Cytotoxic New Nortriterpene from Roots of Potentilla atrosanguinea var. argyrophylla and its UPLC Quantification

Abstract

Potentilla atrosanguinea is well known for its ethnomedicinal uses since ancient times. The present study includes the isolation of a new nortriterpene, 28-methyl-acanthochlamate (1), from the roots of P. atrosanguinea. The structure of 1 was established from HR-ESI-MS and NMR spectroscopic analysis. Its relative stereochemistry was set with the help of NOESY correlation experiment. Compound (1) has been quantified by a new developed UPLC-DAD method (1.1 mg/g). The in vitro cytotoxicity of the new compound (1) was evaluated by sulforhodamine B assay against three cancer cells; human cervical cancer (SiHa), epidermal carcinoma (KB), and human adenocarcinoma (Colo-205). The new isolated compound (1) showed a significantly higher cytotoxicity against all the cells (SiHa, IC₅₀ 30.5 µg/mL; KB, IC₅₀ 22.6 µg/mL and Colo-205, IC₅₀ 18.8 µg/mL).

Key words

Potentilla atrosanguinea - Rosaceae - 28-methyl-acanthochlamate - quantification - cytotoxicity

The therapeutic effects of more than 300 species of the Potentilla genus (Rosaceae) are well mentioned in traditional medicinal systems. Potentilla species are reported for their various pharmacological activities such as antioxidant, antiulcerogenic, antineoplastic, antidiabetic, and hepatoprotective [1], [2], [3], [4], [5]. Mainly polyphenols and triterpenoids have been reported from the Potentilla genus [6]. Potentilla atrosanguinea var. argyrophylla (Wall. ex Lehm.), is commonly known as Himalayan cinquefoil. The root decoction of P. atrosanguinea has been used to cure toothache and leaves paste used in cut and wound healing [7]. Its root parts are used in human food and leaves as healthful tea. There is no report available in the literature regarding the isolation of chemical constituents from P. atrosanguinea except few reports [8], [9], [10].

In the present study, one new nortriterpene type compound has been isolated from a combined chloroform and ethyl acetate fraction of ethanol extract of the root part of P. atrosanguinea ([Fig. 1]). Compound 1 was obtained as fine white crystals having a molecular formula of C₃₀H₄₆O₄, determined by HR-ESI-MS (obsd. [M + H]⁺ at m/z 471.3465, calcd. [M + H]⁺ 471.3474) which suggested eight degrees of unsaturation. Five methyls, elevan methylenes, five methines and eight quaternary carbons were observed from ¹³C NMR and the DEPT spectra. NMR data showed that the molecule contains 30 carbon atoms with one methoxy and two carboxyls groups. Two terminal olefinic bonds with signals at δ _H 4.87 (2H, H-23) and δ _H 4.71, 4.58 (H-29) and five tertiary methyls singlets at δ _H 0.81(H-25), 0.94 (H-26, H-27), 1.71(H-24), 1.65 (H-30) were observed ([Table 1]). The presence of one methoxyl carbon in NMR data suggested that the basic skeleton of the molecule contains 29 carbon atoms. The interpretation of 2D NMR spectra and its comparison with a previous report [11] indicated that the molecule has a nortriterpene (lupane type) skeleton with the attachment of methyl to one of the carboxylic groups. Cross-peak correlations for H-5 to C-4, C-23, C-10 and for H-1 to C-2 and C-10 in the HMBC spectrum ([Fig. 2]) confirmed the position of one exocyclic double bond at C-4 and one carboxylic group at C-2 position. The HMBC correlations of H-18 with C-13, C-17, C-19, C-20 and H-19 with C-29 and C-18 confirmed the presence of another exocyclic double bond at C-20 position. Another carboxyl group positioned at C-17 because of cross peak correlations of H-22 with C-17 (δ _c 56.6) and C-28 (δ _c 176.7) in HMBC spectra. The attachment of a methyl group at the C-28 carboxylic group was determined by three bond correlations between the methyl proton (δ _H 3.64 s) and carbon of C-28 (δ _c 176.7) position. By comparing our interpretations with a previous report [11], the structure of compound 1 was established as 28-methyl-acanthochlamate.

Fig. 1 Structure of 28-methyl-acanthochlamate (1).

Fig. 2 Key HMBC (→) and NOESY (↔) spectral data of compound 1.

Table 1 NMR data of compound 1.

