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Abstract

Purpose of the Research

Peliosanthes micrantha Aver.et N.Tanaka has been found in central highland Dak Nong province, Vietnam, and used locally to treat various diseases. This research studied phytochemicals, spectroscopic analysis, and cytotoxic activities of P. micrantha rhizomes (PM-R).

Methods

In this study, steam-distilled essential oil of P. micrantha rhizome (EO) was analyzed by gas chromatography - mass spectrometry (GC-MS). Phytoconstituents of P. micrantha rhizome ethanolic extract (PM-R-ET) were separated by column chromatography and their structures were determined. Attenuated total reflectance (ATR) – Mid Fourier-transform infrared spectroscopy (FTIR) was used to characterize its dry powder (PM-R-DP).

Major Findings

The results showed that EO contained eleven constituents. Two compounds, pumilaside A (2) and β–sitosterol (4), were successfully isolated from PM-R-ET, and unambiguously elucidated by nuclear magnetic resonance (NMR) and mass spectrometry (HRMS). ATR-FTIR spectrum of PM-R-DP discovered 26 different functional groups of its compounds. PM-R-ET showed IC50 value of 96.14 ± 6.12 µg/mL against HT29 cell lines.

Conclusion

Our findings partly confirm previous Ultra High-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-QTOF-MS) study and show two isolated compounds and EO constituents, spectroscopic properties, and cytotoxic activity from the P. micrantha rhizomes.

Keywords

pumilaside A β–sitosterol essential oil GC-MS ATR-FTIR cytotoxicity

Introduction

The genus Peliosanthes Andrews currently referred to Asparagaceae, is indigenous to Asia.¹ Many studies on the discovery and ecological characteristics of the Peliosanthes species reported that the Peliosanthes grow mainly in subtropical to tropical regions within Asia.² In 2013, the medicinal herb Peliosanthes micrantha Aver.et N.Tanaka belonging to Asparagaceae, was discovered in Vietnam,³ and the same species (called Sam cau Krông Nô or Hue da la nho in Vietnamese) was found in central highland Krong No district, Dak Nong province, Vietnam. According to the indigenous peoples of Dak Nong province, the rhizomes of P. micrantha were decocted or alcohol-extracted and used for tonic, detox, hepatic, body nourishment, and vitality male enhancement. In Cao Bang province, P. teta was found and used for various ailments.⁴ Only a few research of chemistry and biological activities have been published even for the genus Peliosanthes and especially for P. micrantha species. Till today, glycoside J-3, J-4, and three spirostanol glycosides were isolated from P. sinica species.⁵ Our previous research presented that P. micrantha rhizome ethanolic extract (PM-R-ET) showed mild DPPH antioxidant activity.⁶

High-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (HPLC-QTOF-MS) is a powerful tool in both chromatographic separation and mass spectrometric detection of the secondary metabolite compounds in herbal extracts, and establishing the relationship between the profile of compound and the biological activities.⁷ After the analysis of chemical components is carried out, it is essential to apply the methods for the guided separation of the metabolite compounds in the herbal extracts. Zhang L. et al⁸ have successfully applied the Ultra High-Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-QTOF-MS)-guidance strategy including finding, tracking, purity determination, and structural elucidation of targeted compounds as well as HPLC chromatographic conditions transplantation to prepare the sulfur-containing ginsenoside Rg1 derivative from sulfur-fumigated ginseng. Xue J. et al⁹ have developed one integrated data-mining strategy that combines molecular networking, in-house polygonal mass defect filtering, and diagnostic fragment ion filtering to identify phytochemicals in Stephania tetrandra based on LC-MS data; then applied to isolate two new bioflavonoids. In Vietnam, although the application of HPLC-QTOF-MS/MS to identify the metabolites was used in some research groups,¹⁰ the discovery of new compounds in the medicinal plants was mainly carried out by the traditional column chromatography¹¹ or other new instrumental separation methods.¹²

Essential oils are natural volatile fractions distilled from herbal plants and formed by classes of substances such as esters of fatty acids, aldehyde alcohols, ester, aliphatic hydrocarbons, mono and sesquiterpenes, etc¹³ The gas chromatography - mass spectrometry (GC-MS) technique was most frequently used to identify and quantitatively determine the constituents of volatile compounds, hydrocarbons, alcohols, acids, esters, etc, in the given plant. Meher et al¹⁴ have used the GC-MS technique to analyze the essential oil of both the leaf and the rhizome of Zingiber purpureum species.

