Chemical Profile and Anticancer Activity of Polyscias guilfoylei Leaf Essential Oil

The Natural Products Journal, 2019, 9, 1-12 

Chemical Profile and Anticancer Activity of Polyscias guilfoylei Leaf Essential Oil

Rajani Kurup1,2, Ajikumaran Nair S.1, Uthayakumari Kalavathy3 and Sabulal Baby1,*

1Phytochemistry and Phytopharmacology Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Pacha-Palode 695562 Thiruvananthapuram, Kerala, India; 2Manonmaniam Sundaranar University, Abishekapatti, Tirunelveli 627012, Tamil Nadu, India; 3Research Centre for Plant Sciences, Department of Botany, St. Mary’s College (Autonomous), Beach Road, Thoothukudi 628001, Tamil Nadu, India

Abstract: Background: Polyscias guilfoylei, commonly called ‘geranium aralia’, is an erect shrub with dark green leaves. P. guilfoylei has been introduced to tropical countries and is generally cultivated in gardens for ornamental purposes. There are no previous studies on the essential oil of P. guilfoylei and its biological activities.

Objectives: In this study, we report the chemical profile of P. guilfoylei leaf essential oil and its anti- cancer activity tested by various in vitro and in vivo assays.

Methods: The chemical profile of P. guilfoylei leaf oil was elucidated by gas chromatographic analyses (GC-FID, GC-MS). Anticancer activity of P. guilfoylei leaf oil was tested by MTT, morphological observations, DNA ladder, comet, caspase, flow cytometry and in vivo assays.

Results: Gas chromatographic profiling of P. guilfoylei leaf oil identified 50 constituents (β-selinene 49.59%, α-selinene 21.68%, (Z)-falcarinol 11.65%). In MTT assay, P. guilfoylei leaf oil at 50, 25, 10, 5 and 1 μg/ml showed 98.6 ± 1.2, 95.3 ± 0.78, 76.8 ± 1.59, 43.6 ± 0.99 and 39.8 ± 1.17% DLA cell death, respectively (CD50 5.96 μg/ml). In flow cytometry, the majority of P. guilfoylei leaf oil (25 μg/ml) treated DLA cells showed an accumulation/cell arrest in G2M phase (61.7 ± 2.6%). In P. guilfoylei leaf oil treated mice (40 days), 5 animals (83.3%, each) were protected in 25, 50 mg/kg groups.

Conclusion: P. guilfoylei leaf oil, with minimal toxicity to normal cells, exhibited significant anti- cancer activity against lymphoma cells enhancing its potential as an anticancer agent.

Keywords: Polyscias guilfoylei, essential oil, chemical profiling, anticancer activity.

1. INTRODUCTION

Polyscias is a genus of flowering plants in the family Araliaceae, also known as the Ginseng family. Araliaceae comprises 55 genera and more than 1500 species distributed in tropical, subtropical and temperate regions, of which many are used as oriental medicines [1]. Many members of Araliaceae are aromatic. Polyscias J. R. Forst. & G. Forst is the second largest genus of Araliaceae family, with 159 spe- cies distributed in tropical and temperate regions [2]. Polyscias has several suspected species and varieties based on their foliage variations. Most members are in the sterile condition (bloom rarely), and so the origin of these plants are ambiguous. In Asian countries, Polyscias fruticosa leaves are used as tonic, anti-inflammatory, anti-toxin, antibacterial and

*Address correspondence to this author at the Phytochemistry and Phytopharmacology Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Pacha-Palode 695562 Thiruvananthapuram, Kerala, India; E-mails: sabulal@jntbgri.res.in; sabulal@gmail.com

also for digestion. Its roots are used as diuretic, anti- dysenteric and also for neuralgia and rheumatic pain [3]. In Thailand, P. fruticosa is eaten raw together with a spicy dip and is boiled in curries [4]. P. fruticosa is widely used in Vietnam as a tonic for the treatment of ischemia and inflammation and to increase blood flow in the brain. P. fruticosa leaves are also eaten as salad [5]. In Ghana, P. fruticosa leaves are traditionally used as a medicine for asthma. This traditional management of asthma was supported by a study which revealed antiasthmatic, antihistaminic and mast cell stabilization effects of P. fruticosa ethanol extract [6]. GC- MS analysis of P. fruticosa leaf oils from Thailand and Fiji has shown quantitative and qualitative differences between them. About 24 constituents were tentatively identified and β-Elemene, α-Bergamotene, Germacrene D and (E)-g- Bisabolene were the major constituents [7]. P. fulva is used for the treatment of various types of cancers in Cameroonian traditional medicine [8].