Position	δ13C		δ1H	Key HMBC	Key NOESY
1	43.0	CH2	2.42 (d, J = 14.9 Hz) 2.25 (d, J = 15.3 Hz)	C-2, C-10
2	178.0	quat	–
4	147.2	quat	–
5	49.9	CH	2.56 (d, J = 12.7 Hz)	C-4, C-10, C-23	H-9
6	24.1	CH2	1.38 (m), 1.32 (m)
7	32.4	CH2	1.40 (m)
8	40.7	quat	–
9	41.8	CH	1.95 (d, J = 12.4 Hz)
10	41.3	quat	–
11	22.6	CH2	1.54 (m)
12	25.3	CH2	1.69 (m)
13	38.3	CH	2.22 (m) (overlapped)	C-14	H-19
14	42.8	quat	–
15	30.6	CH2	1.88 (m), 1.34 (m) (overlapped)
16	32.1	CH2	2.19 (d, J = 13.3 Hz) 1.03 (d, J = 12.8 Hz)	C-17
17	56.6	quat	–
18	49.4	CH	1.56 (m)	C-13, C-17, C-20
19	47.0	CH	2.97 (m)	C-18
20	150.3	quat	–
21	29.7	CH2	1.17 (m), 1.29 (m)
22	37.0	CH2	1.37 (m), 1.86 (m) (overlapped)	C-17, C-28
23	114.8	CH2	4.87 (d, J = 13.7 Hz)		H-25
24	22.6	CH3	1.71 (s)	C-23
25	19.6	CH3	0.81 (s)	C-2	H-26
26	14.5	CH3	0.94 (s)
27	15.8	CH3	0.94 (s)
28	176.7	quat	–
29	109.8	CH2	4.58 (s), 4.71 (s)	C-19
30	19.3	CH3	1.65 (s)	C-29
OCH3	51.3		3.64 (s)	C-28

The cross peak correlations of β oriented H-25 with H-26 and H-23 were observed. The stereochemistry was assigned at position C-5, C-10 and at C-8 as S, S and R, respectively, similar to the previous report [11]. Therefore, by literature support and NOESY interactions, the stereochemistry at C-5, C-8, C-9, C-10, C-13, C-14, C-17, C-18 and C-19 centers were assigned as S, R, R, S, R, R, S, R and R, respectively. From the observed spectroscopic data and previous literature, the final structure of compound 1 was assigned as 28-methyl-5S, 8R, 9R, 10S, 13R, 14R, 17S, 18R, 19R-acanthochlamate. All the spectral data are available as Supporting Information.

Compound 1 has been quantified by developing a new simple and efficient UPLC method ([Fig. 3]). The sample was analyzed at different temperatures (19, 20, 21, 23, 22, and 24 °C), and better results were observed at 22 °C. An isocratic solvent system, 0.01 % formic acid in acetonitrile (ACN; 91 %) and 0.01 % formic acid in water (9 %) was optimized. For identification of compound 1 in the sample, retention time (t_R 3.24) and UV λ _max 194 were compared with the standard. The calibration curve was prepared by injecting six concentrations (0.0156–0.5 mg/mL) of compound 1. A good linearity range of the method was obtained with regression coefficients (R² 0.999). The other parameters were obtained as limit of detection (LOD) 0.0156 mg/mL, limit of quantification (LOQ) 0.0468 mg/mL, percentage relative standard deviation (%RSD) for intra-day 0.64; inter-day precision (%RSD) 1.25, percentage recovery 80–83.5 %, and %RSD for recovery 2.1 % ([Table 2]). Compound 1 was quantified in the root part of P. atrosanguinea as 1.1 mg/g of dry plant material. Compound 1 showed significant activity on SiHa and KB cells (84.6 % ± 1.3, p < 0.001 and 89.5 ± 1.8, p < 0.08, respectively) at 100 µg/mL. Whereas, against Colo-205 it showed remarkable activity (84.2 ± 2.4, p < 0.004) at 25 µg/mL. The IC₅₀ values of Compound 1 ranges between 18.8 to 30.5 µg/mL ([Table 3]). The percentage cytotoxicity of compound 1 is provided in Table 1 S, Supporting Information. Results were expressed as percentage cell inhibition.

Fig. 3 UPLC-DAD chromatograms. Standard (a) and sample(b).

Table 2 Method validation data of compound 1.

Regression Equation	Linearity*	R2	LOD*	LOQ*	Intraday Precision (n = 6)	Interday Precision (n = 3)	Recovery % ± %RSD
* Data expressed in mg/mL
y = 24 958 x-92.55	0.0156–0.5	0.999	0.0156	0.0468	0.64	1.25	82.2 ± 2.1

Table 3 IC₅₀ (µg/mL) value of compound 1.

Sample	SiHa	KB	Colo-205
IC50 (µg/mL)	30.5	22.6	18.8

Materials and Methods

General

NMR spectra were recorded in CDCl₃, on Bruker Advance-600 spectrometer (600 MHz for ¹H and 150 MHz for ¹³C). IR was measured by using an IRPrestige FT-IR spectrophotometer (Shimadzu). A Waters ACQUITY Ultra High Performance LC system (Waters, Milford, MA, USA) was used for the quantification analysis. The system has been equipped with autosampler, PDA detector, binary solvent delivery pump, and Masslynx v4.1 software. ACN, water and formic acid used were LC/MS/MS grade solvents and purchased from J. T. Baker (Avantor Performance Material Inc.). All the other used chemicals and solvents were of analytical grade.