Fourier-transform infrared spectroscopy (FTIR) between 4000 cm⁻¹ to 400 cm⁻¹ is an established time-saving method to characterize and identify functional groups of metabolites in herbal plants. The absorption band of a specific bond is called the characteristic band of this bond.¹⁵ Canbay et al¹⁶ have used FTIR analysis to identify the functional groups of the bioactive compounds in grapeseed extracts obtained from ethyl acetate, water, and petroleum ether. FTIR analysis can be used with the Attenuated total reflectance (ATR) technique for qualitative functional group analysis of extracts or foods.¹⁷

Plant-based medicines have good potential as a primary source of chemotherapeutic drugs. Cytotoxic compounds such as metabolites including phytosterols are currently used for cancer chemotherapy.¹⁸ A variety of plants have been evaluated to investigate their cytotoxic activities. Many tumor cell lines such as human breast adenocarcinoma (MCF-7), the human promyelocytic leukemia tumor (HL60), human neuroblastoma (SH-SY5Y), African green monkey kidney fibroblast (CV1-P), rat brain glioma (C6), mouse macrophage (RAW264.7), mouse fibroblast (3T6) cells have been tested for in vitro cytotoxic potential.¹⁹

Recently, we reported the use of UHPLC-QTOF-MS to tentatively identify phytochemicals in an ethanol extract of the P. micrantha rhizomes and dereplicated one known compound of glycoside J-3 (1), previously isolated in the P. sinica and three known compounds, pumilaside A (2), pumilaside C (3), and β–sitosterol (4).⁶ In the same plant, our study showed the presence of seven constituents, with 1-nonacosene (5) as the major component (35.56%) along with 1-docosene (6), and two amides, (Z)-9-hexadecenamide (7), and erucylamide (8) and three oxygen-containing compounds, bis (2-ethylhexyl) sebacate (9), vinyl stearyl ether (10), and 2-octadecoxyethanol (11) in the leaves essential oil by GC-MS analysis.

In this paper, we reported the tentative identification of eleven constituents in P. micrantha rhizome essential oil by GC-MS. The isolation and structural identification of two known natural products, pumilaside A (2) and β-sitosterol (4), of ethanol fraction of P. micrantha rhizomes were presented. These compounds are isolated for the first time in Peliosanthes genus. Rhizome powder of P. micrantha was studied by ATR-FTIR method. Afterward, the ethanol extract was tested for cytotoxic activity against A549, MCF-7, HepG2, Hela, and HT-29 cell lines.

Material and Methods

Chemicals and Reagents

All reagents, n-hexane, ethyl acetate, dichloromethane, methanol, anhydrous sodium sulfate, NaHCO₃, L-glutamine, sodium piruvate, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), sulforhodamine B, and ellipticine were purchased from Sigma-Aldrich Chem. Co. (St. Louis, MO., USA). Retention Index Standard Product No. R 8769 was purchased from Sigma-Aldrich Chem. Co. (St. Louis, MO., USA). Silica gel (Qingdao Ocean Chemical Group Co. Ltd, China), C18 columns, and reversed-phase Triart C18 column (YMC Co, Ltd, Japan), silica gel G plates (Qingdao Marine Chemical Inc., China). Deionized water for HPLC; HPLC grade acetonitrile, methanol, reagent grade formic acid (≥98%) were obtained from Fisher (USA). Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium with Eagle salt (MEME), and fetal bovine serum (FBS) were obtained from Life Technologies, Inc., (Gaithersburg, MD, USA). TCA (Trichloroacetic acid), acetic acid were purchased from Bio Basic Inc. (Canada). Tris-base, SRB Dimethyl sulphoxide (DMSO) were obtained from MP Biomedicals (Strasbourg, France). Sulforhodamine B and Ellipticine were purchased from Sigma Aldrich (St. Louis, MO, USA). Human lung carcinoma A549 cells, human breast carcinoma MCF-7 cells, human hepatocarcinoma HepG2 cells, human cervical carcinoma Hela cells, and human colorectal carcinoma HT-29 cells were obtained from Prof. J. M. Pezzuto, Long-Island University (US) and Prof. Jeanette Maier, University of Milan (Italy).

Plant Material

The rhizomes of P. micrantha were collected in Dak Nong Province, Vietnam in October 2021 and identified by Dr Nguyen Sinh Khang, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology (VAST). The voucher specimen (PM-R-2021) has been lodged at the Center for High Technology Research and Development, VAST.

Steam-Distilled Essential oil Preparation and GC-MS Analysis

Powdered P. micrantha rhizomes (300 g) were placed on a stainless steel holed disk over distilled water (3 L) in our 5 L apparatus with a chilled condenser. Essential oil was vaporized by steam distillation for 4 h and separated. The isolated oil was dried over anhydrous sodium sulfate for 12 h to obtain essential oil, P. micrantha rhizomes (PM-R-EO), weighed, and placed in a refrigerator. Experiments were triplicated.

