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. 2023 May 22;13(6):208. doi: 10.1007/s13205-023-03581-4

Antiproliferative, molecular docking, and bioavailability studies of diarylheptanoids isolated from stem bark of Garuga pinnata Rox B.

Srilekha Konakanchi 1, Rajender Vadluri 1, Kireety Sharma Anumula 1, Narashimulu 2, Devendar Banothu 3, Thupurani Murali Krishna 1,✉
PMCID: PMC10203062  PMID: 37229275

Abstract

Diarylheptanoids are a major class of plant secondary metabolites characterized by 1, 7-diphenyl heptanes in a seven-member carbon frame. In the present study, diarylheptanoids (garuganins 1, 3, 4 and 5) isolated from Garuga pinnata stem bark were evaluated for cytotoxic activity against MCF-7 and HCT15 cancer cell lines. Among the tested compounds, garuganin 5 and 3 exhibited the highest cytotoxic activity against HCT15 and MCF-7 with IC50 2.9 ± 00.8 μg/mL, 3.3 ± 0.1 μg/mL and 3.2 ± 0.1 μg/mL, and 3.5 ± 0.3 μg/mL, respectively. The molecular docking of garuganin 1, 3, 4 and 5 exhibited significant affinity toward the tested EGFR 4Hjo protein. The free energy and inhibitory constant of the compounds ranged from − 7.47 to − 8.49 kcal/mol and 3.34 micromolar to 944.20 nM nanomolar, respectively. Based on the results of cytotoxic activity, garuganin 5 and 3 were further evaluated for time- and concentration-dependent intracellular accumulation studies. The time-dependent intracellular concentration of garuganin 3 and 5 after 5 h of incubation increased about 5.5- and 4.5-fold, 204.16 ± 0.02 and 145.4 ± 0.36 nmol/L mg, respectively. The concentration-dependent intracellular concentration of garuganin 3 and 5 at 200 µg/mL increased of about > 12- and ninefold, 186.22 ± 0.05 and 98.73 ± 0.02 nmol/L mg, respectively. The intracellular concentrations of garuganin 3 and 5, in the presence of verapamil, cyclosporine and MK 571, was found to be significant in the basal direction compared to the apical directions. The results indicate that, garuganin 3 and 5 exhibited significant cytotoxic activity against MCF-7 and HCT15 cancer cell lines and also exhibited high binding affinity toward EGFR protein compared to garuganin 1 and 4.

Keywords: Diarylheptanoids, Cytotoxicity, Molecular docking, Verapamil, Cyclosporine, MK 571, Time dependent, Concentration dependent, Basal direction, Apical direction

Introduction

Cytotoxicity assay is a preliminary method used for the evaluation of the anticancer potential of compounds either chemically derived or isolated from natural sources such as medicinal plant extracts. In the current scenario, most of the prescribed drugs for the treatment of different types of cancers have been reported to exhibit adverse effects in humans. In addition, multidrug resistance is also an alarming threatening issue in the field of cancer. Medicinal plants immensely provide good candidates for the treatment of cancer (Avani et al. 2008). Globally, plant-derived anticancer agents such as camptothecin derivatives (camptothecin and irinotecan), epipodophyllotoxins (etoposide and teniposide) vinca alkaloids (vincristine, vinblastine and vindesine), and taxanes (paclitaxel and docetaxel) are extensively used. The structural diversity of plant-derived compounds is a key factor for their attributed anticancer mechanism such as initiation of apoptosis and inhibition of cell proliferation pathways.