Though most species are aromatic, chemical profiling of volatile constituents and biological activity studies of Poly scias species are scanty. In India, there are seven Polyscias species and several cultivars. Polyscias guilfoylei (W. Bull) L. H. Bailey commonly called ‘geranium aralia’ is an erect shrub with dark green leaves. P. guilfoylei has been introduced to tropical countries and are generally cultivated in gardens for ornamental purposes. In Malaysia, P. guilfoylei leaves are used in flavouring, and in Java due to the diuretic property of its roots and leaves, their decoctions are given for kidney stone [9]. Jang and co-workers in a chemical ecology study found that the volatile semiochemicals from P. guilfoylei fresh leaves and leaf extracts are attractants of the mated female oriental fruit fly, Bactrocera dorsalis [10]. Phytochemistry of P. guilfoylei aerial parts yielded seven oleanane saponins, and they were tested for antiproliferative activities against three cell lines (J774.A1, HEK-293 and WEHI-164). 3β-O-[β-D-Glucopyranosyl-(1→2)-α-L-arabino-pyranosyl] echinocystic acid 28-[O-β-D-glucopyranosyl- (1→6)O-β-D glucopyranosyl] ester showed significant activ- ity against all three cell lines with IC50 values 0.19 ± 0.001 μM (J774.A.1), 0.35 ± 0.003 (HEK-293) and 0.64 ± 0.045 (WEHI-164), respectively [11]. Four secondary compounds viz., 3-O-β-D-glucopyranosyl spinasterol, 3-O-β-D- glucopyranosyl oleanolic acid, isophytol and oleanolic acid, were isolated from P. guilfoylei leaves [12]. Again, Tuyet and co-workers isolated five saponins (β-D-glucurono- pyranosyl oleanolic acid, a mixture of two saponins of 3-O- β-D-glucopyranosyl-(1→3)-β-D-glucuronopyranosyl oleano- lic acid and 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucurono- pyranosyl oleanolic acid, 3-O-β-D-glucopyranosyl-(1→3)- [β-D-glucopyranosyl-(1→4)]-β-D-glucuronopyranosyl olea- nolic acid, 3-O-β-D-glucopyranosil-(1→2)-[β-D-glucopyra- nosyl-(1→4)]-β-D-glucuronopyranosyl oleanolic acid and 3- O-β-D-glucopranosyl-(1→4)-β-D-glucuronopyranosyl olea- nolic acid 28-O-β-D-glucopyranosyl ester) from P. guilfoylei leaves [13]. Elgindi and co-workers isolated three saponins (3-O-[β-D-glucopyranosyl-(1→3)-β-D-glucuronopyranosyl- 6’-methyl ester] oleanolic acid-28-O-methyl ester, 3-O-[β-D- glucuronopyranosyl-6’-methyl ester] oleanolic acid-28-O-β- D-glucopyranosyl and 3-O-[β-D-glucopyranosyl(1→3)-β-D- glucuronopyranosyl-6-methyl ester] oleanolic acid 28-O-β- D-glucopyranosyl ester) from P. guilfoylei aerial parts [14]. There are no previous studies on the essential oil constituents of P. guilfoylei and their biological activities.

2. MATERIALS AND METHODS
2.1. Plant Material, Isolation of Essential Oil

Fresh leaves of P. guilfoylei were collected from Palode, Thiruvananthapuram in Kerala, India. Plant specimen was taxonomically identified, herbarium specimen prepared and deposited in JNTBGRI herbarium (TBGT 36171). Fresh chopped P. guilfoylei leaves (270 g) were subjected to hydrodistillation using a Clevenger-type apparatus for 6 h, and the leaf oil obtained was stored at 4 oC for further studies.

2.2. Physical Parameters

Refractive index (J257 Digital Refractometer, Rudolf Research Analytical, USA), specific rotation (Autopol IV Polarimeter, Rudolf Research Analytical, USA) and specific gravity of P. guilfoylei leaf oil were measured.

2.3. GC-FID Analysis

P. guilfoylei leaf oil was diluted (30 μl, dissolved in 1.5 ml acetone) and 1 μl was injected on to a GC-2010 Plus Gas Chromatograph with AOC-20i auto injector and FID (Shimadzu, Japan), fitted with an Rxi-5 Sil MS capillary column (5% phenyl-95% dimethyl polysiloxane, non-polar, 30 m x 0.25 mm i.d, 0.25 μm film thickness; Restek, USA). GC operation conditions: injection mode split; split ratio 50; injector temperature 220 oC; oven temperature programme 60- 250 oC (3 oC/min); carrier gas N2 at 3 ml/min; detector tem- perature 250 oC. Leaf oil injections were repeated thrice un- der similar conditions. Relative percentages of individual components were calculated from the peak area percentage report of volatiles.