GC–MS analysis of the PM-R-EO was carried out as previously described¹¹ on a 456-GC (SCION) equipped with a flame ionization detector, SQ Select (SCION), and a Rxi-5ms column (30 m × 0.25 mm (i.d.), film thickness 0.25 µm df, RESTEK). The injector and detector temperatures were set at 250 °C and 250 °C, respectively. The column temperature progress was initiated at 80 °C for 1 min., followed by an increase to 170 °C at 5 °C/min, then to 280 °C at 50 °C/min, and finally kept isothermally at 280 °C for 3 min. The carrier gas was helium at a flow rate of 1 mL/min. The sample was injected by splitting. The split ratio injected was 30 µL of essential oil. The mass selective detector conditions were as follows: ionization voltage 70 eV, emission current 40 mA, and acquisitions scan mass range 50-500 amu under full scan, scan velocity 1 s/scan. Retention Index was calculated for GC non-isothermal ramp using Van den Dool and Kratz equation (Van den Dool and Kratz RI) areas by the following formula:

I_{x} = 100 n + 100 (t_{x} - t_{n}) / (t_{n + 1} - t_{n})

Where: I_x = retention index of component x

n = carbon number of n-alkane peak eluting immediately before sample peak

t_x = retention time of sample peak

t_n = retention time of n-alkane peak eluting immediately before sample peak

t_n ₊ ₁ = retention time of n-alkane peak eluting immediately after sample peak.

Identification of peaks and constituents was done with the NIST 17 library (resemblance percentage > 90%). Constituent content (%) was calculated as relative peak area to total peak areas by the following formula:

Content (%) = \frac{Peak area of constituent}{\sum Peak areas of all constituent} \times 100

Ethanol Extraction

The fresh rhizomes of P. micrantha (8.700 kg) are washed, sliced, and dried for 5 days in an oven at 80 °C and ground into powder (PM-R-DP). Dried powder of the rhizomes of P. micrantha (2.800 kg) was extracted with 96% ethanol (17.5 L × 3 times) at room temperature in an ultrasonic 20 kHz and 1 kW extractor as described previously.⁶ The combined extract was concentrated to dryness under reduced pressure, yielding a residue of PM-R-ET (123.30 g, brown solid). PM-R-ET was flushed with nitrogen gas and stored at −20 °C for future use.

Isolation and Purification

PM-R-ET was reconstituted in ethanol 96% and sequentially partitioned with n-hexane, ethyl acetate (EtOAc) to give the n-hexane portion H0 (23.1 g), EtOAc portion EA0 (39.1 g) and ethanolic portion E0 (42.6 g). The EtOAc-soluble EA0 (39.1 g) portion was further fractionated on silica gel chromatography column (CC 63-200 μm) eluting with gradient elution of increasing polarity solvent mixture of n-hexane–EtOAc (ie, 100:1→ 90:10 → 80:20 → 60:40 → 40:60 → 20:80 → 0:100, 2 L each) to obtain 7 fractions (EA₁ → EA₇). The EA₁ fraction was separated on CC (40-63 μm) and then eluted by n-hexane–EtOAc (20: 1, v/v) to give 3 subfractions (EA_1.1 → EA_1.3). The EA_1.2 subfraction was again separated on CC (40-63 μm) and then eluted by n-hexane–EtOAc (15: 1, v/v) for 2 subfractions (EA_1.2.1, HĐ-1). A white powder (E1) (30 mg) was recovered after concentration under vacuum from the PM-R-1 subfraction. The EA₆ fraction was separated on CC (40-63 μm) and then eluted by n-hexane–EtOAc (5: 1, v/v) to give 5 subfractions (EA_6.1 → EA_6.5). Then, the EA_6.2 subfraction was sequently eluted by diclomethane:methanol (10:1, v/v) to give 4 subfractions (EA_6.2.1 → EA_6.2.4). The EA_6.2.2 subfraction was purified on a reversed-phase YMC-Triart C18 column (5 µm × 250 × 4.6 mm-TA12S05-2546WT) by methanol–water (4: 1, v/v) to obtain a colorless solid (E2) (61.3 mg) after concentration under vacuum.

Structural Characterization

Nuclear magnetic resonance (NMR) spectra for structural elucidation of isolated compounds were measured on a Bruker Avance-500 spectrometer (Bruker, Karlsruhe, Germany) in DMSO-D6 operating at 500 MHz for ¹H NMR, HMBC, and HMQC, 150 MHz for ¹³C NMR, respectively. Tetramethylsilane (TMS) was used as an internal standard. EI mode MS spectra of isolated compounds were measured on an X500R QTOF mass spectrometer (AB SCIEX, USA).