Diarylheptanoids are a major class of plant secondary metabolites characterized by 1, 7-diphenyl heptanes in a seven-member carbon frame. With reference to the structure, diarylheptanoids are grouped into cyclic and linear diarylheptanoids. These diarylheptanoids are apparently gaining importance because of their remarkable pharmacological activities. Approximately, 400 diarylheptanoids have been identified so far from the plants of different families. The presence of linear diarylheptanoids has been reported in plants such as Alpina, Aingiber and Curcuma of the Zingiberaceae family and Betula and Alnus from Betulaceae, whereas cyclic diarylheptanoids are rich in plants such as Garuga (Burseraceae), Acer (Aceraceae), Juglans (Juglandaceae), and Myrica (Myricaceae) (Vidakovic et al. 2017; Alberti et al. 2018). The linear diarylheptanoids isolated especially from Curcuma and Zingiber have been extensively using as Asian traditional medicines for the treatment of various human alignments (Kunnumakkara et al. 2017). The cyclic diarylheptanoids are reported to possess antimicrobial activities (Keseru and Nogradi 1993; Khatun et al. 2013).

Bioavailability is an important phenomenon of any drug for its action. The availability of a drug is directly proportional to its action. Bioavailability is a variable parameter which directly depends on the route of drug administration. The drugs administrated parentally possess 100% bioavailability compared to the drugs administrated orally. The measurement of bioavailability is important to standardize the dose of any drug. Garuga pinnata belonging to family Burseraceae has been reported to exhibit various pharmacological properties such as antioxidant, antibacterial, antifungal, anti-inflammatory, antidiabetic, and anticancer properties (Thupurani et al. 2012, 2013a, b, c). In the context of the pharmacological properties of diarylheptanoids, the present study aims to evaluate the anticancer activity and bioavailability of diarylheptanoids isolated from Garuga pinnata stem bark.

Materials and methods

Bioassay-guided fractionation of stem bark extract of Garuga pinnata (G. pinnata)

Based on the previous reports (Thupurani et al. 2012, 2013a, b, c), the stem bark extracted with methanol was selected for the separation of compounds using column chromatography. The methanol extract was placed on the top of the silica gel (100–200 mesh) and eluted with methanol and acetonitrile (10:90 and 90:10). The collected fractions were examined for antibacterial activity (Tabe S1 in supplementary files) to eliminate non-active fractions and to use active fractions for further studies. The active fractions with a single spot on TLC sheets were elucidated for its structure using 1HNMR.

Cell viability assay

The antiproliferative activity of the previously isolated compounds garuganin 1 (14,16,6-trimethoxy-2-oxa-1(1,3),3(1,4)-dibenzenacyclodecaphan-6-en-8-one), garuganin 3 (15,6-dihydroxy-16-methoxy-2-oxa-1(1,3),3(1,4)-dibenzenacyclodecaphan-6-en-8-one), garuganin 4 (16,6-dimethoxy-2-oxa-1(1,3),3(1,4)-dibenzenacyclodecaphan-6-en-8-one), and garuganin 5 (16-hydroxy-26,7-dimethoxy-1,2(1,3)-dibenzenacyclononaphan-6-en-5-one) from the stem bark of Garuga pinnata (Fig. 1) (spectral data shown as supplementary file (Annexure-1)) was evaluated against MCF-7 (human breast cancer cell lines) and HCT15 (human colon cancer cell lines) by the method described elsewhere (Plumb et al 1989). Prior to the cytotoxic determination, the cell lines were grown in RPMI1640 (Hyclone, South Logan, UT, USA). In brief, cell lines at 80% confluence were detached using 0.25% trypsin and transferred on to 96-well microtiter plates maintaining 5 × 1043 cells per well. The stock solutions (10 mg/mL) of garuganins (1, 3, 4 and 5) and standard cyclophosphamide were prepared in 0.1% DMSO and serially diluted (1000–0 µg/mL) and added to the 96-well plate containing the selected cancer cell lines. Plates were incubated at 37 °C in a CO2 (5%) incubator for 48 h. Control cells were cultured in medium containing 0.1% DMSO. Thereafter, 100 μL of 0.4% MTT solution in PBS was added to each well and incubated for an additional 4 h. The formazan crystals formed were dissolved in DMSO 100 μL/well and 25 μL of Sorensen’s glycine buffer for 10 min on a plate shaker. The optical density was measured in a microplate reader (Biochrom, Cambourne, UK) at 550 nm. The inhibition concentration (IC50) values are represented as mean ± S.D.

