) δH 10.69 (br. s, OH), 7.50 (s, 2H, H-2/H-2′); 13C NMR (151MHz, DMSO-d6) δC: 159.5 (2C, C-7, C-7′), 148.5 (2C, C-4/C-4′), 140.00 (2C, C-2/C-2′), 136.8 (2C, C-3/C-3′), 112.7 (2C, C-6/C-6′), 110.6 (2C, C-5/C-5′), 108.0 (2C, C-1/C-1′); ESIMS m/z 301 [M − H]− [17].
  • Heterophylliin A (5): Off-white amorphous powder; 1H NMR (600 MHz, (CH3)2CO-d6: D2O; 9:1) δH 7.23, 7.03 (each 2H, s, gal H-2/H-6), 6.61, 6.48 (each 1H, s, HHDP H-3, H-3′), 6.39 (1H, d, J = 4.2 Hz, glc H-1), 5.64 (1H, t, J = 10.2 Hz, glc H-3), 5.22 (1H, dd, J = 13.2, 6.6 Hz, glc H-6), 5.05 (1H, t, J = 10.2 Hz, glc H-4), 4.55 (1H, br. dd, J = 6.6, 10.2 Hz, glc H-5), 4.20 (1H, dd, J = 4.2, 10.2 Hz, glc H-2), Hz, 3.75 (1H, br. d, J = 13.2 Hz, glc H-6) [18].
  • Gemin D (6): Off-white amorphous powder, 1H NMR (600 MHz, (CH3)2CO-d6: D2O; 9:1) (α- and β-anomers) δH: 7.01, 7.00 (each s, 2H in total, gal H-2/H-6), 6.59, 6.58 (each s, 1H in total, HHDP H-3), 6.44, 6.43 (each s, 1H in total, HHDP H-3ʹ), 5.46, 5.28 (1H in total, each t, J = 10.2 Hz, glc H-3α, β), 5.24 (1/2H, d, J = 4.2 Hz, glc H-1α), 5.21, 5.18 (1H in total, each dd, J = 6.6, 10.2 Hz, H-6α, β), 4.95, 4.92 (1H in total, each t, J = 10.2 Hz, H-4α, β), 4.72 (1/2H, d, J = 7.2 Hz, H-1β), 4.52, 4.06 (1H in total, each ddd, J = 1.2, 6.6, 10.2 Hz, H-5α, β), 3.81 (1/2H, dd, J = 4.2, 10.2 Hz, H-2α), 3.78, 3.71 (1H in total, each dd, J = 1.2, 13.2 Hz, H-6α, β), 3.57 (1/2H, dd, J = 7.2, 10.2 Hz, H-2β) [19].
  • Lythracin D (7): Off-white amorphous powder, 1H NMR (600 MHz, (CH3)2CO-d6: D2O; 9:1) δH: 7.08, 6.57, 6.60 (each 1H, s, valoneoyl-H), 6.73, (1H, s, flavogallonyl-H), 6.91, 6.61, 6.54 (each 1H, s, HHDP-H); 4.90 (1H, d, J = 1.8 Hz, glc-1 H-1), 4.81 (1H, t, J = 1.8 Hz, glc-1 H-2), 5.08 (1H, t, J = 1.8 Hz, glc-1 H-3), 5.62 (1H, dd, J = 1.8, 8.4 Hz, glc-1 H-4), 5.38 (1H, ddd, J = 1.2, 4.8, 9.6 Hz, glc-1 H-5), 4.92 (1H, dd, J = 2.4, 12.6 Hz, glc H-6), 3.73 (1H, d, J = 12.6 Hz, glc-1 H-6), 4.75 (1H, d, J = 1.8 Hz, glc-2 H-1), 5.01 (1H, t, J = 1.8 Hz, glc H-2), 4.50 (1H, dd, J = 1.2, 6.6 Hz, glc-2 H-3), 5.12 (1H, t, J = 7.2 Hz, glc-2 H-4), 5.55 (1H, dt, J = 1.8, 7.2 Hz, glc-2 H-5), 4.93 (1H, dd, J = 2.4, 12.6 Hz, glc-2 H-6), 3.89 (1H, d, J = 12.6 Hz, glc-2 H-6) [13].
  • Pedunculagin (8): Off-white amorphous powder, 1H NMR [600 MHz, (CH3)2CO-d6: D2O; 9:1] (α and β-anomer mixture) δH: 6.64, 6.63, 6,60, 6.59, 6.54, 6.50, 6.323, 6.321 (each, s, 4H in total, HHDP-H), 5.43 (1/H, t, J = 9.6 Hz, glc H-3α), 5.41 (1/2H, d, J = 3.6 Hz, glc H-1α), 5.24, 5.20 (1H in total, each dd, J = 6.6, 12.6 Hz, glc H-6 α, β), 5.19 (1/2H, t, J = 9.6 Hz, glc H-3β), 5.033, 5.027 (1H in total, each t, J = 9.6Hz, glc H-4 α, β), 5.01 (1/2H, d, J = 7.8 Hz, glc H-1β), 4.82 (1/2H, dd, J = 7.8, 9.6 Hz, glc H-2β), 4.57, 4.18 (1H in total, each ddd, J = 1.8, 6.6, 9.6 Hz, glc H-5α, β), 3.83, 3.76 (each 1H, dd, J = 1.2, 12.6 Hz, glc H-6α, β] [13].
  • Flosin B (9): Off-white amorphous powder, 1H NMR [600 MHz, (CH3)2CO-d6: D2O; 9:1] δH: 7.57, 7.16, 7.12 (each 1H, s, valoneoyl dilactone-H), 6.84, 6.45, 6.35 (each 1H, s, HHDP-H), 5.51 (1H, dd, J = 2.4, 8.4 Hz, glc H-4), 5.18 (1H, br.dd, J = 4.8, 9.6 Hz, glc H-5), 4.98 (1H, t, J = 2.4 Hz, glc H-3), 4.97 (1H, d, J = 2.4 Hz, glc H-1), 4.78 (1H, t, J = 1.8 Hz, glc H-2), 4.70 (1H, dd, J = 3.6, 13.2 Hz, glc H-6), 3.71 (1H, d, J = 12.6 Hz, glc H-6) [13].
  • Lagerstroemin (10): Off-white amorphous powder, 1H NMR [600 MHz, (CH3)2CO-d6: D2O; 9:1] δH: 7.57, 7.16, 7.13 (each 1H, s, valoneoyl dilactone-H), 6.76, 6.53, 6.36 (each 1H, s, HHDP-H), 5.57 (1H, d, J = 4.8 Hz, glc H-1), 5.38 (1H, t, J = 1.8 Hz, glc H-3), 5.36 (1H, dd, J = 2.4, 9 Hz, glc H-5), 5.19 (1H, dd, J = 3.6, 9 Hz, glc H-4), 4.64 (1H, dd, J = 3, 12.6, glc H-6), 4.61 (1H, dd, J = 1.8, 4.8, glc H-2), 3.72 (1H, d, J = 12.6, glc H-6) [13].