ATR-FTIR Analysis

ATR-FTIR was one of the technologies used to investigate the chemistry of the P. micrantha rhizomes. Before ATR-FTIR analysis, dry P. micrantha rhizomes were ground to a fine powder using an agate mortar, then sifted using sieves with mesh sizes ranging between no.100 and no. 120, aimed at isolating particles with dimensions falling within the 0.125-0.150 mm range. The collected sample was PM-R-DP. This sieving process minimized the potential impact of radiation scattering attributable to variable particle sizes, thus ensuring analytical consistency. The ATR-FTIR measurements were conducted using an FTIR Spectrometer (Nicolet Apex, Thermo Scientific), covering a wavelength spectrum between 4000 cm⁻¹ and 400 cm⁻¹ as previously described.²⁰ Each sample was subjected to 128 scans with a spectral resolution of 4 cm⁻¹, and measurements were replicated six times to affirm the reliability of the data. As a first step, the dry ground rhizome powders were analyzed for spectral band positions to identify the signatures of the major functional groups. Assignments of the main bands were carried out by analyzing the acquired spectra and comparing them with the literature.

Cell Culture and Cytotoxicity Assay

MTT was dissolved in PBS (pH 7.4). A549, MCF-7, HepG2, Hela, and HT-29 cells were were kept in DMEM or MEME supplemented with L-glutamine, sodium piruvate, NaHCO₃, penicillin/streptomycin, 10% FBS, Trypsin-EDTA (0.05%). They were maintained in 25 cm² flasks at 37 °C in a humidified incubator with 5% CO₂. The cytotoxic effects of PM-R-ET on cancer cells were evaluated by measuring the metabolic activity using the MTT assay.

MTT assay, as previously described by Skehan et al,²¹ was performed to assess the cytotoxicity of the extract of P. micrantha. The test extract, PM-ET, was dissolved in DMSO. The stock samples were dissolved in DMSO 100% at a concentration of 20 mg/mL. 10 µL of six serial dilutions of test extract (ie, 0.8, 4.0, 20.0, 100, 200, and 500 µg/mL) were brought in 96-well plates in the culture mediums (without FBS). The cells were grown and regulated to appropriate concentrations, and then 190 µL of the cell medium was brought into the 96-well plates containing 10 µL of test extracts. Ellipticine as a reference was used at different concentrations of 0.08, 0.4, 2.0, and 10.0 μg/mL. DMSO 10% mL was used as the negative control. The blank negative control without cell and sample was arranged and measured in the well plates of the same test serial. Following incubation for 72 h, the medium was removed, 10 μL MTT (5 mg/mL in PBS) was added to each well, and the cells were incubated for an additional 4 h at 37 °C at 5% CO₂. After 4 h, the supernatant was removed, and 150 μL DMSO was added to each well. The absorbance at 540 nm was recorded using a BIO-TEK spectrophotometer. The formula used to calculate the inhibition percent (%) was:

Inhibition (%) = 100 - \frac{OD (sample) - OD (blank)}{OD (DMSO) - OD (blank)} \times 100

IC50 was calculated by TableCurve 2Dv4 software. Experiments were performed in triplicate.

Statistical Analysis

Packages of Excel (Microsoft Co, Redmond, WA, USA) and SPSS (version 19.0 statistical software, SPSS Inc, Chicago, USA) were used for statistical analysis. Results represented means ± sd of replicated determinations. Comparison and separation of the mean values were performed using Tukey's multiple comparison test.

Results

GC–MS Analysis

The essential oil of PM-R-EO was obtained at a yield of 0.01% based on the fresh material weight. Eleven components of the essential oil were found using GC–MS (Figure 1).

Figure 1.

GC-MS chromatogram of PM-R-EO sample.

The major constituents present in the PM-R-EO were tentatively identified as a high alkane, 1-docosene (19), as the main component (21.71%), followed by two high alcohols, octacosanol (16.84%) (20) and eicosanol (14.03%) (18), and an ester, octatriacontyl pentafluoropropionate (11.16%) (22) (Table 1).

Table 1.

Chemical Constituents of PM-R-EO Sample.

T_R (min)	Compounds	Compound Type	Functional Group	Van den Dool and Kratz RI	Content (%)	RI calc.	RI lit.
20.172	ar-tumerone (12)	Sesquiterpene	aromatic C=C, C=O, C–H bending	1291	5.06	1652	1667²²
21.134	cycloprop[e]inden-2(1H)-one, octahydro-3-hydroxy-1a,5-bis(hydroxymethyl)-5,6b-dimethyl (13)	Alcohol	OH	1313	1.92	1572	NL
21.554	1-propylundecyl trifluoroacetate (14)	Ester	C=O, C-O-C, C-F	1323	2.45	1753	NL
22.554	aromadendrene oxide-(1) (15)	Oxygenated sesquiterpene	C-O-C	1347	6.13	1457	1439²³
22.638	8S,14-cedrandiol (16)	Terpenoid	OH	1349	6.06	1890	1891²⁴
22.862	palmitic acid (17)	Acid	C=O, C-O-C	1354	8.35	2012	2003²⁵
23.06	eicosanol (18)	High alcohol	OH	1359	14.03	2267	2273²⁶
24.165	1-docosene (19)	High alkane	C=C	1385	21.71	2152	2148²⁷
25.139	octacosanol (20)	High alcohol	OH	1409	16.84	3104	3110²⁸
26.113	mono(2-ethylhexyl) phthalate (21)	Phthalate ester	C=O, C-O-C, OH	1433	6.29	2163	2152²⁹
26.311	octatriacontyl pentafluoropropionate (22)	Ester	C=O, C-O-C, C-F	1438	11.16	2475	NL
Total identiﬁed					100.00
Terpenoids					17.25
Hydrocarbons					21.71
Oxygen-containing compounds, including:					73.23
- alcohol					38.85
- acid					8.35
- ester					19.9