2.4. GC-MS Analysis

P. guilfoylei leaf oil was diluted (30 μl, dissolved in 1.5 ml acetone), 1 μl was injected into a GC-Saturn 2200 fitted with a VF-5 capillary column (5% phenyl, 95% dimethyl polysiloxane, non-polar, 30 m x 0.25 mm i.d., 0.25 μm film thickness; Varian Inc., USA). GC-MS operation conditions: injector temperature 220 oC, transfer 240 oC, detector temperature 250 oC, oven temperature programme 60-250 oC (3 oC/min), carrier gas He 1.4 ml/min., mass spectra-electron impact (EI+) mode 70 eV and ion source temperature 240 oC.

2.5. LRI Determination, Data Analysis

Linear Retention Indices (LRI) of oil constituents were determined on the VF-5 column using standard C5-C30 straight chain hydrocarbons (Aldrich Chemical Company, USA). Individual compounds in P. guilfoylei leaf oil were identified by NIST database matching, comparison of LRI’s and comparison of mass spectra with Adam's database [15] (Table 1).

2.6. Animals

Animals used for the experiments were inbred Swiss Albino mice (25 to 30 g weight) reared in Jawaharlal Nehru Tropical Botanic Garden and Research Institute (JNTBGRI) Animal House. All animals were caged in uniform hygienic conditions and fed with standard pellet diet (Krish Scientists Shoppe, Bangalore) and water ad libitum as per the guide- lines of Institute Animal Ethics Committee (IAEC) (B- 01/02/2015/PPD-01), which is governed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

2.7. Cell lines

Dalton’s Lymphoma ascitis (DLA) cells, originally obtained from Amala Cancer Research Centre, Trissur, India were maintained as transplantable tumors in the peritoneal cavity of Swiss Albino mice.

2.8. Thymocytes, Peritoneal Macrophages, Bone Marrow Cells

Male mice were sacrificed by cervical dislocation, thymus glands were carefully excised without adjoining lymph nodes, separated thymus glands were transferred to sterile RPMI-1640 medium and a single cell suspension of thymocytes was prepared. To isolate peritoneal macrophages male mice were sacrificed by cervical dislocation and immediately injected with 5 ml chilled sterile RPMI-1640 medium to the peritoneal cavity and peritoneal exudate cells were collected. The glass adherent cell population (macrophages) was separated. To collect bone marrow cells, male mice were sacrificed by cervical dislocation, femur bones were separated carefully, separated femur bones were injected and fleshed with 5 ml of sterile RPMI-1640 medium using a syringe fitted with 25 gauge needle onto a 70 μm nylone cell stainer and single cell suspension of bone marrow cells were prepared. Viability of thymocytes, peritoneal macrophages and bone marrow cells were assessed by trypan exclusion method using a Neubauer counting chamber.

Table 1. Chemical profile of the leaf essential oil of Polyscias guilfoylei. 

2.9. In Vitro Cytotoxicity of P. guilfoylei Leaf Oil, MTT Assay

MTT assay was performed essentially as described earlier [16]. Briefly, sterile RPMI medium supplemented with 10% fetal bovine serum, 100 units of penicillin, 100 μg/ml of streptomycin containing 1 x 106 DLA cells/ml were seeded in 24 well plates and incubated with 0.1% DMSO, various doses of P. guilfoylei leaf oil (50, 25, 10, 5 and 1 μg/ml) or standard anticancer drug, vincristine (5 μg/ml), for 48 h in a Galaxy 170R carbon dioxide incubator (Eppendorf, Germany) at 37 oC, 5% CO2, 95% air and 95 relative humidity. After 48 h incubation, the spent medium was replaced with fresh RPMI medium with 20 μl of 5 mg/ml MTT and cells were incubated for an additional 4 h. MTT formazan product was dissolved in DMSO and optical density was measured at 570 nm using an ELISA plate reader (PR 601, Qualisystem gsk). In comparison test, thymocytes or macrophages or bone marrow or DLA cells were seeded at a concentration of 1 x 106 cells per ml in RPMI medium and MTT assays were performed as described.

2.10. Morphological Observations by Phase Contrast and Fluorescent Microscopy

Morphological observation of the treated DLA cells was performed as described elsewhere [17]. Briefly, 1 x 106 per ml of DLA cells were treated with DMSO (0.1%), P. guilfoylei leaf oil (25 μg/ml) or vincristine (5 μg/ml) in sterile RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 units per ml of penicillin and 100 μg/ml of streptomycin in a CO2 incubator at 37oC, 5% CO2, 95% air and 95 relative humidity in 24 well plate. After 48 h incubation, DLA cells were harvested and washed with PBS (pH 7.4) and observed under a phase-contrast microscope to assess their nuclear condensation and membrane blebbing. To determine the morphological changes and viability, treated DLA cells were harvested and washed with PBS (pH 7.4) and mixed with 25 μl of acridine orange-ethydium bromide stain (100 μg/ml) and observed under a CKX41 inverted phase-contrast-fluorescent microscope (Olympus, USA) using blue filter and photographed with a CAMEDIA C-4000 Zoom digital compact camera (Olympus, USA). Non-viable cells were observed in orange-red, while live cells were observed in green colour.