2.5. Cytotoxicity

2.5.1. Cell Culture

Four oral squamous cell carcinoma (OSCC) cell lines (Ca9-22 derived from gingiva; HSC-2, HSC-3, and HSC-4 derived from tongue) were purchased from RIKEN Cell Bank, Tsukuba, Japan. Three human oral mesenchymal cell types, [gingival fibroblast (HGF), periodontal ligament fibroblast (HPLF), and pulp cell (HPC)], at 10–18 population doubling level were cultured at 37 °C in DMEM supplemented with 10% heat-inactivated FBS (56 °C, 30 min), 100 U/mL penicillin G, and 100 µg/mL streptomycin sulfate under a humidified 5% CO2 atmosphere [20].
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.

2.5.2. Cytotoxicity Assay

Cells were detached from the culture dishes using 0.25% trypsin, inoculated at 2×103 cells per 100 μL in a 96-microwell plate and incubated for 48 h to ensure complete cell attachment to the well. The culture medium was replaced with 100 μL of fresh medium containing different concentrations of the test compounds (7 (0.9, 1.7, 3, 6.7, 13, 27, 54, 107 μM); 8 (2, 4, 8, 16, 32, 64, 128, 255 μM); 9 (1.3, 2.5, 5.10, 20, 40, 81, 162 μM); 10 (1.3, 2.5, 5.10, 20, 40, 81, 162 μM); DXR (0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 μM); 5-FU (8, 16, 31, 63, 125, 250, 500, 1000 μM); and DMSO vehicle (0.008, 0.016, 0.031, 0.063, 0.125.0.25, 0.5, 1%)). Cells were further incubated for 48 h, and the number of viable cells relative to the control was determined via the MTT assay, which is based on MTT metabolism to formazan by viable cells, as described previously [20]. In brief, cells were incubated in the microplate for 2 h with 0.2 mg/mL MTT, the purple formazan precipitate was solubilized by adding 100 μL DMSO, OD560 was recorded on a microplate reader (Infinite F50R; TECAN, Männedorf, Switzerland), and the readings were adjusted for cytotoxicity caused by the vehicle. The concentration of test compound that produced cytotoxicity by 50% (CC50) was determined in triplicate from the dose–response curves.
CC50 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 CC50 values for the normal and tumor cell lines were calculated and the ratio of the means gives the tumor specificity (TS):
TS = mean CC50 normal cell lines/mean CC50 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.