RI calc., calculated Van den Dool and Kratz retention index; RI lit., Van den Dool and Kratz retention index from the literature; T_R, retention time; NL, Non literature identified.

Isolation and NMR Characterization

Our isolation protocol for PM-R-ET has given two compounds. Compound (E1) was isolated from the P. micrantha as a white amorphous powder and its structure is assigned by spectroscopic data (NMR and MS). In Figure S1, its molecular formula was determined to be C₂₁H₃₈O₈ by EI negative ion peak at m/z 463.2525 for [M + HCOO]⁻ (calculation 463.2543) in the electrospray ionization mass spectrometry. Structural elucidation of (E1) is based on NMR data summarized in Table S1 and visually described in Figure S2. The ¹H-NMR of compound (E1) presented the signals of three methyl groups at δH 1.44 (3H, d, J = 6.5 Hz, H-12), 1.01 (3H, d, J = 6.5 Hz, H-13), 1.24 (3H, s, H-14), 1.75 (3H, s, H-15) (Figure S3). ¹³C-NMR and HSQC spectra of compound (E1) (Figures S4 and S5) displayed 21 carbon signals including one β-glucopyranosyl, two tert-methyls, two sec-methyls, four methylenes, five methines (two of them were oxygenated) and two quaternary carbons (one of two methyl groups was oxygenated). The anomeric proton was at δH 4.53 ppm which correlated with C-6 (δC 78.55) in the HMBC spectrum suggesting that the sugar moiety was attached at C-6 (Figure S6). Detailed HMBC analysis showed correlations from H-1/C-9, C-10 and C-14; H-5/C-3, C-4, C-6, C-7, C-9, C-10 and C-14; H-6/C-7, C-10 and C-11; H-7/C-5 and C-12; H2-9/C-5, C-7 and C-10; H3-12/C-7, C-11 and C-14; H3-13/C-7, C-11 and C-12; H3-14/C-1, C-5, C-9 and C-10; H3-15/C-3, C-4 and C-5. ¹H-¹H COSY data revealed H-1/H2; H-6/H-5 and H-7; H-7/H-8 (δ 1.63) and H-11 (Figure S7). All the data showed a partial structure as described in Figure S8. Then, (E1) was suggested to be a glucoside of eudesmane-type sesquiterpenoid having three hydroxyl groups at C-1, C-4, and C-6. The position of the glycosyl unit was ascertained to be C-6 from the HMBC correlation of glucosyl H-1’/C-6. Key NOESY correlations of H-4/H-7, and H-6/H-3 and H-5 indicated that H-4, H-7 were on the same side of the lactone ring while H-3, H-5, and H-6 were placed on the other side. So, (E1) could be assumed to be structured with one known structure, (1S,4S,5R,6R,7S,10S)-1,4,6-trihydroxyeudesmane 6-O-b-D-glucopyranoside. The data showed that the structure of (E1) could be assigned as pumilaside A.^30,31

Compound (E2) was obtained in the form of colorless prismatic crystals. The EI-HRMS spectrum displayed an adduct ion [M-H]⁻ at m/z 413.3247 (calculation 413.3783) on negative mode (Figure S9). It corresponded to the molecular formula C₂₉H₅₀O. Structural elucidation of (E2) based on NMR data was summarized in Table S2 and visually described in Figure S10. The ¹H NMR spectrum of (E2) recorded in DMSO-d6 (Figure S11) showed the presence of one olefinic proton at δH 5.35) (H-6) suggesting the presence of a tri-substituted double bond at C-5/C-6 in its structure. Its ¹H NMR spectrum showed one multiplet proton (δH 3.53) (H-3) corresponding to a proton connected to the C-3 hydroxy group. The ¹H NMR spectrum of (E2) also showed the presence of six methyl signals which appeared as two angle methyl singlets at δH 0.68 (CH3-19) and at δH 1.01 (CH3-18), three methyl doublets that appeared at δH 0.81, 0.83, and 0.93; and a methyl triplet at δH 0.84, all indicating the presence of steroid frame. The ¹³C NMR spectrum (Figure S12) together with the DEPT spectrum of (E2) in DMSO-d6 (Figure S13 and Table S2) displayed 29 carbon signals with two olefin carbons at δC 140.74 (C5) and 121.71 (C6). A carbon signal at δC 71.80 ppm suggested the presence of an oxygenated carbon (corresponding to C3-OH of steroid frame). Thus, the structure of (E2) was assigned as β-sitosterol (4) that was consistent to the reported spectral data with known structure, β-sitosterol or (3S,8S,9S,10R,13R,14S,17R)-17-[(2R,5R)-5-ethyl-6-methylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol.³²

ATR-FTIR Spectroscopy

ATR-FTI spectrum of PM-R-D was shown in Figure 2 and presented 28 vibrations.