2.11. DNA Ladder Assay

DNA ladder assay was carried out using commercially available DNA ladder kit purchased from Life Technologies, USA (Cat. No. KH01021). Briefly, DLA cells (1 x 106 cells/ml) were treated with DMSO (0.1%), P. guilfoylei leaf oil (25 μg/ml) or vincristine (5 μg/ml) in 24 well plate for 48 h in sterile RPMI medium supplemented with 10% FBS, streptomycin (100 μg/ml) and penicillin (100 units/ml) in a CO2 incubator with 5% CO2 at 37 oC. After 48 h of incubation DLA cells were harvested, washed and suspended in PBS (pH 7.4). DNA from the DLA cells treated with vehicle, P. guilfoylei leaf oil or vincristine were extracted following the procedure given by the manufacture of the DNA ladder kit. Isolated DNA was suspended in DNA suspension buffer and 30 μl of each sample or 5 μl of DNA ladder (Thermo Scientific, Cat. No. SM0243, Gene ruler 100 bp-1 kbp DNA ladder) were loaded into the wells of 1.2% agarose gel containing 0.5 μg/ml ethidium bromide in a horizontal electrophoresis unit (Bio-Rad, USA). The loaded agarose gel was run at 5V/cm for 2 h and images of DNA ladder was captured using Bio Rad Gel Doc XR+ imaging system loaded with Image Lab Software (Bio-Rad, USA).

2.12. Comet Assay

Comet assay was carried out essentially as described earlier [18]. Briefly, DLA cells (1 x 106 cells/ml) were treated

with DMSO (0.1%), P. guilfoylei leaf oil (25 μg/ml) or vincristine (5 μg/ml) in 24 well plate for 48 h in sterile RPMI medium supplemented with 10% FBS, streptomycin (100 μg/ml) and penicillin (100 units/ml) in a CO2 incubator with 5% CO2 at 37 oC. After 48 h incubation, DLA cells were harvested, washed and suspended in PBS (pH 7.4). Cell suspension (10 μl) (10,000 cells) were added to 75 μl of low melting agar, mixed thoroughly and immediately spread uniformly over the normal melting agar, coated in frosted slide. A third layer of normal melting agar was added over the cell paved layer and a cover slip was placed. Solidified slides after removing the cover slips were dipped in lysis buffer (pH 10.0) for 2 h and kept in electrophoresis buffer (pH 13.0) for 20 min. Treated slides were electrophoresed in a horizontal electrophoresis unit (Bio-Rad, USA) for 30 min. After electrophoresis, the slides were added with neutralizaion buffer (pH 7.5) drop wise and stained with ethidium bromide (20 μg/ml). Stained slides were observed under a CKX41 inverted phase-contrast-fluorescent microscope (Olympus, USA) using green filter and photographed with a CAMEDIA C-4000 Zoom digital compact camera (Olympus, USA).

2.13. Caspase Assay

Caspase 3 activity was determined by commercially available caspase 3 colorimetric assay kit from Sigma, USA (Cat. No. CASP-3-C). Briefly, DLA cells (1 x 106 cells/ml) were treated with DMSO (0.1%), P. guilfoylei leaf oil (25 μg/ml) or vincristine (5 μg/ml) in 24 well plate for 24 h in RPMI medium supplemented with 10% FBS, streptomycin (100 μg/ml) and penicillin (100 units/ml) in a CO2 incubator with 5% CO2 at 37 oC . After 24 h incubation, DLA cells were harvested, washed with PBS (pH 7.4) and lysed in 1 x lysis buffer (pH 7.4). DLA cell lysates (10 μl) and peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD- pNA) (10 μl) were mixed and made up to 1 ml with caspase 3 assay buffer (pH 7.4) and incubated for 2 h at 37 oC. Along with the samples, reagent blanks containing 990 μl of caspase 3 assay buffer (pH 7.4) and 10 μl of Ac-DEVD-pNA were also incubated as described above. After 2 h, the optical densities of the incubated samples were measured at 405 nm using a UV-Visible spectrophotometer (UV-1650PC, Shimadzu, Japan). The concentration of p-nitroanilide released from the substrate was calculated following the instruction manual provided with the assay kit.

2.14. In Vivo Anti-tumor Activity in DLA Challenged Mice

In vivo anti-tumor activity of P. guilfoylei leaf oil was determined as described previously [16]. Briefly, 30 male Swiss Albino mice (25-30 g body weight) were divided into 5 groups of 6 animals each. First group of six animals was kept as a vehicle control group, received 0.1% of DMSO, second, third and fourth groups of six animals (each) were kept as test groups, which received 10, 25 or 50 mg/kg of P. guilfoylei leaf oil. Fifth group of animals was the standard anticancer drug control group, which received 10 mg/kg of vincristine. All mice were challenged with 1 x 106 DLA cells per ml intraperitoneally. Vehicle or drug treatment started from the second day of DLA challenge and continued for 15 days. Animals in control, test and vincristine groups received vehicle or drugs in specified doses once daily (0.1 ml per animal, intraperitoneally) for 15 days and animals were observed for 40 days for lethality.

