The Chemical Investigation of Four Balanophora Species and Cytotoxicity with Inhibition of NO Production
Nguyen Thanh Tung1, Nguyen Viet Than1, Nguyen Quang Hung2,3, Ain Raal4*
1Department of Pharmacognosy, Hanoi University of Pharmacy, Le Thanh Tong 15, 11000 Hanoi, Vietnam.
2Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology (VAST), Hoang Quoc Viet 18, 11300 Ha Noi, Vietnam.
3Graduate University of Science and Technology, VAST, Hoang Quoc Viet 18, 11300 Ha Noi, Vietnam.
4Institute of Pharmacy, Faculty of Medicine, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
*Email: [email protected]
ABSTRACT
The phytochemistry and biological activity of four rare Balanophora species (B. subcupularis P.C. Tam (BS), B. tobiracola Makino (BT), B. fungosa var. globosa (Jungh.) B.Hansen (BFG), and B. fungosa subsp. indica (Arn.) B.Hansen (BFI)) growing in Vietnam have been studied. The six pentacyclic triterpenoids including β-amyrin, β-amyrone, lupenone, lupeol, β-amyrin acetate, and lupeol acetate could be identified in all GC-MS chromatograms of four species but very different area percentage. The chemical investigation of B. subcupularis and B. tobiracola led to the isolation of coniferyl aldehyde, gallic acid, cinnamic acid, and caffeic acid. Gallic acid and coniferyl aldehyde were isolated from B. subcupularis for the first time, Among isolated compounds, coniferyl aldehyde showed moderate cytotoxicity activity at cancer cell lines MCF-7, A549, Hep3B, and PC3 at the concentration of 100 µg/ml (CS% = 24.05 – 46.61%). Besides, this compound also significantly inhibited NO production (I% = 101.96 and 114.29 at two respectively concentrations of 30 µg/ml and 100 µg/ml).
Key words:Balanophora subcupularis, Balanophora tobiracola, Isolation, Biological activity, HPTLC, GC-MS
INTRODUCTION
In Vietnam, Genus Balanophora J.R. & G.Forst. consisted of eight species, one subspecies, and one variety with three species including B. subcupularis, B. harlandii, and B. tobiracola were recently recorded to Flora of the country [1-5]. Local people used some species belonging to this genus to treat some different diseases as body aches, abdominal pain or to help strengthen muscles and bones [6]. Chemical investigation of Balanophora species led to the isolation of some different classes of compounds such as hydrolyzable tannins, phenylpropanoids, and triterpenes [7]. High-speed counter-current chromatography was used to separate and purify galloyl, caffeoyl, and hexahydroxydiphenoyl esters of glucoses from the aerial parts of the parasitic plant B. simaoensis (syn. B. fungosa subsp. indica) [8]. Recently, two new butenolides were isolated from aerial parts of B. fungosa while three new muurolane-type sesquiterpene glycosides were isolated from the whole plant of B. fungosa subsp. indica [9, 10]. Lately, some lignans and phenolic compounds have been isolated from ethyl acetate fraction of B. fungosa var. globosa (Jungh.) B.Hansen growing in Vietnam [11]. The comparative HPTLC analysis of five Balanophora species including B. simaoensis, B. spicata, B. laxiflora, B. dioica, and B. polyandra in China led to the identification of some triterpenoids including β-amyrin palmitate (balanophorin A), lupeol palmitate (balanophorin B), β-amyrin acetate and lupeol acetate in all chromatograms of five species [12].
Our study aimed to investigate the chemical composition and to develop HPTLC and GC-MS to compare the chemical compositions of the aforementioned species, as well as evaluate their biological activity, along with isolation and identification of some compounds from B. subcupularis and B. tobiracola. This is the first time the chemical composition of B. subcupularis was investigated, as well as some triterpenoids, were identified in four Balanophora species using GC-MS, and some biological activity of four Balanophora species were evaluated.
MATERIALS AND METHODS
Plant material
Whole plants of B. fungosa subsp. indica (Arn.) B.Hansen (BFI) (Lao Cai prov., January 2017, 22°19’35” N 103°48’21” E, Voucher specimens (VS) number HNU 024069), B. fungosa var. globosa (Jungh.) B.Hansen (BFG) (Lam Dong prov., January 2016, 12°05’23”N 108°27’10”E, VS number HNU 024066), and two recently recorded species for Flora of Vietnam including B. tobiracola Makino (BT) (Lang Son prov., January 2018, 21°53’33’’N 106°22’57’’E, VS number HNU 024056) and B. subcupularis P.C. Tam (BS) (Dien Bien prov., November 2017, 22°03’56” N 103°06’13” E, VS number HNU 024068) were collected and authenticated by Prof. Phan Ke Loc and MSc. Nguyen Anh Duc, Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi, Vietnam. Voucher specimens of studied species were deposited at the Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi, Vietnam. The sliced plant material was dried in a ventilated oven and stored in sealed PE bags.