3. Results and Discussions

3.1. Structure Determination of the Isolated Compounds

A 70% aq acetone extract of L. inermis leaves was fractionated using a Diaion HP-20 gel (2.3. Extraction and Isolation). The multistep isolation using gel chromatography combined with preparative HPLC purification afforded a new megastigmane glucoside gallate (1, Figure 2), along with the known benzyl 6′-O-galloyl-β-D-glucopyranoside (2) from the Diaion 75% aq MeOH fraction, and benzoic acid (3) and ellagic acid (4) from the Diaion MeOH fraction. In addition, two O-glycosidic ellagitannins, heterophylliin A (5) and gemin D (6), and four C-glycosidic ellagitannins, lythracin D (7), pedunculagin (8), flosin B (9), and lagerstroemin (10) were purified from the Diaion 50% aq MeOH eluate.

3.1.1. Structure of the New Megastigmane (1)

Compound 1 was isolated as a colorless gummy solid, and its molecular formula, C26H36O12, was established from the HRESIMS molecular ion peak at m/z 563.2072 [M + Na]+ (calculated for C26H36O12Na, 563.2099) and m/z 539.2128 [M − H]− (calculated for C26H35O12, 539.2134), as well as the 13C NMR data (Table 1). The 1H NMR spectrum of 1 (Figures S3–S5) displayed spectroscopic features typical of a galloyl moiety [22]; an aromatic 2H singlet (δH 7.13, H-2″/H-6″) that exhibits HSQC correlation with a non-oxygenated aromatic carbon (δC 109.6, 2C, C-2″/C-6″) and HMBC correlations with oxygenated aromatic carbon (δC 146, 2C, C-3″/C-5″, and δC 137.7, C-4″), a quaternary aromatic carbon (δC 120.5, C-1″), and a carbonyl carbon (δC 167.2, C-7″). The 1H NMR and 1H–1H COSY spectra of 1 (Table 1, Figures S6–S8) exhibited a resonating system of seven aliphatic proton spins at δH 4.49–3.19 (Table 1), with a large coupling pattern of the glucose H-2′–H-5′ (JH-1′–H-2′ = 7.8, JH-2′–H-3′ = JH-3′–H-4 = JH-4′–H-5′ = 9.6 Hz), highlighting the 4C1 conformation of the glucopyranose moiety [23]. A large coupling constant of the glucose H-1′ proton signal (δH 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 13C chemical shifts (δC 102.2, 74.5, 77.5, 71.3, 74.8, 64.8) to the glucose carbons C-1′–C-6′, respectively (Table 1). The 13C NMR spectrum of 1 (Figure S9) also exhibited a system of thirteen carbons (Table 1), characteristic of the megastigmane moiety [24]. Aided by the 1H–1H 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 δC 72.3 (C-3) (correlated in the HSQC spectrum to a 1H multiplet signal at δH 4.14 (H-3)), two non-oxygenated methylene carbons at δC 41.6 (C-2) (correlated in the HSQC spectrum to the H2-2 proton signals at δH 1.21 (1H, t, J = 8.4 Hz, H-2ax) and 2.25 (1H, ddd, J = 2.4, 5.4, 14.4 Hz, H-2eq) and at δC 39.2 (C-4) (correlated in the HSQC spectrum to the H2-4 proton signals at δH 1.94 (1H, br.dd, J = 9.6, 16.2 Hz, H-4ax) and δH 2.30 (1H, dd, J = 5.4, 16.2 Hz, H-4eq)), one oxygenated methylene carbon at δC 68.6 (C-12) (correlated in the HSQC spectrum to δH 3.39 and 3.28 (each 1H, d, J = 11 Hz)), two tetrasubstituted olefinic carbons at δC 128.8 (C-5) and 136.6 (C-6), and a quaternary carbon at δC 43.5 (C-1). The remaining two methyl carbons of this component appear at δC 24.3 and 19.8, and one of them is correlated in the HSQC with a 3H-singlet at an up-field shift of 0.91 (H3-11), and the other is correlated with the proton signal at a relatively low-field shift δH 1.54 (3H, brs, H3-11). The 1H chemical shift of the latter corresponds to the methyl group on an olefenic carbon, and the broadening of the signal is explained by the W-shaped 1H–1H coupling with the H-4 proton signal (δH 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 (1H–1H COSY, Figures S6–S8) and HSQC (Figures S10–S12) spectra substantiated the presence of two methylene groups (δH 2.14 and 2.2 (2H, dd, H2-7), correlated with a carbon signal at δC 22.3 (C-7) and δH 2.48 (2H, t, H2-8), correlated with a carbon signal at δC 44.2 (C-8)). The other two carbons were identified as methyl and ketone carbons: the methyl proton signal at δH 2.08 (3H, s, H3-10) exhibits an HSQC correlation with the carbon peak at δC 29.8 (C-10) and an HMBC correlation with a ketonic carbonyl carbon peak at δC 209.3. The same carbonyl carbon is also correlated with the H2-8 signal at δH 2.48 in the HMBC spectrum. This four-carbon segment, proposed as –CH2–CH2–CO–CH3, was placed at the olefenic carbon C-6 (δC 133.6), as deduced from the HMBC correlations of the H-7 signal at δH 2.14 with C-6 (δC 133.6) and the neighboring carbons C-1 (δC 43.5) and C-5 (δC 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 (δC 64.8) in addition to a weak HMBC correlation of glucose-H-6 (δH 4.16) with the galloyl carbonyl carbon peak (δC 167.2). The down-field shift of the glucose anomeric carbon (δC 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′ (δH 4.49) and the megastigmane moiety C-3 (δC 72.8).
The 1H NMR spectrum showed the H-3 signal at δH 4.14 as a multiplet in the 1H NMR spectrum, where the coupling constants of J2ax-3, J2eq-3, J4ax-3, and J4eq-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 1H-1H nuclear Overhauser effect spectroscopy (NOESY) correlations between the axially oriented glucose H-1′ proton (δH 4.49) and both H-3 (δH 4.14) and H-4eq (δH 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 H2-11 and NOESY correlations of both H-2ax (δH 1.21, t) and H-4ax (δH 1.94) with H3-12 (δH 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 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 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.
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.

