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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

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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 37^oC 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 37^oC, 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 37^oC 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 (R_f 0.47) could be detected in the chromatogram of BS as a bright blue band at white light while cinnamic acid (R_f 0.51) could be detected in the chromatogram of BT at UV 254 nm (B. tobiracola).

a)	b)	c)
Figure 1. HPTLC chromatogram of four species developed with the eluent system toluene – ethyl acetate – formic acid (14:10:0.5) (v/v/v)

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.

a)	b)	a)	b)
A)		B)
Figure 2. HPTLC chromatogram of n-hexane fractions from four species belonging to genus Balanophora developed under two different conditions. (A) HPTLC silica gel plate developed with n-hexane – ethyl acetate (5:1) at visual illumination (a) and 366 nm (b); (B) C18 RP-HPTLC plate developed with ethyl acetate – acetonitrile (3:2) at visual illumination (a) and 366 nm (b)

 

GC-MS analysis of pentacyclic triterpenes

Total ion chromatograms of n-hexane fractions of four Balanophora species and chemical structures of some identified compounds are shown in Figure 3. Six pentacyclic triterpenes including β-amyrone, β-amyrin, lupenone, lupeol, β-amyrin acetate, and lupeol acetate were identified in all chromatograms of four species but in different area percentage (Table 1).

β-Amyrone (βAm1) was eluted a little bit sooner than β-amyrin. The mass spectrum of this metabolite presented fragment ions at m/z 218, 203, 189 which are similar to β-amyrin. However, there was an additional fragment ion at m/z 424 corresponding to molecular formula C₃₀H₄₈O instead of the fragment ion at m/z 426 of β-amyrin. B. fungosa var. globosa (12,31 %) và B. subcupularis (13,06 %) have the high are the percentage of this component. β-Amyrin (βAm2) could be easily identified in chromatograms of four species with the base peak at m/z 218 in the mass spectra which is characterized for Δ-12 ursane or olean skeleton, the relative abundance of fragment ion at m/z 203 is three times higher than of fragment ion at m/z 189; the parent ion peak at m/z 426 suggested a molecular formula of C₃₀H₅₀O. Among four species, B. subcupularis possesses the highest area percentage of this component in the chromatogram (10,41 %).

Lupenone (L1) (1,65 – 11,27 %) and lupeol (L2) (1,20 – 11,25 %) were also identified in all chromatograms of 4 species. Lupenone had a base peak at m/z 205 and some other fragment ion at m/z 189, m/z 409 [M⁺ - CH₃]; the presence of the parent ion at m/z 424 suggested the molecular formula of C₃₀H₄₈O. On the other hand, lupeol had a base peak at m/z 207 and some other characteristic fragment ion at m/z 411 [M⁺ - CH₃], m/z 408 [M⁺ - H₂O], m/z 393 [M⁺ - CH₃ - H₂O], and the parent ion m/z at 426 suggesting the molecular formula of C₃₀H₅₀O. The highest is the percentage of lupeol and lupenone in chromatogram belonged to B. subcupularis.

β-amyrin acetate (βAm3) was eluted 3 min later than β-amyrin. The fragmentations of β-amyrin acetate were quite similar to β-amyrin but there was an additional present of fragment m/z 43 equivalent to acetyl. This component was the main constituent in chromatograms of B. tobiracola, B. fungosa subsp. indica, B. fungosa var. globosa (31,96 – 39,82 %), however in very low concentration in chromatogram of B. subcupularis (0,50 %). Lupeol acetate (L3) is the latest eluted compound among identified pentacyclic triterpenes. This is the main component in all chromatograms of four Balanophora species (23,28 – 36,19 %). The mass spectrum of lupeol acetate showed a base peak at m/z 189. Some other fragments included m/z 468 [M⁺], m/z 453 [M⁺ - CH₃], m/z 408 [M⁺ - CH₃COO].