Figure 2.

ATR-FTIR spectrum of PM-R-D sample.

The vibration assignments are in Table 2. The FTIR absorptions at 603 cm⁻¹, 652 cm⁻¹, and 677 cm⁻¹ in the region below 800 cm⁻¹ were known as the skeletal bands relating to the carbohydrate skeletal and/or the benzene ring stretching vibrations.^19,33 An absorption peak at 926 cm⁻¹ in the region between 900-1200 cm⁻¹ defined the coupled C-C and C-O stretching and C-OH bending vibrations.³⁴ Two peaks at 1314.00 cm⁻¹ and 1027 cm⁻¹ were assigned to C=O vibration and C–O absorptions respectively.³⁵ The absorption peaks at at around 1013 cm⁻¹ in the region of 1010-1060 cm⁻¹ and 1615 cm⁻¹, corresponding to double bonds, C=O and S=O of polysaccharides.^36,37 Two absorption peaks at 1641 cm⁻¹ and 1635 cm⁻¹ were appeared in the 1623-1640 cm⁻¹ region, characteristic of double bonds stretching. Three absorption peaks at 1013 cm⁻¹, 1027 cm⁻¹, and 1030 cm⁻¹ in the region between 1000-1200 cm⁻¹ might indicate the pyranose forms of the polysaccharides.^38,39 IR bands at 814 cm⁻¹, 828 cm⁻¹, and 922 cm⁻¹ could relate to the α-anomeric carbon configurations of the polysaccharide.^37,39,40 FTIR spectrum presented the peaks at 1029 cm⁻¹, 1248 cm⁻¹, and 1732 cm⁻¹ which were assigned as polysaccharide characteristic bands due to different acetyl stretching vibrations.^41,42 A small peak at 1414 cm⁻¹ in the 1200-1500 cm⁻¹ region was assigned to O–H/C–H vibrations.⁴³

Table 2.

Assignments of ATR-FTIR Functional Groups and Constituents of P. micrantha Rhizomes.

Wavelength (cm⁻¹)	Range (cm⁻¹)	Type of Vibration and Compounds	IR Functional Group Vibration
3841	3800-3955	Stretching: alcohol, various volative compounds	O-H
3697	3610-3700	Stretching: alcohol, various volative compounds	O-H
3437	3400-3500	Stretching: alcohol, various volative compounds	O-H
3437	3400-3500	Stretching: β-sitosterol	O-H
925	900-1200	Bending: alcohol	C-OH
3743	3740-3780	Stretching: amide	N-H₂
3332	3327-3334	Stretching: amine	N-H
2918	2910-2940	Stretching: flavonoid, saponin, phenolic	C-H and alkyl (–CH₂- and –CH₃)
2855	2840-2860	Stretching: cyclic hydrocarbon	C-H
2855	2840-2860	Stretching: β-sitosterol	C-H
2832	2830-2860	Stretching: double bonds, β-sitosterol	C=C
2366	2300-2400	Stretching: double bonds, β-sitosterol	C=C
1641	1623-1645	Stretching: aliphatic	Conjugated C=C
1635	1623-1645	Stretching: cyclic alkene, β-sitosterol	-C=C
1314	1300-1380	Stretching: double bonds	-C=C or -C=O
1248	1050-1270	Stretching: S=O of sulfonyl group, glycoside J-3	S=O of sulfonyl group
1027	1000-1200	Stretching: alcohols, carboxylic acids, esters, and ethers	C–O
652	650-900	Stretching: benzene ring
3282	3240-3320	Stretching: O-H sym./asym. polysaccharide	O-H
3282	3240-3320	Stretching: symmetrical/asymmetrical polymeric (O–H)	O-H
2918	2910-2940	Stretching: polysaccharide	C-H and alkyl (–CH₂- and –CH₃)
1615	1600-1620	Stretching: double bond, polysaccharide	-C=O symmetrical deformation
1732	1730-1740	Stretching: acetyl	acetyl C=O
1248	1050-1270	Stretching: acetyl	acetyl C–O
1414	1388-1423	Stretching: O–H/C–H deformation and C–H/C–H₂	C-H (CH₂)
1029	1026-1035	Stretching: asymmetric tensile, polysaccharide	C-O-C and C-O-H
1013	1000-1200	Stretching: glycoside J-3, glucopyranoside pumilaside A, polysaccharide	Pyranose form
1029	1000-1200		Pyranose form
926	900-1200	Stretching: coupled single C-bond, polysaccharide	Coupled C-C and C-O
926	900-1200	Bending: C-OH, polysaccharide	C-OH glycosides
814	800-930	Stretching: α-anomer	α-glycosides
827
922
603	Blow 800	Stretching: carbohydrate	Skeletal bands
677	Blow 800	Stretching: carbohydrate	Skeletal bands