Chemicals and reagents
The solvents and chemicals used for extraction, isolation, and TLC analyses are of analytical standard. The reference compounds including caffeic acid, cinnamic acid, coniferyl aldehyde, gallic acid, and lupeol were obtained from Biopurify phytochemicals Ltd. (China).
Extraction and isolation
The dried and powdered whole plants of BFI, BFG, BT, and BS were ultrasonically extracted with methanol (3 times for 30 min). The total extract of each species was concentrated in vacuo. Then the crude extract was suspended in water and partitioned between n-hexane and ethyl acetate successively. The obtained fractions were evaporated in vacuo and used for further analysis.
The ethyl acetate residue of B. subcupularis (8 g) was chromatographed on a silica gel column eluted by 1, 10, 20, 40, 80, and 100% of methanol in dichloromethane to give 6 fractions BSE1-6. The fraction BSE1 by a silica gel column eluted by dichloromethane to obtain compound S1 (3.3 mg) (1). The fraction BSE-5 was separated by a silica gel eluted by 0-90% of methanol in dichloromethane to afford S2 (3.5 mg) (2).
The ethyl acetate residue of B. tobiracola (10 g) was separated by silica gel eluted by 1, 10, 20, 40, 80, and 100% of methanol in dichloromethane to give 6 fractions BTE1-6. The fraction BTE2 by a silica gel column eluted by 10, 20, 30, and 40 % of methanol in dichloromethane afforded two sub-fractions BTE2.1 and BTE2.2, combined based on TLC analysis. BT3.1 (4.2 mg) (3) was purified from BTE2.2. Fraction BTE5 was chromatographed by Sephadex LH-20 CC with methanol to give 3 sub-fractions BTE5.1-BTE5.3. BT6.1 (3.1 mg) (4) was isolated from BTE5.2 using preparative TLC.
The identification of the structure of compound base on MS and NMR spectral analysis, as well as comparison to commercial standards. 1H NMR spectra were recorded using Bruker Avance III HD spectrometer at 500 MHz. Methanol-d4 or DMSO was used as solvents. Structural assignments were performed by comparing the spectra with literature data.
High-performance thin layer chromatography
High-performance Thin layer chromatography is still a very useful method in the research of medicinal plants because of its simplicity and great flexibility. In our study, the HPTLC of samples was developed using an HPLTC system (CAMAG, Switzerland). The plate was developed in Auto Developing Chamber (ADC-2, CAMAG) using various Eluent Systems with different polarizations. The chromatographic evaluations were done with vision CATs software.
In our previous study, gallic acid, caffeic acid, and methyl caffeate have been identified in the HPTLC chromatogram of four species [11]. In this study, for the identification of cinnamic acid and coniferyl aldehyde, these two compounds were dissolved in methanol to a concentration of 1.0 mg/mL. The crude residues of each species (0.1 g) were dissolved in methanol (10 mL). The same volume (10 µl) of the samples and reference solutions were applied to the same HPTLC plate using Linomat V (CAMAG, Switzerland). After development, the plate was taken images at a wavelength of 254 nm and 366 nm using TLC-Visualizer (CAMAG). Then the plate was derivatized with anisaldehyde – sulphuric acid (AS) reagent and taken the image in white light.
For the HPTLC analysis of triterpenoids, n-hexane residues of each species extract (50 mg) were dissolved in methanol (5 mL). The same volume of each solution (10 µL) was applied on the same HPTLC silica gel 60 F254 plate and C18 RP-HPTLC plate using Linomat 5 applicator (CAMAG) and respectively developed using two eluent systems n-hexane – ethyl acetate (5:1, v/v) and ethyl acetate – acetonitrile (3:2, v/v) using ADC-2. After development, the plates were sprayed with AS reagent, heated at 120°C for 5-10 min, then evaluated in vis. or UV-366 nm.