3.1.2. Structure of Known Compounds (2–10)

Structure of Compounds 2–4

  • Benzoic acid (3), together with the tannin-related metabolite benzyl 6′-O-galloyl-β-D-glucopyranoside (2), and ellagic acid (4) are isolated for first time from henna, and their structures (Figure 4) were determined from the NMR and ESIMS data referenced to the literature values as follows:
  • Compound 2 was isolated as a colorless crystalline solid. Its structure was identified from the 1H NMR pattern of a monosubstituted benzene [δH 7.95 (2H, dd, J = 1.8, 7.2 Hz, H-2/H-6), 7.62 (1H, tt, J = 1.8, 7.2 Hz, H-4), and 7.50 (2H, dd, J = 7.2, 7.2 Hz, H-3/H-5)] (Figure S18) and the 13C NMR data δC 132.89 (C-4), 130.7 (C-1), 129.2 (C-2/C-6), and 128.5 (C-3/C-5) (Figure S20), which are consistent with a monosubstituted benzene as well. The presence of a carboxyl group was recognized by a broad proton signal in the low-field region (δH 12.9) and a carbonyl carbon peak at δC 167.23 (C-7). The ESIMS molecular ion peak at m/z 121 [M–1]– confirmed the identification of 2 as a benzoic acid [15].
  • Compound 3 was isolated as a white amorphous powder. Its 1H NMR spectrum (Figure S22) exhibited proton signals of a 1C4 β-D-glucopyranose core {δH 4.55 [1H, dd, J = 1.8, 12 Hz, glc H-6], 4.39 (1H, d, J = 8.4 Hz, glc H-1), 4.34 (1H, dd, J = 6.6, 12.6 Hz, glc H-6), 3.29 (1H, dd, J = 8, 9 Hz, glc H-2), 3.44 (1H, t, J = 9 Hz, glc H-3), 3.44 (1H, t, J = 9 Hz, glc H-4), and 3.57 (1H, ddd, J = 1.8, 6, 9 Hz, glc H-5)} [12,13,22]. The spectrum also exhibited a 2H singlet at δH 7.13 (2H, s, galloyl H-2/H-6), which exhibited a HSQC correlation with the 2C peak at δC 109.7 (2C, galloyl C-2/C-6). These are characteristic of a galloyl moiety [12,13,22]. The spectrum also exhibited a proton signal of a benzyl moiety at δH 7.33 (2H, dd, J = 1.2, 7.8 Hz, H-2/H-6), 7.26 (2H, dt, J = 1.2, 7.8 Hz, H-3/H-5), 7.20 (1H, dt, J = 1.2, 7.8 Hz, H-4), and 4.79, 4.59 (each 1H, d, J = 12 Hz, H-7). The 13C NMR spectrum (Figure S24) exhibited carbon peaks corresponding to the structural components of 2 (viz, benzyl, galloyl, and glucose). The HMBC correlations between the galloyl 2H singlet (δH 7.13) and the glucose H2-6 (δH 4.55 and 4.34) with the galloyl carbonyl carbon (δC 167.2) evidenced the placement of the galloyl moiety at C-6 of the glucose core. The HMBC correlations of the benzyl H2-7 at δH 4.79 and 4.59 (each 1H, d, J = 12 Hz) with the glucose anomeric carbon (δC 102.7), as well as the HMBC correlation of the glucose anomeric proton (δH 4.39) with the benzylic carbon C-7 (δC 70.9), indicated the placement of the benzyl moiety at the anomeric center of the glucose, as shown by the structural formula of 2 (Figure 4). The ESIMS negative ion peak at m/z 421 [M − H]− confirmed the identification of 2 as benzyl 6′-O-galloyl-β-D-glucopyranoside [16]. Compound 4 was isolated as a pale yellow amorphous powder. Its 1H NMR spectrum (Figure S27) exhibited a 2H singlet signal at δH 7.50 (H-2/H-2′) and a broad singlet at δH 10.69 (br. s, OH). The 13C NMR spectrum exhibited seven carbon peaks, each equivalent to two carbons [δC 159.5 (2C, C-7/C-7′), 148.5 (2C, C-4/C-4′), 140.0 (2C, C-2/C-2′), 136.8 (2C, C-3/C-3′), 112.7 (2C, C-6/C-6′), 110.6 (2C, C-5/C-5′), and 108.0 (2C, C-1/C-1′). 