In high wavenumber region of double bonds stretching between 2300-2940 cm⁻¹, the vibrations at 2366 cm⁻¹, 2832 cm⁻¹, and 2936 cm⁻¹ were also assigned to C=C stretching.⁴⁴ An absorption peak at 2855 cm⁻¹ was assigned to CH₃ cyclic hydrocarbon stretching or to C-H stretching.⁴⁵ In high wavenumber region of O-H vibrations, there are small O-H stretching vibrations at 3697 cm⁻¹, 3841 cm⁻¹, and 3437 cm⁻¹.^45,46 The FTIR spectrum showed a broad intensity peak at 3332 cm⁻¹, and a relatively weak band at 3743 cm⁻¹, both are the characteristic N-H stretching absorptions of secondary amine and amides, respectively.³³

Peak at round 2918 cm⁻¹ was assigned to C-H and alkyl bonds of the polysaccharides.^37,47 A small peak at 3282 cm⁻¹ was assigned to O-H vibration of the polysaccharides.⁴²

Cytotoxic Activity

The cytotoxic activity of ethanolic extract from P. micrantha rhizomes, PM-ET, was presented in Table 3. The sample inhibition increased while HT29 cell concentrations increased from 0.8 to 500 µg/mL. Its IC50 cytotoxic value against HT29 cells was 96.14 ± 6.12 µg/mL. It did not show significant cytotoxicity against HepG2, A549, Hela, and MCF-7 cells (IC50 > 100 µM).

Table 3.

Cell Cytotoxicity of Alcohol Extract from P. micrantha Rhizomes.

Concentration (µg/mL)	Alcohol Extract from P. micrantha Rhizomes
	Hela		HT29		MCF7		HepG2		A549
	Inhibition %	SD	Inhibition %	SD	Inhibition %	SD	Inhibition %	SD	Inhibition %	SD
500	105.24	1.03	91.24	1.98	41.40	0.97	51.48	1.12	40.21	0.83
200	71.03	0.98	78.70	0.64	30.45	1.53	40.32	0.71	31.49	0.46
100	45.11	0.96	46.49	2.56	22.16	2.13	33.48	2.44	23.83	1.57
20	18.49	1.70	12.54	1.70	13.72	0.98	20.37	1.76	9.55	0.54
4	12.85	1.07	9.43	0.85	6.02	0.61	12.92	1.06	3.88	0.14
0.8	2.58	0.28	1.98	0.16	4.33	0.37	7.26	0.64	1.63	0.16
IC50	>100		96.14 ± 6.12	>100		>100		>100
	Ellipticine
IC50	0.34 ± 0.03		0.36 ± 0.03	0.39 ± 0.02		0.32 ± 0.02		0.40 ± 0.03

*Values are the mean of three replicates ± standard deviation.

Discussion

GC–MS Analysis of Essential Oil

Among eleven constituents tentatively identified in PM-R-EO, the presence of oxygen-containing compounds was highest (total 73.23%), followed by one hydrocarbon (21.71%), and finally three terpenoids (total 17.25%). Three terpenoids were of the same concentration of around 6.00%. One aromatic sesquiterpene, ar-tumerone (5.06%) (12), as main volatile oil constituent of Curcuma longa, has been shown to suppress tumor, bacterial and fungal growth, platelet aggregation, oxidative stress, senile dementia, and metabolic syndrome.⁴⁸ Second oxygenated sesquiterpene, aromadendrene oxide-(1) (6.13%) (15), had been found in the chloroform extract of Andrographis paniculata.⁴⁹ Another terpenoid, 8S,14-cedrandiol (6.06%) (16), had been identified in Hybanthus enneaspermus.⁵⁰ The high alkane, 1-docosene (19) (21.71%), was found in the essential oils from both P. micrantha rhizomes and leaves in our research,³⁰ and in the essential oils from T. dioica flowers and leaves.⁵¹