Fig. (1). Cytotoxicity of P. guilfoylei leaf oil on DLA cells determined by MTT assay. DMSO, Dimethyl sulphoxide, zero percentage cell death was observed in vehicle control (0.1% DMSO). Values are mean ± S.D of three separate determinations, * P ≤ 0.05, ** P ≤ 0.01 (com- pared to standard anticancer drug vincristine). 

2.15. Cell Cycle, Apoptotic Analysis of DLA Cells by Flow Cytometry

Cell cycle analysis was carried out as described earlier [16]. Briefly, DLA cells (1 x 106 cells/ml) were treated with DMSO (0.1%), P. guilfoylei leaf oil (25 μg/ml) or vincristine (5 μg/ml) in 24 well plate for 48 h in RPMI medium supplemented with 10% FBS, streptomycin (100 μg/ml) and penicillin (100 units/ml) in a CO2 incubator with 5% CO2 at 37 oC . After 48 h incubation, DLA cells were harvested, washed with PBS (pH 7.4) and fixed in 70% ethanol over- night. These fixed DLA cells were added with propidium iodide matrix mix containing 100 μg/ml RNase, 40 μg/ml propidium iodide in PBS (pH 7.4) containing 0.105% Triton X-100 and incubated in darkness at 37 oC for 30 min. After incubation, the cells were analyzed for cell cycles with a flow cytometer (BD Bioscience, USA) and data were acquired with the DIVA software.

Apoptosis in DLA cells was analyzed by flow cytometry using Alexa Fluor 488 annexin V/Dead cell apoptosis kit from Invitrogen, USA (Cat No. V13241). Briefly, DLA cells (1 x 106 cells/ml) were treated with DMSO (0.1%), P. guilfoylei leaf oil (25 μg/ml) or vincristine (5 μg/ml) for 48 h as described and cells were washed with PBS twice. Harvested cells were suspended in 100 μl 1x annexin-binding buffer and added with 5 μl of Alexa Fluor 488 annexin V, 1 μl of 100 μg/ml propidium iodide and incubated at room temperature for 15 min in darkness. After incubation, cells were added with 400 μl of 1x annexin-binding buffer and cells were analyzed immediately with a flow cytometer (BD Bioscience, USA) and data were acquired with DIVA software.

2.16. Statistical Analysis

Statistical analyses of the data were carried out using one way ANOVA followed by Dunnett's post hoc comparisons. P values ≤ 0.05 were considered to be significant.

3. RESULTS AND DISCUSSION

P. guilfoylei leaf essential oil yield was 0.05% (v/fr. wt.) and its physical parameters showed specific gravity of 0.84, refractive index 1.5064 and specific rotation {α} D 20.6 = +0.714. Gas chromatographic profiling of P. guilfoylei leaf oil led to the identification of 50 constituents, with β- Selinene (49.59%), α-Selinene (21.68%) and (Z)-Falcarinol (11.65%) as the major ones (Table 1). Sesquiterpene hydro- carbons (80.38%) are the major constituents in P. guilfoylei leaf oil followed by others (13.27%), Oxygenated Sesquiterpenes (4.45%) and Diterpenes (0.47%) (Table 1).

In MTT assay (Fig. 1), P. guilfoylei leaf oil showed varying levels of cytotoxicity in 48 h culture; 50, 25, 10, 5 and 1 μg/ml showed 98.6 ± 1.2, 95.3 ± 0.78, 76.8 ± 1.59, 43.6 ± 0.99 and 39.8 ± 1.17% DLA cell death, respectively. Stan- dard drug (vincristine) at 5 μg/ml showed 68.5 ± 1.1% DLA cell death, which is comparable to the DLA cell death observed for P. guilfoylei leaf oil at 10 μg/ml. CD50 of P. guilfoylei leaf oil is 5.96 μg/ml. Comparison of the cytotoxicities of 0.1% DMSO, P. guilfoylei leaf oil or vincristine on DLA cells, macrophages, thymocytes and bone marrow cells is given in Table 2. DMSO (0.1%) was devoid of any cytotox- icity on various types of cells tested. At 25 μg/ml, P. guilfoylei leaf oil showed 97.8 ± 2.0%, 19.2 ± 1.1%, 20.1 ± 2.3% and 21.2 ± 1.8% cytotoxicity on DLA, macrophages, thymocytes and bone marrow cells, respectively. In 25 μg/ml, vincristine treated DLA, macrophages, thymocytes and bone marrow cells showed 100 ± 1.0%, 40.8 ± 2.9%, 44.1 ± 3.2% and 34.1 ± 2.9% cytotoxicity, respectively. When compared to the cytotoxicity of vincristine treated cells, the higher dose (25 μg/ml) of the leaf oil showed only mild toxicity to normal cells. P. guilfoylei leaf oil didn’t show any concentration-dependent increase in cytotoxicity in normal cells but vincristine showed significant cytotoxicity to normal cells concentration dependently (Table 2).