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 (δH 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 1H NMR spectrum (Figure S29) exhibited aromatic proton signals at δH 7.23, 7.03 (each 2H, s, galloyl H-2/H-6) of two galloyl units and two 1H singlets (δH 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 1H–1H COSY correlations (Figure S30), were assigned for the 4C1 D-glucopyranose core as follows: δH 6.39 (1H, d, J = 4.2 Hz, glc H-1), 5.64 (1H, t, J = 10.2 Hz, glc H-3), 5.22 (1H, dd, J = 13.2, 6.6 Hz, glc H-6), 5.05 (1H, t, J = 10.2 Hz, glc H-4), 4.55 (1H, br. dd, J = 6.6, 10.2 Hz, glc H-5), 4.20 (1H, dd, J = 4.2, 10.2 Hz, glc H-2), and Hz, 3.75 (1H, br. d, J = 13.2 Hz, glc H-6). The up-field shift of the glucose H-2 signals (δH 4.20) indicated the deacylation of the glucose 2-OH, whereas the small coupling constant (J = 4.2 Hz) of the anomeric proton signal indicated the alpha-oriented C-O bond at the glucose’s anomeric center [14,18]. The large difference in the chemical shifts of the glucose H2-6 signals (δH 4.20 and 3.75) indicated the placement of the HHDP group at O-4/O-6 of the glucose, and therefore, the remaining O-1 and O-3 should be acylated by the galloyl units [14]. These data, together with the comparison with the literature values [18], led to the identification of 5 as heterophylliin A (5, Figure 5).
  • Compound 6 was isolated as an off-white amorphous powder. Its 1H NMR spectrum exhibited double resonances of all proton signals (Section 2.4. Spectroscopic Data of Isolated Compounds), which indicated the existence of 6 as a mixture of α- and β-anomer. The aromatic proton signals [δH 7.01, 7.00 (each s, 2H in total), δH 6.59, 6.58 (each s, 1H in total), and δH 6.44, 6.43 (each s, 1H in total) are consistent with the galloyl H-2/H-6, HHDP H-3, and HHDP H-3ʹ, respectively [22]. Proton signals of a glucose core [δH: 5.46, 5.28 (1H in total, each t, J = 10.2 Hz, glc H-3α, β), 5.24 (1/2H, d, J = 4.2 Hz, glc H-1α), 5.21, 5.18 (1H in total, each dd, J = 6.6, 10.2 Hz,), 4.95, 4.92 (1H in total, each t, J = 10.2 Hz, H-4α, β), 4.72 (1/2H, d, J = 7.2 Hz, H-1β), 4.52, 4.06 (1H in total, each ddd, J = 1.2, 6.6, 10.2 Hz, H-5α, β), 3.81 (1/2H, dd, J = 4.2, 10.2 Hz, H-2α), 3.78, 3.71 (1H in total, each dd, J = 1.2, 13.2 Hz, H-6α, β), and 3.57 (1/2H, dd, J = 7.2, 10.2 Hz, H-2β) were detected in the NMR spectrum of 6. These double resonances indicate the presence of the unacylated OH group of the anomeric center [14]. Resonances of the glucose H-2 signals with the up-field shifts (δH 3.81 and 3.57) also evidenced the existence of the free OH-2 on the glucose core. Similar to heterophylliin A (5), the wide difference in the chemical shifts of the glucose H2-6 signals (δH 5.21, 5.18, H-6α, β, and δH 3.78, 3.71, H-6α, β) indicated the bridging of the HHDP moiety at O-4/O-6 of the glucose core, leaving the glucose O-3 for the galloyl moiety. These spectroscopic data, which are reasonably identical with those previously published, confirmed the identity of 6 as gemin D (Figure 5) [19].