Fig. 1.

Fig. 1

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Diarylheptanoids (garuganin 1, 3, 4, 5) isolated from the stem bark of Garuga pinnata Roxb

Molecular docking studies

Docking parameters

The Auto Dock 4.2 was used for molecular docking studies. The target protein EGFR was obtained from protein data bank having PDB ID 4HJO 2D (Palabindela et al. 2023) structures of all the compounds generated with the help of ChemBioDraw Ultra 12.0 software (www.cambridgesoft.com), whereas the energy-minimized 3D structures were generated by using Gaussian 09 (Frisch et al. 2009) with basic set hf/3-21 g* (Frisch et al. 2009). The crystallographic 3D structure of EGFR protein was extracted from RCSB Protein Data Bank with PDB ID: 4HJO. The previously associated ligands and water molecules of the downloaded protein were eliminated using the Discovery Studios. The molecular docking studies were carried out using AutoDock Tools version 1.5.6 and AutoDock 4.2 package suite (http://autodock.scripps.edu/resources/references). A cubic grid box was built by taking 60 points along the three axes joint spacing of 0.3750 Å. In this docking protocol, the population size, 150 were used to optimized the binding mode of ligands. Cygwin interface was used to get the dlg file from where the results such as binding energy and inhibition constant were obtained and 2D and 3D images were rendered using Schrodinger’s maestro v9.5 vizualizer interface.

Intracellular drug absorption studies

Compounds that exhibit significant antiproliferative properties were selected for the intracellular drug absorption studies. Caco-2 cells ATCC (Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% l-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 15% fetal bovine serum. The culture plates were maintained at 37 °C under a humidified atmosphere of 5% CO2 till the cells attained 80% of confluency. The cells were detached and subcultured onto a fresh culture medium for transport characteristic studies. The ability of transportation and intracellular accumulation of diarylheptanoids were evaluated using a published method (Vaidyanathan and Walle 2003). Briefly, Caco-2 cells were seeded at a cell density of 0.01 × 106 (approximately 10,000 cells) per well on 96-well microtiter plates containing incubation medium including Hank’s balanced salt solution, pH 7.4 (HBSS; 5.4 mM KCl, 137 mM NaCl, 0.8 mM MgCl2, 1.3 mM CaCl2, 0.4 mM KH2PO4, 0.3 mM NaH2PO4, and 10 mM HEPES/Tris) and incubated at 37 °C for 3–4 h. Thereafter, the incubation medium was removed and the cells were cultured  in DMEM media along with different concentrations (from 5 to 300 μg/mL) of compounds for different times (from 10 min to 6 h) to determine the concentration-dependent and time-dependent drug absorption.

Cellular transport characteristics

To measure the ability of compounds to cross the Caco-2 cell membrane, the cells were seeded at a density of 1 × 104 on Millicell Cell Culture Inserts (Millipore, Billerica, MA, USA). The cells were subjected to transepithelial electrical resistance till reaching the resistance value 300 Ω⋅cm2 or slightly more (Ma and Kim 2010). The compounds were transferred via bilateral direction of cell membrane (basolateral (BL) and apical (AP) direction) to determine the transport efficiency in the presence of P-gp, MRP, and MRP2- selective inhibitors such as verapamil, cyclosporine and MK 571, respectively. At the end of the experiment, the cell lysates were obtained by freeze-thawing to determine the intracellular concentration of compounds.

Sample preparation and quantitative analysis

The compounds were extracted from 0.2 mL of cell suspension for absorption and transport studies using 1 mL acetone. The mixture is vortexed and subjected for centrifugation (3000 × g) for 10 min. The supernatant was transferred to a fresh Eppendorf tube and reconstituted with 50 μL acetonitrile and evaporated under N2 atmosphere to collect compounds and their metabolite compounds. Further separation and purification of compounds was done using a reverse-phase HPLC system (Symmetry C18 column, 50 × 2.1 mm, 5 μm, Waters Corp., USA) with a 0.1% acetic acid/acetonitrile (10:90, v/v) mobile phase.