Table 2. In vitro cytotoxic effect of P. guilfoylei leaf oil on different cells in MTT assay. 

Fig. (2). (A) DLA cells treated with 0.1% DMSO (vehicle control) for 48 h and photographed under fluorescent microscope using acridine orange-ethidium bromide staining. Live cells appeared in green colour with intact cell membrane; (B) DLA cells treated with P. guilfoylei leaf oil (25 μg/ml) for 48 h and photographed under fluorescent microscope using acridine orange-ethidium bromide staining. Dead cells appeared in orange red colour with membrane blebbing; (C) DLA cells treated with vincristine (5 μg/ml) for 48 h and photographed with fluorescent microscope using acridine orange-ethidium bromide staining. Dead cells appeared in orange red colour with membrane blebbing; (D) Control DLA cells treated with 0.1% DMSO for 48 h and photographed under phase-contrast microscope. Live cells appeared with intact cell mem- brane and without nuclear condensation. (E) DLA cells treated with P. guilfoylei leaf oil (25 μg/ml) for 48 h and photographed under phase- contrast microscope. Dead cells appeared with membrane blebbing and nuclear condensation; (F) DLA cells treated with vincristine (5 μg/ml) for 48 h and photographed with phase-contrast microscope. Dead cells appeared with membrane blebbing and nuclear condensation. 

DLA cells treated with 0.1% DMSO, 25 μg/ml P. guilfoylei leaf oil or 5 μg/ml vincristine for 48 h, in morphological observations under phase contrast microscope (Fig. 2D- F) which showed membrane blebbing with nuclear condensation (in leaf oil and vincristine treated DLA cells), while intact cell membrane with a normal nucleus was ob- served in DMSO treated control cells. In fluorescent microscopy, DLA cells treated with 0.1% DMSO, 25 μg/ml of P. guilfoylei leaf oil or 5 μg/ml of vincristine for 48 h, stained with acridine orange-ethidium bromide dye, the live control cells appeared in green colour but P. guilfoylei oil or vincristine treated cells appeared in orange red colour with membrane blebbing, nuclear condensation and disorganized cell membranes, which are the hallmarks of apoptosis (Fig. 2A- C).

In DLA cells treated with 0.1% DMSO, 25 μg/ml of P. guilfoylei leaf oil or 5 μg/ml of vincristine for 48 h, the isolated DNA in ladder assay showed intact and non-degraded nuclear DNA in control DLA cells (Fig. 3A-D). P. guilfoylei leaf oil, as well as vincristine treated DLA cells showed significant DNA damage as expressed in DNA ladder assay. In comparison with 100 bp-1Kbp DNA ladder (run along with), DNA isolated from P. guilfoylei leaf oil or vincristine treated DLA cells showed significant degradation in nucleosomal region as well as in the linker DNA.

Fig. (3). (A) DNA ladder of 100 bp-1kbp size; (B) Intact nuclear DNA, from DLA cells treated with 0.1% DMSO (vehicle control) for 48 h; (C) Degraded nuclear DNA obtained from DLA cells treated with P. guilfoylei leaf oil (25 μg/ml) for 48 h; (D) Degraded nuclear DNA ob- tained from DLA cells treated with vincristine (5 μg/ml) for 48 h; (E) Intact nuclear DNA of control DLA cells treated with 0.1% DMSO (vehicle control) for 48 h in comet assay, (F) Comet formation of degraded nuclear DNA from DLA cells treated with P. guilfoylei leaf oil (25 μg/ml) for 48 h in comet assay. (G) Comet formation of degraded nuclear DNA from DLA cells treated with vincristine (5 μg/ml) for 48 h. 

Fig. (4). Caspase 3 activity in DLA cells treated with P. guilfoylei leaf oil. DMSO, Dimethyl sulphoxide (vehicle control), DLA cells (1 x 106 cells/ml) incubated with DMSO (0.1%), P. guilfoylei leaf oil (25 μg/ml) or vincristine (5 μg/ml) for 24 h and caspase 3 activities were deter- mined. Values are mean ± S.D, n = 3, * P ≤ 0.05 (compared to DMSO control). 

Comet assay (Fig. 3E-G), substantiates the observation made in the ladder assay, wherein the DLA cells treated with 0.1% DMSO showed intact nucleus (without any DNA degradation) and appeared in round shape without any comet formation. P. guilfoylei leaf oil (25 μg/ml) or vincristine (5 μg/ml) treated DLA cells in fluorescent microscopy showed significant DNA degradation with comet formation.