Structure of C-Glycosidic Ellagitannins (7–10)

The C-glycosidic ellagitannins 7–10 (Figure 6) are known compounds which were previously isolated from the same plant resource. Their structures were identified from NMR data (Section 2.4. Spectroscopic Data of Isolated Compounds and Figures S33–S40) and compared with the published NMR data as lythracin D (7), pedunculagin (8), flosin B (9), and lagerstroemin (10) [13].

3.2. 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 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.

3.3. Previously Reported Bioactivities of the Known Compounds 2–10

L. inermis is a medicinal plant that is popular in Unani medicine and ancient Egyptian medical papyri, with promising biological attributes: strong fungicidal, antibacterial, virucidal, antiparasitic, antiamoebiasis, astringent, and antihemorrhagic [4,5]. In addition, henna’s plant extracts were found to be effective sedative, hypotensive, anti-Alzheimer’s, antioxidant, hepatoprotective, immunomodulatory, and anticancer agents [3,6,7,8,10,43]. In this study, we report on the isolation and structural identification of a megastigmane (1) as well as several ellagitannins and tannin-related phenolics. Reviewing biological studies conducted on these compounds (Table 3) emphasized the above-mentioned biological qualities of henna.

4. Conclusions

To further our search for the exploration of new phytomolecules from polyphenolic-rich 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.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antiox12111951/s1, Figure S1: Positive-mode HRESIMS spectrum of compound 1; Figure S2: Negative-mode HRESIMS spectrum of compound 1; Figure S3: 1H NMR spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S4: Expanded 1H NMR spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S5: Expanded 1H NMR spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S6: 1H-1H COSY spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S7: Expanded 1H-1H COSY spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S8: Expanded 1H-1H COSY spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S9: 13C NMR spectrum of compound 1 [151 MHz, acetone-d6 + D2O (9+1)]; Figure S10: HSQC spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S11: Expanded HSQC spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S12: Expanded HSQC spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S13: HMBC spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S14: Expanded HMBC spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S15: NOESY spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S16: Expanded NOESY spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S17: Expanded NOESY spectrum of compound 1 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S18: 1H NMR spectrum of compound 2 (600 MHz, DMSO-d6); Figure S19: 1H-1H COSY spectrum of compound 2 (600 MHz, DMSO-d6); Figure S20: 13C NMR spectrum of compound 2 (151 MHz, DMSO-d6); Figure S21: HSQC spectrum of compound 2 (600 MHz, DMSO-d6); Figure S22: 1H NMR spectrum of compound 3 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S23: 1H-1H COSY spectrum of compound 3 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S24: 13C spectrum of compound 3 [151 MHz, acetone-d6 + D2O (9+1)]; Figure S25: HSQC spectrum of compound 3 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S26: HMBC spectrum of compound 3 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S27: 1H NMR spectrum of compound 4 (600 MHz, DMSO-d6); Figure S28: 13C NMR spectrum of compound 4 (151 MHz, DMSO-d6); Figure S29: 1H NMR spectrum of compound 5 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S30: 1H-1H COSY spectrum of compound 5 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S31: 1H NMR spectrum of compound 6 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S32: 1H-1H COSY spectrum of compound 6 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S33: 1H NMR spectrum of compound 7 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S34: 1H-1H COSY spectrum of compound 7 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S35: 1H NMR spectrum of compound 8 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S36: 1H-1H COSY spectrum of compound 8 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S37: 1H NMR spectrum of compound 9 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S38: 1H-1H COSY spectrum of compound 9 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S39: 1H NMR spectrum of compound 10 [600 MHz, acetone-d6 + D2O (9+1)]; Figure S40: 1H-1H COSY spectrum of compound 10 [600 MHz, acetone-d6 + D2O (9+1)]; Experimental procedures of TD-DFT calculation of the ECD spectrum of 1; Figure S41: Three-dimensional structures of possible stereoisomers of compound 1; Figure S42: Calculated (A) and practically measured (B) ECD spectra of compound 1 [56,57,58,59].

Author Contributions

Conceptualization, M.A.A.O. and T.H.; methodology, H.S. and T.H.; software, E.A.O.; validation, M.A.A.O., H.S. and T.H.; formal analysis, M.A.A.O., H.S. and E.A.O.; investigation, M.A.A.O. and H.S.; resources, A.A.A.A. and M.M.A.; data curation, M.A.A.O. and E.A.O.; writing—original draft preparation, M.A.A.O., E.A.O., A.A.A.A., M.M.A., H.S., B.A.A.-W. and T.H.; writing—review and editing, M.A.A.O., H.S. and T.H.; visualization, T.H.; supervision, T.H.; project administration, M.A.A.O. and B.A.A.-W.; funding acquisition, B.A.A.-W. and M.A.A.O. All authors have read and agreed to the published version of the manuscript.