 

Table 1. Retention indices, formula, and MS data of compounds identified in n-hexane fractions of four Balanophora species

Compound	RI*	Formula	M+	Production (relative abundance %)	% Area
					BFI	BFG	BT	BSP
β-Amyrone (βAm1)	3249	C30H48O	424 (10)	218 (100), 203 (62), 44 (26), 219 (19), 189 (16), 207 (14), 83 (14), 95 (14), 41 (13), 205 (13), 409 (6) (M+-15)	2.8	12.3	1.0	13.1
β-Amyrin (βAm2)	3278	C30H50O	426 (6)	218 (100), 203 (48), 44 (20), 83 (19), 207 (18), 219 (17), 85 (14), 189 (14), 95 (11), 69 (10), 411 (2) (M+-15)	3.9	1.8	1.5	10.4
Lupenone (L1)	3298	C30H48O	424 (42)	205 (100), 109 (72), 83 (66), 44 (64), 107 (59), 95 (57), 93 (54), 121 (53), 81 (52), 189 (44), 409 (28) (M+-15), 381 (6) (M+- 43)	4.3	4.2	1.7	11.3
Lupeol** (L2)	3326	C30H50O	426 (40)	207 (100), 83 (70), 189 (69), 95 (67), 44 (65), 135 (62), 109 (61), 107 (60), 121 (58), 93 (58), 411 (18) [M+-15], 393 (7), 383 (6)	3.0	1.2	1.7	11.3
β-Amyrin acetate (βAm3)	3375	C32H52O2	468 (2)	218 (100), 203 (48), 219 (18), 189 (17), 43 (16), 204 (10), 69 (10), 95 (10), 119 (10), 135 (9)	39.0	32.0	39.8	0.5
Lupeol acetate (L3)	3426	C32H52O2	468 (28)	189 (100), 43 (69), 121 (57), 95 (56), 107 (54), 93 (53), 135 (52), 109 (46), 81 (46), 190 (42), 453 (12) [M+-15], 408 (10)	23.3	30.7	30.7	36.2
Total					76.3	82.2	76.4	82.8

^*RI: retention indices calculated on a DB-5MS column using a homologous series of straight-chained alkanes (C8-C40) analyzed under the same condition; ^**Structure of lupeol was confirmed with commercial standard

 

Figure 3. Total ion chromatograms of four Balanophora species and chemical structures of identified triterpenes Note:  β-amyrone (βAm1), β-amyrin (βAm2), lupenone (L1), lupeol (L2), β-amyrin acetate (βAm3), lupeol acetate (L3)

Isolated compounds

The ethyl acetate residues of BS and BT were chromatographed via various chromatography columns to obtained four compounds. These isolated compounds were identified as coniferyl aldehyde (1), gallic acid (2), cinnamic acid (3), and caffeic acid (4) based on NMR spectral data and MS data, as well as comparison to commercial standards of gallic acid, cinnamic acid, caffeic acid, and coniferyl aldehyde (Figure 4). (see supplementary file)

 

Figure 4. Isolated compounds from ethyl acetate fractions of B. subcupularis and B. tobiracola

Bioassay

Coniferyl aldehyde showed a significant inhibitory effect on NO production, whereas the inhibitory ability of two taxa of B. fungosa (BFI and BFG) at a concentration of 100 µg/mL was 51.26 and 65.01 % (Table 2). On the other hand, B. subcupularis and B. tobiracola showed very weak activity.

Table 2. Inhibitory effect of the total residues and isolated compounds on NO production

Samples	Concentration (µg/mL)	% Inhibitory	±SD	% Cell survival	±SD
BFI	30	10.08	1.13	99.22	0.84
	100	51.26	0.52	93.03	0.44
BFG	30	25.21	1.26	80.77	0.38
	100	65.01	0.9	69.62	2.33
BT	30	26.75	2.61	86.47	1.75
	100	47.3	1.36	85.21	2.4
BSP	30	29.19	1.59	92.3	2.08
	100	36.27	2.7	91.27	2.18
Coniferyl aldehdyde	30	101.96	0.14	75.25	0.24
	100	114.29	0.14	63.65	0.24
Cardamonin*	0.3µM	30.25	0.38	91.45	0.33
	3µM	79.83	1.01	82.96	1.37

*The positive control.

B. subcupularis (BS) is the only species that inhibited weak cytotoxicity against the cancer cell line A549 (human lung carcinoma) at a concentration of 100 µg/mL (CS% = 48.29 %). Total residues of other species demonstrated weak cytotoxicity. Among isolated compounds, coniferyl aldehyde showed cytotoxicity against all four cancer cell lines at a concentration of 100 µg/mL (CS% = 24.05 – 46.61%) (Table 3).