Statistical analysis

The results of cytotoxicity, drug absorption, and transport characteristics were represented as mean ± SD using MS Excel 10 version (n = 3). Student’s “t” test was conducted to find the probability of the means.

Results

We isolated four active compounds from the methanol extract of G. pinnata stem bark. The isolated compounds were garuganin 1, 3, 4, and 5.

1HNMR spectral analysis of the isolated compounds (the spectra are shown in Fig.S1 in supplementary files)

Garuganin 1 ((Z)-14,16,6-trimethoxy-2-oxa-1(1,3),3(1,4)-dibenzenacyclodecaphan-6-en-8-one)

1H NMR (400 MHz, CDCl3) δ 7.32 (d, J = 7.9 Hz, 2H), 6.95 (d, J = 7.9 Hz, 2H), 6.66 (s, 1H), 6.38 (s, 1H), 5.28 (s, 1H), 3.87 (s, 6H), 3.31 (s, 3H), 3.18 (t, J = 6.4 Hz, 2H), 2.45–2.33 (m, 4H), 2.18 (t, J = 6.1 Hz, 2H) ppm.

Garuganin 3 (Z)-15,6-dihydroxy-16-methoxy-2-oxa-1(1,3),3(1,4)-dibenzenacyclodecaphan-6-en-8-one

1H NMR (400 MHz, CDCl3) δ 7.23 (d, J = 7.8 Hz, 1H), 7.06–7.01 (m, 3H), 6.67 (s, 1H), 5.24 (s, 1H), 5.19 (s, 1H),), 3.88 (S, 1H), 3.16 (t, J = 6.5 Hz, 2H), 2.42–2.34 (m, 4H), 2.16 (t, J = 6.2 Hz, 2H).

Garuganin 4 (Z)-16,6-dimethoxy-2-oxa-1(1,3),3(1,4)-dibenzenacyclodecaphan-6-en-8-one

1H NMR (400 MHz, CDCl3) δ 7.35 (d, J = 7.7 Hz, 2H), 7.05–6.98 (m, 3H), 6.80–6.77 (m, 2H), 5.27 (s, 1H), 3.84 (s, 3H), 3.37 (s, 3H), 3.15 (t, J = 6.2 Hz, 2H), 2.43–2.34 (m, 4H), 2.23 (t, J = 6.3 Hz, 2H) ppm.

Garuganin 5 (E)-16-hydroxy-26,7-dimethoxy-1,2(1,3)-dibenzenacyclononaphan-6-en-5-one

1H NMR (400 MHz, CDCl3) δ 7.42 (s, 1H), 7.31 (d, J = 7.7 Hz), 7.12 (d, J = 7.4 Hz, 1H), 6.98 (d, J = 7.7 Hz, 1H), 6.92 –6.85 (m, 2H), 5.29 (s, 1H), 5.16 (s, 1H), 3.87 (s, 6H), 3.42 (s, 3H), 3.18 (t, J = 6.3 Hz, 2H), 2.44–2.31 (m, 4H), 2.15 (t, J = 5.9 Hz, 2H) ppm.

MTT cell viability assay

The diarylheptanoids (garuganin 1, 3, 4, and 5) exhibited cytotoxic activity against HCT-15 and MCF-7 cell lines. Among the tested compounds, garuganin 5 exhibited the highest cytotoxic activity against HCT-15 and MCF-7, with IC50 2.9 ± 00.8 μg/mL and 3.3 ± 0.1 μg/mL, respectively. Garuganin 3 was also noted to have significant activity against HCT15 and MCF-7, with IC50 3.2 ± 0.1 μg/mL and 3.5 ± 0.3 μg/mL, respectively. On the other hand, the cytotoxicity of garuganin 1 and 4 was found to be tenfold less compared to the cytotoxicity of garuganin 5 and 3. The IC50 of garugnin 1 recorded against HCT15 and MCF-7 was 25.4 ± 0.5 and 32.4 ± 0.6 μg/mL, respectively (Table 1).