Funding

The authors are thankful to the Deanship of Scientific Research at Najran University for funding this work under the Distinguished Research Program (grant code NU/DRP/MRC/12/3).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

C:\Users\ASUS\Downloads\NMRSpectroscopic data of the reported compounds are included in the Supplementary Material File and available from the corresponding author upon request.

Acknowledgments

The authors are thankful to the Deanship of Scientific Research at Najran University for funding this work under the Distinguished Research Program (grant code NU/DRP/MRC/12/3).

Conflicts of Interest

The authors declare no conflict of interest.

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Antioxidants 12 01951 g001
Figure 1. Summary of major bioactivities of henna metabolites.
Figure 1. Summary of major bioactivities of henna metabolites.
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Figure 2. Structure of new megastigmane (1). (A) Key HMBC correlations. (B) Important NOESY correlations.
Figure 2. Structure of new megastigmane (1). (A) Key HMBC correlations. (B) Important NOESY correlations.
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Figure 3. Suggested biosynthesis of compound 1 from β-ionone and gallic acid.
Figure 3. Suggested biosynthesis of compound 1 from β-ionone and gallic acid.
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Figure 4. Structures of the compounds 2–4, first isolated from henna.
Figure 4. Structures of the compounds 2–4, first isolated from henna.
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Figure 5. Structure of the ellagitannins 5 and 6.
Figure 5. Structure of the ellagitannins 5 and 6.
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Figure 6. Structures of the known ellagitannins 7–10.
Figure 6. Structures of the known ellagitannins 7–10.
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Table 1. The 1D (1H, 13C NMR) and 2D (HMBC and NOESY) spectroscopic data of compound 1.
Table 1. The 1D (1H, 13C NMR) and 2D (HMBC and NOESY) spectroscopic data of compound 1.
PositionδHδCHMBCNOESY
1 43.5
2ax1.21, 1H, t (12.6)41.6C-2, C-3, C-4, C-11, C-12H-11, H-2ex
2eq2.25, 1H, br.dd (8.4, 14.4)C-1H-2aq
34.14, 1H, m72.8 H-1′, H-4eq, H-12
4ax1.94, 1H, br.dd (9.6, 16.8)39.2C-3, C-5, C-6H-11, H-4eq
4eq2.30, 1H, br. dd (5.4, 16.8)C-2, C-5, C-6H-4ax
5 128.8
6 133.6
72.14, 1H, dd (8.4, 14.4) a,
2.20, 1H, dd (8.4, 14.4) a		22.3C-5, C-6, C-8, C-9H-11, H-13
82.48, 2H, t (8.4)44.2C-7, C-9H-11, H-13
9 209.3
102.08, 3H, s29.8 bC-8, C-9H-7
11
11		3.39, 1H, d (11),
3.28, 1H, d (11)		68.6C-1, C-2, C-11H-3
120.91, 3H, s24.3C-1, C-2, C-6, C-12H-2ax, H-4ax, H-7, H-8
131.54, 3H, br.s19.8C-4, C-5, C-6H-7, H-8
1′4.49, 1H, d (7.8)102.2C-3H-3, H-4eq
2′3.19, 1H, dd (7.8, 9.6)74.5C-1′, C-3′
3′3.44,1H, t (9.6)77.5C-2′, C-4′
4′3.35,1H, t (9.6)71.3C-3′, C-5′, C-6′
5′3.61, 1H, ddd (1.8, 9.6,74.8C-3′, C-4′
6′4.61,1H, dd (1.8, 13.8)64.8C-5′, C-7″
4.16, 1H, dd (7.2, 12.0)
1″ 120.5
2″/6″7.13, 2H, s109.6
3″/5″ 146
4″ 137.7
7″ 167.2
a Approximate multiplicity because proton signal overlapped with solvent impurities; b Hidden under the solvent peaks and identified from HSQC correlation with δH 2.08 (H3-10), which together with H2-8 (δH 2.48, 2H, t) exhibited HMBC correlations with C-9 (δC 209.3).
Table 2. Cytotoxicity of ellagitannins 7–10 against human OSCC cell lines and normal oral cells a.
Table 2. Cytotoxicity of ellagitannins 7–10 against human OSCC cell lines and normal oral cells a.
CC50 (μM) bCC50 (μM) b
Ca9-22HSC-2HSC-4MeanHGFHPLFHPCMean TS c
Lythracin D (7)89 ± 467 ± 848 ± 1068 157 ± 36 152 ± 5 152 ± 4 154 2.3
Pedunculagin (8)93 ± 548 ± 448 ± 363 178 ± 11187 ± 4157 ± 7174 2.8
Flosin B (9)180 ± 18 66 ± 880 ± 4109 200 174 ± 22169 ± 14181 1.7
Lagerstroemin (10)185 ± 1475 ± 1187 ± 2115 200 159 ± 9155 ± 5171 1.5
Doxorubicin0.5 ± 0.050.16 ± 0.040.17 ± 0.010.29 10 ± 010 ± 010 ± 010>35
5-FU33 ± 1210 ± 235 ± 626 1000 ± 01000 ± 01000 ± 01000>38.0
a Ca9-22, HSC-2, and HSC-4 are oral squamous cell carcinoma (OSCC) cell lines. HGF, HPLF, and HPC are the normal oral cells. b Results are expressed as the mean ± SD of three independent experiments. c TS = [CC50 (HGF) + CC50 (HPLF) + CC50 (HPC)]/[CC50 (Ca9-22) + CC50 (HSC-2) + CC50 (HSC-4)].
Table 3. Reported pharmacological activities of isolated compounds 2–10.
Table 3. Reported pharmacological activities of isolated compounds 2–10.
CompoundReported Biological Activity
benzyl-6′-O-galloyl-β-D-glucopyranoside (2)Antifungal activity against C. albicans clinical isolates and reference strains [44].
Moderate inhibitory effects against lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells [45].
Benzoic acid (3)Antibacterial and antifungal activity [46].
Ellagic acid (4)Antioxidant, anti-inflammatory, antimutagenic, antiproliferative, antiallergic, antiatherosclerotic, cardioprotective, hepatoprotective, nephroprotective, and neuroprotective properties [47].
Heterophylliin A (5)Antidiabetic (moderate inhibitory effect against dipeptidyl peptidase IV and α-glucosidase) activity [48]. Antioxidant, antiallergic, and anti-inflammatory [49].
Gemin D (6)Cytotoxic and chemo-preventive, in vitro anti-HIV activity, anti-Leishmania donovani amastigote, a potent growth-inhibitor of sarcoma 180 cells in mice. A potent inhibitory effect as a 3-hydroxy-3-methylglutaryl-coenzyme-A reductase [50].
In vivo antigenotoxic activity [51].
Lythracin D (7)Anticholinesterase [13], cytotoxic to OSCC cell lines [12].
Pedunculagin (8)Anti-acne vulgaris (mediated by anti-inflammatory activity and 5α-reductase inhibition) [52], antibacterial, and antihemolytic [53]. Antitumor activity [38,39,40].
Flosin B (9)Contributes to the antidiabetic activity of Lagerstroemia speciosa by increasing glucose uptake of rat adipocytes [54].
Anti-acetylcholinesterase [13].
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].
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Orabi, M.A.A.; Orabi, E.A.; Awadh, A.A.A.; Alshahrani, M.M.; Abdel-Wahab, B.A.; Sakagami, H.; Hatano, T. New Megastigmane and Polyphenolic Components of Henna Leaves and Their Tumor-Specific Cytotoxicity on Human Oral Squamous Carcinoma Cell Lines. Antioxidants 2023, 12, 1951. https://doi.org/10.3390/antiox12111951