Plant material

Plant material was collected from Kunjam Pass, situated at an elevation of 4590 m in Lahaul and Spiti district of Himachal Pradesh, India in the month of August 2013 and identified by taxonomist Dr. Brij Lal. A specimen was deposited in the herbarium at CSIR-IHBT, Palampur, India (voucher number-PLP 16 515).

Extraction and isolation

The dried underground part (1.5 kg) of P. atrosanguinea was percolated with 95 % EtOH at room temperature (3.5 L × 1, followed by 1.5 L × 7, 95 : 5, v/v). The extracts were combined and dried under vacuum at 45 °C with yield 67 g. A part of the extract (57 g) was suspended in distilled water followed by successive partitions with CHCl₃ (500 mL × 3, 8 g), EtOAc (500 mL × 3, 11.6 g) and n-butanol (500 mL × 3, 20 g). The CHCl₃ and EtOAc extracts were combined on the basis of similar TLC profiling.

16 g (pooled CHCl₃ and EtOAc extract) was applied to a dry column packed with silica 230–400 mesh size and eluted with step gradients EtOAc-Hexane (up to 10 %), EtOAc : CHCl₃:MeOH : H₂O (15 : 8 : 2 : 0.5 followed by 15 : 8 : 4 : 1). Seventeen major fractions were collected. One compound (1) was isolated from fraction II. Compound 1 was crystallized after elution from normal phase silica gel (230–400) column by 10 % EtoAc in hexane.

28-Methyl acanthoclamate (1): isolated as white fine needles (40 mg); [α]_D ^18.5 + 25.0 (c 0.001, MeOH); IR (neat): 2958, 2920, 1728 cm⁻¹; MP: 153–155 °C; HRMS/ESI, m/z: obsd. [M + H]⁺ 471.3465, calcd. [M + H]⁺ 471.3474.

UPLC quantification of compound 1

The stock solutions of compound 1 and sample were prepared using methanol with concentrations of 0.5 mg/mL and 5 mg/mL, respectively, and filtered through a Whatman PTFE syringe filter (size 0.2 µm). For analysis, UPLC BEH C18 (Waters Acquity; 2.1 mm × 100 mm with particle size 1.7 µm) was used. The sample was analyzed at 205 nm PDA detection wavelength with 0.5 µL of injection volume. For the validation of the developed method, all the parameters were evaluated (see Supporting Information).

Sulforhodamine B assay

SiHa, KB and Colo-205 were obtained from National Centre for Cell Science, Pune, India. SiHa and KB cells were grown in Dulbeccoʼs modified Eagleʼs medium (DMEM; Invitrogen Sciences) while Colo-205 cultured in F-12 HAMS medium (Invitrogen Biosciences), supplemented with 10 % heat-inactivated fetal bovine serum (Invitrogen Biosciences) and 1 % antibiotic and antimycotic solutions (Invitrogen Biosciences). Cells were maintained at 37 °C with 5 % CO₂ and 95 % humidity in a CO₂ incubator [12], [13].

Cells were trypsinized, washed twice with phosphate-buffered saline (PBS) and grown at a density of 2 × 10⁴ cells/well in 96 well plates. Different concentrations (10, 25, 50 and 100 µg/mL) of compound 1, in complete medium were added. Vinblastine (1 µM; Sigma-Aldrich) was used as positive control, whereas cells supplemented with complete medium only were used as negative control. Plates were incubated at 37 °C for 48 h in a CO₂ incubator. After 48 h, 50 µL of 50 % trichloroacetic acid (Merck) was added and the plates were kept at 4 °C for 1 h. The plates were flicked and washed five times with water and air-dried. Subsequently, 100 µL SRB solution (in 1 % GAA) was added and incubated for 30 min at room temperature. After incubation, plates were washed six times with 1 % GAA, air dried and 10 mM Tris base (Sigma Aldrich) was added. The absorbance was measured using a microplate reader (BioTeK Synergy H1 Hybrid Reader) at 540 nm [14]. The growth inhibition rate was calculated as percentage of parallel negative controls. Data is the results of three independent experiments with mean ± SD. P-values were calculated with the help of GraphPad Quick-Calcs: t test calculator (http://www.graphpad.com/quickcalcs/ttest1/?Format=SD).

Supporting Information

UPLC-DAD method validation and ESI-MS analysis details as well as spectral data of the compound are available as Supporting Information.

Acknowledgements

Authors are grateful to the Director, CSIR-IHBT, Palampur (HP), India for continuous encouragement and for providing necessary facilities. Authors would also like to thanks CSIR, New Delhi, India for funding BSC-106 and BSC-209 project under which this work was carried out. IHBT Communication No: 3932.

Conflict of Interest

None. The authors declare that they have no competing interests.

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