Table 1.

Cytotoxic activity (IC50) of the compounds isolated from the stem bark of Garuga pinnata Roxb

Compounds Cytotoxic activity (IC50) μg/mL
HCT15 MCF-7 Normal collateral cell Normal breast cell
Garuganin 1 25.4 ± 0.5 32.4 ± 0.6 – –
Garuganin 3 3.2 ± 0.1a 3.5 ± 0.3a – –
Garuganin 4 29.0 ± 0.4 37.5 ± 0.4 – –
Garuganin 5 2.9 ± 00.8 3.3 ± 0.1 – –
Cyclophosphamide 2.3 ± 0.2a 3.0 ± 0.2a – –
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n = 4, results are mean ± SD, Student’s ‘t’ test was performed to compare the means. aP < 0.5

Molecular docking

Based on the molecular docking study result, the diarylheptanoids (garuganin 1, 4, 3, and 5) exhibited significant affinity toward tested EGFR 4Hjo protein (Table 2). The free energy of the tested compounds ranged from − 7.47 to − 8.49 kcal/mol. The inhibitory constant of the compounds tested ranged from 3.34 micromolar to 944.20 nM nanomolar. Among the tested compounds, garuganin 3 and 5 showed immense interaction with EGFR 4Hjo protein with free binding energy − 8.22 kcal/mol and − 8.49 kcal/mol, respectively. The inhibition constant of garuganin 3 and 5 revealed to possess 944.20 nM and 594.91 nM, respectively. Garuganin 3 formed four hydrogen bonds with MET769, MET769, ASN818, and ASP831 with bond length 1.71 Å, 2.11 Å, 2.23 Å, and 2.06 Å, respectively (Fig. 2A–I). Garuganin 5 also formed three hydrogen bonds with ALA698, ARG817, and LYS851with bond length 1.82 Å, 2.19 Å, and 1.65 Å, respectively (Fig. 2a A–F and bA–D). The interaction of the remaining compounds, garuganin 1 and 4, was found to be moderate with the binding free energy − 7.47 kcal/mol and − 7.71 kcal/mol, respectively. Finally, the standard drug, cyclophosphamide, also docked with EGFR where it showed − 6.61 kcal/mol binding energy and 14.29 micromolar, inhibition constant. It also formed two hydrogen bonds with the same residue PHE 832, with bond lengths 1.99 Å and 2.06 2.25 Å. The theoretical KI (inhibition constants) of garuganin 3 and 5 produced in silico are directly proportional to their high significant IC50 values in in vitro studies of antiproliferative activity.

Table 2.

Molecular docking interaction parameters of compounds with the EGFR 4HJO protein

Compound Binding energy (kcal/mol) Inhibition constant (µM)/Nm No. of hydrogen bonds Residues involved in hydrogen bonding (bond length in A0)
Garuganin 1 − 7.47 3.34 µM 1 ALA847 (2.12),
Garuganin 3 − 8.22 944.20 nM 4 MET769(1.71), MET769(2.11), ASN818(2.23), ASP831(2.06)
Garuganin 4 − 7.71 2.23 µM 1 PHE699(2.45)
Garuganin 5 − 8.49 594.91 nM 3

ALA698(1.82), ARG817(2.19)

LYS851(1.65)
Cyclophosphamide − 6.61 14.29 µM 2 PHE 832 (1.99)
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Fig. 2.