AMA Style

Orabi MAA, Orabi EA, Awadh AAA, Alshahrani MM, Abdel-Wahab BA, Sakagami H, Hatano T. New Megastigmane and Polyphenolic Components of Henna Leaves and Their Tumor-Specific Cytotoxicity on Human Oral Squamous Carcinoma Cell Lines. Antioxidants. 2023; 12(11):1951. https://doi.org/10.3390/antiox12111951

Chicago/Turabian Style

Orabi, Mohamed A. A., Esam A. Orabi, Ahmed Abdullah Al Awadh, Mohammed Merae Alshahrani, Basel A. Abdel-Wahab, Hiroshi Sakagami, and Tsutomu Hatano. 2023. "New Megastigmane and Polyphenolic Components of Henna Leaves and Their Tumor-Specific Cytotoxicity on Human Oral Squamous Carcinoma Cell Lines" Antioxidants 12, no. 11: 1951. https://doi.org/10.3390/antiox12111951

APA Style

Orabi, M. A. A., Orabi, E. A., Awadh, A. A. A., Alshahrani, M. M., Abdel-Wahab, B. A., Sakagami, H., & Hatano, T. (2023). New Megastigmane and Polyphenolic Components of Henna Leaves and Their Tumor-Specific Cytotoxicity on Human Oral Squamous Carcinoma Cell Lines. Antioxidants, 12(11), 1951. https://doi.org/10.3390/antiox12111951

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