Gas chromatography-mass spectrometry
Triterpenoids have been investigated by some chromatographic techniques such as gas chromatography (GC) [13, 14], high-performance thin layer chromatography (HPTLC) [15], and mixed-mode liquid chromatography-tandem mass spectrometry [16]. In this study, the GC-MS analysis has been performed to confirm the presence of triterpenoids in n-hexane fractions of four Balanophora species. The n-hexane fractions (10 mg) were dissolved in the mixture of n-hexane – chloroform (1:4, v/v) (5 mL) and submitted to filtration over a small silica column gel to obtain the solutions used for GC-MS analysis. The GC-MS studies were performed on a GC system Agilent 7890A coupled with an Agilent 5975C inert MSD and equipped with a DB-5 MS capillary column (30m x 250 µm x 0.25 µm). Column constant flow was set at 1 mL/min. The system was operated under the following conditions: inlet temperature 300 °C; MSD transfer line temperature 300 °C; oven temperature program 200 (3 min) then 2 °C/min to 300 °C, run time 53 min. The mass spectrometer was monitored to scan m/z 40-550 with an ionizing voltage at 70 eV. The individual peaks were identified by their fragmentation patterns to those in library NIST 08 thanks to NIST MS search 2.0 software. Some minor peaks in the chromatograms remained unidentified because of the lack of library mass spectra of the corresponding compounds.
Cytotoxic assay
The cytotoxicity of the total residues of four aforesaid species and some isolated compounds were evaluated by using the previously described method [11], using MTT cell proliferation assay kits. The following four cancer cell lines have been studied: MCF-7 (human breast carcinoma), A549 (human lung carcinoma), Hep3B (human liver cancer), PC3 (human prostate gland carcinoma). The cells were cultured in RPMI-1640 or DMEM at 37oC with 5% CO2, supplemented with 10% FBS, penicillin, and streptomycin sulfate. They were grown in a 96-well flat-bottom plate (200 µL, 1 × 105 cells/well, in triplicated) and incubated for 24 h. After that, DMSO solutions of the residues at concentrations of 30 and 100 µg/ml were added to the wells. Then the treated cells were incubated for 48 h at 37oC, followed by the MTT assay. The absorbance (OD; λ = 570 nm) was recorded and the cell survival was determined using non-linear regression analysis. Camptothecin (Sigma-Aldrich, St. Louis, Missouri, USA) was used as a positive control.
NO production in LPS-stimulated RAW264.7 cells
Besides, the inhibitory effect of residues on the NO production in LPS-stimulated RAW 264.7 cells was also evaluated using the previously described method [11]. Cells were cultured in DMEM, at 37oC with 5% CO2, supplemented with 10% FBS, penicillin (100 units/mL) and streptomycin sulphate (100µg/mL). Then they were seeded in a 96-well plate at 2.5 × 105 cells/well, in triplicate. The cells were treated with two concentrations of samples prepared in DMSO (30 and 100 µg/ml), followed by incubation for 24 h. The Griess method was used to estimate the nitrite concentration in the culture supernatant. Besides, the remaining cell solutions were evaluated by measurement of cell viability with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cardamonin (Sigma-Aldrich, St. Louis, Missouri, USA) was used as a positive control.
RESULTS AND DISCUSSION
HPTLC of balanophora species
For the identification of cinnamic acid and coniferyl aldehyde, the combination of toluene, ethyl acetate, and formic acid (14:10:0.5) (v/v/v) as mobile phase gave well-separated, compact, and symmetrical bands (Figure 1). Some similar profiles of separated bands of Balanophora species could be observed in both λ = 254 nm and λ = 366 nm before derivatization as well as white light after derivatization. Besides, coniferyl aldehyde (Rf 0.47) could be detected in the chromatogram of BS as a bright blue band at white light while cinnamic acid (Rf 0.51) could be detected in the chromatogram of BT at UV 254 nm (B. tobiracola).
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Figure 1. HPTLC chromatogram of four species developed with the eluent system toluene – ethyl acetate – formic acid (14:10:0.5) (v/v/v)
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Note: a) plate observed at 254 nm, b) Plate observed at 366 nm, c) Plate observed at white light after derivatization. BFG - Balanophora fungosa var. globosa, BFI - B. fungosa subsp. indica, BS - B. subcupularis, BT - B. tobiracola, Co - Coniferyl aldehyde, Ci - Cinnamic acid
The comparative HPTLC chromatograms of n-hexane fractions from four species developed on HPTLC silica gel 60 F254 plates showed six similar bands (Figure 2A) while chromatograms developed on the C18 RP-HPTLC plate had five similar bands (Figure 2B). The purple color of the bands indicated that they could be triterpenoids. These results suggested that there were similar components in n-hexane fractions of four species.
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