Fig. 2

Fig. 2

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a (A) 2D interaction of garuganin 1with EGFR 4Hjo protein, (B) 3D interaction of garuganin 1 with EGFR 4Hjo protein, (C) 2D interaction of garuganin 3 with EGFR 4Hjo protein, (D) 3D interaction of garuganin 3 with EGFR 4Hjo protein, (E) 2D interaction of garuganin 4 with EGFR 4Hjo protein, (F) 3D interaction of garuganin 4 with EGFR 4Hjo protein. b (A) 2D interaction of garuganin 5 with EGFR 4Hjo protein, (B) 3D interaction of garuganin 5 with EGFR 4Hjo protein, (C) 2D interaction of cyclophosphamide with EGFR 4Hjo protein, (D) 3D interaction of cyclophosphamide with EGFR 4Hjo protein

Docking scores of at least ten conformations for each compound are provided in Table S2 in Supplementary files.

Detection of gauganin 3 and 5 parent and their product ions

Garuganin 3 and 5 and their product ions were detected using mass spectrometer. The garuganin 3 parent ions were found at m/z 509.35 and their product ions at m/z, 325.11. The garuganin 5 parent ions were found at m/z, 400.11, and their product ions were found at m/z, 650.11. The mass spectra are shown in Fig S2 in Supplementary files.

Time- and concentration-dependent intracellular absorption of garuganin 3 and 5

Based on the significant antiproliferative results exhibited by the compounds, garuganin 3 and 5 were selected for time- and concentration-dependent intracellular absorption studies. Varied concentrations of garuganin 3 and 5 were incubated with Caco-2 cells at different time intervals. The time-dependent intracellular accumulation of compounds was steadily raised over 5 h of incubation (Fig. 3a). After 2 h of incubation, the concentration of garuganin 3 increased about 3.0-fold compared with the first 30 min (37.12 ± 0.02 versus 111.36 ± 0.12 nmol/L mg), whereas the intracellular concentration of garuganin 5 after 2 h of incubation increased of about 2.5-fold compared with the first 30 min (32.33 ± 0.01 versus 80.82 ± 0.031 nmol/L mg). However, the intracellular concentration of garuganin 3 after 5 h of incubation increased about 5.5-fold compared with the first 30 min (37.12 ± 0.02 versus 204.16 ± 0.02 nmol/L mg). On the other hand, the intracellular concentration of garuganin 3 after 5 h of incubation was increased by about 4.5-fold compared with the first 30 min (32.33 ± 0.01 versus 145.4 ± 0.36 nmol/L mg) (Fig. 3a). Simultaneously, we also determined the concentration-dependent intracellular concentration of garuganin 3 and 5 (Fig. 3b). It is noted that the intracellular concentration of garuganin 3 at 100 µg/mL was increased by about sixfold compared to the intracellular concentration recorded at 10 µg/mL (15.31 ± 0.01 versus 91.85 ± 0.02 nmol/L mg), whereas the intracellular concentration of garuganin 5 at 100 µg/mL was increased by about fourfold compared to the intracellular concentration noted at 10 µg/mL (10.97 ± 0.01 versus 60.33 ± 0.16 nmol/L mg) (Fig. 3b). However, the intracellular concentration of garuganin 3 at 200 µg/mL increased about > 12-fold compared to the intracellular concentration observed at 10 µg/mL (15.31 ± 0.01 versus 186.22 ± 0.05 nmol/L mg). On the other hand, the intracellular concentration of garuganin 5 at 200 µg/mL was increased by about ninefold compared to the intracellular concentration found at 10 µg/mL (10.97 ± 0.01 versus 98.73 ± 0.02 nmol/L mg) (Fig. 3b).

Fig. 3.