Caspase 3 activity in DLA cells treated with DMSO (0.1%), P. guilfoylei leaf oil (25 μg/ml) or vincristine (5 μg/ml) for 24 h were 0.0018 ± 0.00003, 0.0038 ± 0.00022 and 0.0035 ± 0.00027 μmol pNA/min/ml/ 1 x 10 6 DLA cells, respectively (Fig. 4). DLA cells treated with P. guilfoylei leaf oil (25 μg/ml) and vincristine (5 μg/ml) showed 111.1% and 94.4% increase in caspase 3 activity, respectively, compared to caspase 3 activity of 0.1% DMSO treated control DLA cells.

Fig. (5). (A) DLA cells treated with 0.1% DMSO (vehicle control) for 48 h; (B) DLA cells treated with P. guilfoylei leaf oil (25 μg/ml) for 48 h; (C) DLA cells treated with vincristine (5 μg/ml for 48 h; (D) Graph representing the cell cycle distribution of DLA cells treated with 0.1% DMSO, P. guilfoylei leaf oil (25 μg/ml) or vincristine (5 μg/ml); Values are mean ± SD, n = 3, *P ≤ 0.05, **P ≤ 0.01 (compared to control). 

In cell cycle analysis of DLA cells by flow cytometry (Fig. 5A-D), 0.1% DMSO treated control DLA cells showed normal cell cycle, 40.3 ± 2.4% of the cells which appeared in G0G1 phase, 35.8 ± 1.4% in S phase and 23.6 ± 1.1% in G2M phase, respectively. P. guilfoylei leaf oil (25 μg/ml) treated DLA cells showed an accumulation and arrest of cells in G2M phase and the distribution of G0G1, S and G2M phase were 14.0 ± 1.2%, 24.3 ± 2.0% and 61.7 ± 2.6%, respectively. The accumulation of leaf oil treated cells in G2M further triggers an internal signal switch on the apoptotic cycle which culminates in cell death. In case of vincristine (5 μg/ml) treated cells, the cell arrest appeared in G2M phase of the cell cycle. Different percentage of cells in G0G1, S and G2M phases was 18.2 ± 1.3, 21.6 ± 1.8 and 60.2 ± 1.4%, respectively.

In apoptotic analysis of DLA cells (flow cytometry) (Fig. 6A-D), 0.1% DMSO treated control cells showed normal population of live cells in the third quadrant (Q3) of the graph, which showed 96.7 ± 1.1% of unstained live cell population. In P. guilfoylei leaf oil (25 μg/ml) treated DLA cells, the cell population is distributed majorly in late apop- totic quadrant (Q2), wherein the percentage of late apoptotic

cells corresponds to 97.0 ± 2.0%. DLA cells arrested in G2/M phase initiated apoptosis and culminated in cell death. In vincristine (5 μg/ml) treated DLA cells, 93.2% of the cells were distributed in early apoptotic (37.4 ± 1.8%) and late apoptotic (55.8 ± 4.0%) stages.

Mice challenged with DLA cells treated with 0.1% DMSO, P. guilfoylei leaf oil or vincristine is given in Table 3. Animals in the control group started dying from 14th day, and complete death was observed in 20 days. In 40 days of the experimental period, in P. guilfoylei leaf oil treated animals, 4 (66.6%) animals were protected in 10 mg/kg treated group and 5 (83.3%) animals each were protected in 25 mg/kg and 50 mg/kg received groups. Even when the dose doubled (25 to 50 mg/kg), the efficacy was the same and hence 25 mg/kg was considered as the therapeutic dose. In standard drug (vincristine, 10 mg/kg) treated group only 3 animals were protected in the experimental period. On com- paring the efficacy of P. guilfoylei leaf oil with vincristine, 10 mg/kg of the leaf oil showed equal efficacy with vincris- tine in the same dose. Moreover, vincristine treated animals exhibited mild alopecia and sluggish activity due to the toxicity associated with vincristine, while the protected animals in P. guilfoylei leaf oil treated groups didn’t show any toxic symptoms during the experimental period.

Fig. (6). (A) DLA cells treated with 0.1% DMSO (vehicle control) for 48 h; (B) DLA cells treated with P. guilfoylei leaf oil (25 μg/ml) for 48 h; (C) DLA cells treated with vincristine (5 μg/ml) for 48 h; (D) Graph representing the stages (live/early apoptotic/late apoptotic/necrotic) of DLA cells treated with 0.1% DMSO, P. guilfoylei leaf oil (25 μg/ml) or vincristine (5 μg/ml); Values are mean ± SD, n = 3, *P ≤ 0, *P ≤ 0.001 (compared to control). 

Table 3. Antitumor activity of P. guilfoylei leaf oil in DLA challenged mice. 