Table 3. Cytotoxicity of the total residues and isolated compounds

Samples	Concentration (µg/mL)	% Cell survival (CS%)
		MCF-7		A549		Hep3B		PC3
		CS%	±SD	CS%	±SD	CS%	±SD	CS%	±SD
BFI	30	80.41	1.19	66.48	1.86	67.56	1	84.48	0.14
	100	66.9	1.93	50.32	1.61	45.9	0.84	65.22	0.23
BFG	30	94.35	1.92	68.09	1.72	75.34	1.97	90.51	2.49
	100	78.08	1.72	60.01	2.53	63.73	1.34	68.12	1.9
BT	30	88.36	1.52	64.3	1.97	85.83	1.81	68.39	2.38
	100	65.29	2.05	62.47	1.38	70.68	2.06	59.07	4.25
BS	30	76.47	3.41	62.33	2.06	93.3	1.59	84.67	13.34
	100	72.58	1.95	48.29	2.78	88.63	3.11	67.55	3.89
Coniferyl aldehdyde	30	64.22	0.41	64.56	2.95	65.11	0.71	56.9	2
	100	24.72	1.2	46.61	1.96	43.07	3.12	24.05	1.13
Camptothecin*	0.1µg/mL	54.46	2.37	50.05	2.1	71.1	1.23	53.81	0.14
	10µg/mL	19.87	1.97	26.52	0.86	26.04	2.19	12.3	0.62

* The positive control. MCF-7 (human breast carcinoma), A549 (human lung carcinoma), Hep3B (human liver cancer), PC3 (human prostate gland carcinoma)

Generally, HPTLC and GC-MS profiles of the Balanophora species were analogous with a varied intensity which suggested the presence of similar phytochemicals with different concentrations. Among identified triterpenoids using GC-MS, some compounds possessed important pharmacological activity: lupeol (lupane type) has been shown to possess some significant biological activity such as anti-inflammatory, antiarthritic, antigeogenic, antioxaluric and anticalciuric, and metastatic melanoma inhibitory for human [17], while lupeol acetate dose-dependently inhibited nitric oxide (NO) production, iNOS and COX-2 expression in LPS-stimulated macrophage RAW 264.7 cells and may take part in the antinociceptive and anti-inflammatory effect of B. spicata [18].

All six triterpenoids (lupenone, lupeol, lupeol acetate, β-amyrin, β-amyrin acetate, and lupeol acetate) were isolated in different species belonging to the genus Balanophora J.R.&G. Forst. in previous studies, especially from B. fungosa subsp. indica (syn. B. simaoensis, B. indica) with lupenone, lupeol acetate, β-amyrin, β-amyrin acetate, and β-amyron were isolated and identified from this species [7]. Besides, lupeol was isolated from B. tobiracola [19]. However, there was no report on isolation or identification of these compounds in B. fungosa var. globosa and B. subcupularis. This is the first time these compounds were identified in these species with the same triterpenoids were identified in GC-MS chromatograms of four species studied by us. Thus, the content of triterpenes is typical to all Balanophora species studied by us and other authors, but their different proportions may affect the pharmacological effects of different Balanophora species.

Our previous study showed that gallic acid and caffeic acid could be detected in HPTLC chromatograms of the four above-stated species [11]. In this paper, the qualitative HPTLC of coniferyl aldehyde and cinnamic acid showed that there were some differences in their chromatograms with coniferyl aldehyde could be detected in B. subcupularis while cinnamic acid could be detected in B. tobiracola. The results obtained from GC-MS analyses of four previously mentioned Balanophora species are compatible with the HPTLC analysis of n-hexane fractions from these species. Thus, the content of triterpenoids is typical to all Balanophora species studied by us and other authors [7], but their different proportions may affect the pharmacological effects of different Balanofora species.

 

CONCLUSION

Gallic acid and coniferyl aldehyde were isolated from B. subcupularis for the first time, whereas cinnamic acid and caffeic acid were isolated from B. tobiracola growing in Vietnam. Besides, six pentacyclic triterpenoids were identified in all chromatograms of four Balanophora species but different area percentage. The differences in the proportion of compounds could affect the pharmacological effects of different Balanophora species. Among four species, B. subcupularis showed weak cytotoxicity against the cancer cell line A549. On the other hand, two taxa of B. fungosa (B. fungosa subsp. indica and B. fungosa var. globosa) possessed a better inhibitory effect on NO production than two B. tobiracola and B. subcupularis. Besides, coniferyl aldehyde which is a principal compound of B. subcupularis showed cytotoxicity against all four cancer cell lines at a concentration of 100 µg/ml as well as a significant inhibitory effect on NO production.

ACKNOWLEDGMENTS: The authors of the article thank the Institute of Pharmacy, Faculty of Medicine, University of Tartu for paying the publication fee.

CONFLICT OF INTEREST: None

FINANCIAL SUPPORT: None

ETHICS STATEMENT: The study do not involve humans and animals and do not include any other ethical conflicts.