Fig. 3

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a Time-dependent intracellular concentration of garuganin 3 and 5. b Concentration-dependent intracellular accumulation of garuganin 3 and 5. c Intracellular concentration of garuganin 3 and 5 via the basolateral and apical directions in the presence of verapamil drug transporter. d Intracellular concentration of garuganin 3 and 5 via the basolateral and apical directions in the presence of cyclosporine drug transporter. e Intracellular concentration of garuganin 3 and 5 via the basolateral and apical directions in the presence of MK-571 drug transporter, f intracellular concentration of garuganin 3 and 5 via the basolateral and apical directions in the absence of drug transporters

Transport characteristics of garuganin 3 and 5 through Caco-2 cells

The transport efficiency and subsequent intracellular accumulation of garuganin 3 and 5 from apical and basolateral directions was tested in the presence of drug transporters such as verapamil, cyclosporine, and MK 571. The intracellular concentration of garuganin 3, transferred via the basolateral and apical direction in the presence of verapamil, was found to be 77.09 ± 0.02 and 67.21 ± 0.02 nmol/L mg, respectively, whereas the intracellular concentrations of garuganin 5 administered in the presence of vermapil via the basolateral and apical directions were 66.98 ± 0.04 and 61.39 ± 0.06 nmol/L mg, respectively (Fig. 3c). The intracellular concentration of garuganin 3 and 5 in the presence of cyclosporine was 73.0 ± 0.05 nmol/L mg, 59.07 ± 0.06 nmol/L mg and 60.31 ± 0.10 nmol/L mg, 48.62 ± 0.05 nmol/L mg, noted via the basolateral and apical directions, respectively (Fig. 3d). In contrast to verapamil and cyclosporine, the third drug transporter MK571 was found to be less efficient in the transportation of garuganin 3 and 5. The intracellular concentration of garuganin 3 and 5 in the presence of MK571 was 58.0 ± 0.09 nmol/L.mg, 56.3 ± 0.30 nmol/L.mg and 51.32 ± 0.04 nmol/L.mg, 46.11 ± 0.06 nmol/L.mg, noted via the basolateral and apical directions, respectively (Fig. 3e). The intracellular concentration of concentration of garuganin 3 and 5 was found to be poor in the absence of drug transporters (Fig. 3f).

Discussion

The current research work is an outcome of our previously reported therapeutic functions of Garuga pinnata crude extracts (Thupurani et al. 2012, 2013a, b, c). In the current investigation, we have isolated four bioactive compounds (diarylheptanoids) from stem bark extracted with methanol. All the compounds were identified as garuganin 1, 3, 4, and 5 using 1HNMR. The isolated compounds were evaluated for cytotoxic activity against HCT-15 (human colorectal cancer) and MCF-7 (Michigan Cancer Foundation-7) cell lines. The cytotoxicity of these compounds might be due the presence of the methoxy and hydroxyl functional groups substituted at different positions on the basic moiety. The research in the development of different types of drugs includes substitution of the methoxy and hydroxyl functional groups because of their antiangiogenesis (inhibition of cell migration and invasion) property (Ki et al. 2020). In addition, the frequency and occurrence percentage of hydroxyl group in the approved drug database for the treatment of various cancers is found to be very high, 41.4% (Fei et al. 2016). With reference to the above, we understand that the cytotoxic activity of the isolated compounds may probably be attributed to the methoxy and hydroxyl functional groups. However, the variations in the activity of the isolated compounds might be due to the presence of these functional groups at different positions on the basic ring. Garuganin 5 with two methoxy groups and one hydroxyl group occupied the first place and garuganin 3 with two hydroxyl and one methoxy groups occupied the second place in the cytotoxic activity against HCT15 and MCF-7 cancer cells. On the other side, garuganin 1 with three methoxy groups occupied third and garuganin 4 with a single hydroxyl group occupied the fourth place in the activity against tested cancer cell lines. It has been reported that compounds with three or more than three methoxy groups exhibit poor cytotoxic activity. The poor cytotoxic activity of garuganin 1 could become one of our major discussion points. With the combination of the methoxy and hydroxyl groups, the activity was found to be significant. In the case of garuganin 4, there were only methoxy groups but not any hydroxyl groups on the basic ring.