P. guilfoylei leaf oil constituents were found to accelerate the accumulation of DLA cells in its G2/M phase of the cell cycle. It may be due to the disruption of one or more intermediaries in the normal regulation of spindle apparatus and microtubules activity. This accumulation of DLA cells in

G2/M phase of the cell cycle activates the caspase cascade of apoptosis through caspase 3, which is evident by 111.1% increase in caspase 3 activity in DLA cells treated with P. guilfoylei leaf oil. Essential oils are reported to induce both the mitochondria-dependent and death receptor-dependent apoptotic pathways [19]. Sharma and co-workers investigated the cytotoxic activity of Cymbopogon flexuosus (lemon grass variety) essential oil against various human cancer cell lines by in vitro and in vivo models. C. flexuosus oil showed promising anticancer activity and caused a loss in tumor cell viability by activating the apoptotic processes [20]. P. guil- foylei leaf oil significantly increased DNA strand breaks in DLA cells as reflected in the alkaline comet assay and similar result substantiating the DNA degradation was observed in the DNA ladder assay. DNA ladder assay revealed the strand breaks occurred both in nucleosomal region as well as in the linker DNA. Morphological observation of P. guilfoylei leaf oil treated DLA cells in phase contrast-fluorescent microscopy revealed membrane blebbing, nuclear condensation and cell death, which are the hallmarks of apoptosis. Essential oil constituents (such as carvacrol, citral) showed anticancer activity in various cancer cell lines by inducing apoptosis. Carvacrol-induced apoptosis in the metastatic breast cancer cell MDA-MB-231, by the release of cytochrome c from mitochondria due to the membrane permeabilization followed by the activation of caspases cascade culminated in poly ADP ribose polymerase (PARP) cleavage, DNA fragmentation and cell death [21]. Membrane blebbing, DNA condensation, along with the DNA strand break and degradation revealed the anticancer activity of P. guilfoylei leaf oil and these events are mediated through caspase 3 activation which leads to the PARP cleavage of the typical intrinsic caspase cascade of apoptosis. In flow cytometric analysis (of DLA cells treated with P. guilfoylei leaf oil or vincristine for 48 h treatment in vitro), 97.0 ± 2.0% of leaf oil treated DLA cells were in the late apoptotic quadrant, but vincristine treated DLA cells showed cell distribution both in early (37.4 ± 1.8%) and late apoptotic (55.8 ± 4.0%) stages. From this, it is evident that the efficacy of P. guilfoylei leaf oil is much better than the standard drug vincristine in inducing apoptosis. Many essential oils are strongly cytotoxic to cancer cell lines but less toxic to normal cells. An example is, Amomum tsao-ko fruit oil showed significant cytotoxic activity against cancer cell lines HepG2, Bel-7402, HeLa, SGC-7901 and PC-3, but relatively less cytotoxicity in nor- mal hepatocytes HL-7702 and umbilical vein endothelial (HUVEC) cell lines [22]. Similar trend is also observed in P. guilfoylei leaf oil, wherein the essential oil showed potent cytotoxicity on DLA cells but lesser toxicity to the thymocytes, peritoneal macrophages and bone marrow cells. Cytotoxicity on normal cells was lesser than that of the standard drug vincristine, and this interesting fact attracts P. guilfoylei leaf oil as a candidate for the development of new generation of anticancer agents.

Synergism of P. guilfoylei leaf oil constituents is respon- sible for its anticancer activity. Major constituents in P. guilfoylei leaf oil (β-Selinene (49.59%), α-Selinene (21.68%) and (Z)-Falcarinol (11.65%)) are to be individually investigated for their anticancer potentials. Falcarinol, a fatty alcohol and a natural pesticide, is found in carrots (Daucus carota), Panax ginseng and ivy. It is known to have a protecive effect on certain types of cancers, and more specifically, it reduced the risk of cancer development in rats by one third. Kobaek-Larsen and co-workers encouraged eating carrots due to their Falcarinol (cancer preventive constituent) content [23]. α-Selinene/β-Selinene are known as essential

precursors of defense compounds against various biotic stresses in maize [24]. So far, there are no similar (anticancer) reports on the two major constituents (β-Selinene (49.59%), α-Selinene (21.68%) of P. guilfoylei leaf oil. Other Polyscias species also are least studied for their anti- cancer potentials.

CONCLUSION

This study reveals the anticancer potential of one Polyscias species, P. guilfoylei, against lymphoma both by in vitro and in vivo models. The protection of mice from lymphoma by P. guilfoylei leaf oil is a significant finding towards its anticancer drug potentials.

ETHICS APPROVAL AND CONSENT TO PARTICI- PATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No Animals/Humans were used for studies that are base of this research.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

Not applicable.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

The authors acknowledge funding from the Plan Project Scheme of the Government of Kerala. We express our sincere thanks to Dr. A. Jayakumaran Nair, Department of Bio- technology, University of Kerala, Thiruvananthapuram for flow of cytometric analysis.

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