Based on the cytotoxic activity results, we selected garuganin 3 and 5 for further studies of bioavailability using Caco2 cell lines. This study includes the determination of intracellular concentration of garuganin 3 and 5 in a time-dependent and concentration-dependent manner. Moreover, we also tested the transport efficiency of garuganin 3 and 5 drug penetration trough Caco 2cell membrane in the basolateral and apical directions in the presence and absence of drug transporters. The transport efficiency was found to be slightly higher when the compounds were transported through the basolateral direction in the presence of drug transporters. Bioavailability determination is an important assay for any drug to know its ability of transportation via the cancer cell membrane. Caco-2 is most widely used for this purpose.

Molecular docking studies were carried out using the epidermal growth factor (EGFR) receptor protein. The EGFR is an important transmembrane protein receptor belonging to the tyrosine kinase family (ErbB family) (Wieduwilt and Moasser 2008). This receptor plays a vital role in cell proliferation (Yun et al. 2007), apoptosis (Högnason et al. 2001), cell migration (Andl et al 2004) and cell invasion. The overstimulation and expression of this receptor may lead to the generation of cancer cells. In addition, the activation of EGFR will also cause further stimulation and activation of VEGF (vascular endothelial growth factor), which plays a key role in tumor angiogenesis (Tabernero 2007; Arora and Scholar 2005). Therefore, the EGFR and VEGF transmembrane receptors have been important targets for the development of new drugs for the treatment of different types of cancers. The selection of the EGFR receptor protein from the protein database is based on the number of methoxy functional groups present on garuganin 3 and 5. The number of methoxy groups reduce the cytotoxicity property of the compounds. Thus in the inhibition of metastasis and angiogenesis, we selected garuganin 3 and 5. In accordance with the molecular docking results, both compounds exhibited significant interaction with EGFR receptor protein and exhibited good docking score or band.

Diarylheptanoids are the emerging class of bioactive molecules in the field of cancer therapy. Evidently, the anticancer activity of diarylheptanoids has been well proven in various human cancer cell lines and murine melanoma models (Ravez et al. 2012; Lee et al. 2013; Xi et al. 2015). In a recent publication, the authors reported that the cyclic diarylheptanoids exhibited anitiproliferative and topoisomerase I and IIα inhibitory activities (Uto et al. 2015). The well-known cyclic diarylheptanoid, myricanone isolated from the stem bark of M. rubra was reported to exert anticancer activity (Yang et al. 2020). Plants and their cyclic diarylhepatanoids can become the most important area for drug discovery against different types of cancer treatments in the upcoming years (Ishida et al 2002). In future, this study can be extended for induction of apoptosis, caspase3/7 activities and detection of P-gp and p-Akt protein expression in cancer cells.

Conclusion

In conclusion, the isolated compounds exhibited cytotoxic activity against MCF-7 and HCT-15 cell lines. However, among the four tested garuganins 1, 3, 4 and 5, garuganin 3 and 5 exhibited high antiproliferative activity. Moreover, docking studies proved that garuganin 3 and 5 possess significant binding affinity toward the EGFR protein. The time-dependent and concentration-dependent intracellular concentration of garuganin 3 and 5 was found high in the presence of drug transporters. The basal direction of drug administration into Caco2 cells was found to be better compared to drug administration via the apical direction.

Acknowledgements

The authors are grateful to the Chancellor, Chaitnya Deemed to be University, for his cooperation and encouragement and also thankful to the Department of Biotechnology, NIT Warangal, for providing facilities for this research work.

Funding

The research leading to these results received funding from the Science and Engineering Research Board (SERB), New Delhi, India, under the project of TARE (Teachers Associateship for Research Excellence). Grant. FILE NO.TAR/2018/000561.

Data availability statement

Data are available on request only due to ethical, legal, or commercial reasons.

Declarations

Conflict of interest

The authors disclose no conflict of financial or nonfinancial interest.

Ethical statements

The authors do not have any potential conflict of interest. This study does not involve any human beings or animals.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data are available on request only due to ethical, legal, or commercial reasons.

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