Some compounds identified in the PM-R-EO were previously extracted. From the extract of one edible mushroom mutant of Schizophyllum commune strains,⁵² 1-docosene and some triacontyl pentafluoropropionate derivatives such as triacontyl pentafluoropropionate and dotriacontyl pentafluoropropionate could be identified by GC-MS. However, we could not find any documentation in the literature of the natural source of octatriacontyl pentafluoropropionate (22) accounting for up to 11.16% of all identified EO constituents. Eicosanol (18) (14.03%), was found in the extract of Hypericum carinatum.⁵³ Octacosanol (20) (16.84%), abundant in nature,⁵⁴ was found in the waxy extract of Moricandia sinaica.⁵⁵ Such results suggested that the PM-R-EO contained nonvolatile or semi-volatile components that are not typically dominant in essential oils. Such lipophilic constituents could be distilled by hot and strong steam flow in our experiment. Among the oxygen-containing compounds, mono-(2-ethylhexyl) phthalate (21) (MEHP) was found at a considerable concentration of 6.29%. It induced apoptosis in the human first-trimester placental cell line HTR-8/SVneo.⁵⁶ However, MEHP is one active metabolite of bis(2-ethylhexyl) phthalate (DEHP). These phthalates are the most frequently used plasticizers, and are found in floor coverings, food and drink packaging materials, … ⁵⁷ Such occurrences in the PM-R-EO should be studied in-depth.

Isolation and NMR Characterization

Only two phytoconstituents, pumilaside A (2) and β–sitosterol (4), were isolated by selective solvent fractionations and column chromatography from P. micrantha rhizome extract, although four compounds were previously found, namely, glycoside J-3 (1), pumilaside A (2), pumilaside C (3), and β–sitosterol (4) by UHPLC-QTOF MS,⁶ and eleven EO constituents were found in this study. The successful isolation of two phytoconstituents might be due to their high concentrations and appropriate choice of selective solvent mixtures. None of the constituents present in EO could be isolated.

ATR-FTIR Spectroscopy of P. micrantha Rhizome Powder

Infrared spectroscopy was used to investigate the chemical composition of the P. micrantha rhizomes powder. At first, the samples were analyzed concerning the spectral band positions to identify the signatures of the major functional groups. Then, the assignments of the main bands were carried out by analyzing the acquired spectra and comparing them with the literature. We first assigned IR vibrations to the metabolites in the rhizomes. ATR-FTIR vibrations showed a small peak at 1641 cm⁻¹ that could be of aliphatic C=C stretching in 1-Docosene, the main component of the EO (21.71%),³⁵ while one other small peak at 1635 cm⁻¹ that could be of C=C cyclic alkene stretching which might come from β-sitosterol,⁴⁵ and ar-tumerone.⁴⁸ In the higher wavenumber double bonds region, the vibrations at 2366 cm⁻¹, 2832 cm⁻¹, and 2936 cm⁻¹ were also assigned to C=C stretching of β-sitosterol.⁴⁶ The presence of a benzene ring stretching at 652 cm⁻¹ might come from pumilaside A and pumilaside C.³² The FTIR peaks at 1314 cm⁻¹ and 1027 cm⁻¹ could present C=O vibration and C–O absorption of alcohols, carboxylic acids, esters, and ethers specific functional groups in the rhizomes of P. micrantha, such as 1-propylundecyl trifluoroacetate, palmitic acid, mono(2-ethylhexyl) phthalate, octatriacontyl pentafluoropropionate, and/ or ar-tumerone in the essential oil, respectively.³⁵ An absorption peak at 2855 cm⁻¹ was assigned to CH₃ cyclic hydrocarbon stretching or to C-H stretching of β-sitosterol.⁴⁵ β-sitosterol isolated from Momordica charantia and Aristolochia indica showed a small peak at 3437 cm⁻¹ assigned as OH group stretching.^45,46

The results of FT-IR spectroscopy could be used to elucidate the presence of the related pyranose molecular structures and polysaccharides. The FTIR absorptions at 603 cm⁻¹ and 677 cm⁻¹ in the region below 800 cm⁻¹ were known as the skeletal bands relating to the carbohydrate skeletal vibrations. A relatively extensive absorption peak at 926 cm⁻¹ defined the coupled C-C and C-O stretching and C-OH bending vibrations of characteristic absorptions of polysaccharide,³³ or glycosidic linkages of different carbohydrate configurations.³⁴ The absorption peak at 1615 cm⁻¹ was caused by the C=O polysaccharide symmetrical deformation.³⁶ The FTIR broad intensity peak at around 1013 cm⁻¹, corresponding to asymmetric stretching of S=O to confirm the attachment of sulfonyl groups possibly to sulfated steroidal glycoside J-3, glucopyranoside pumilaside A, and polysaccharides.^5,31,37 Such information indicated the presence of polysaccharides in the water extract of P. micrantha rhizomes. As O-linked acetyl groups commonly in plant-derived polysaccharides show O-linked C=O stretching vibration at around 1740 cm⁻¹, and O-linked C–O stretching vibration at around 1245 cm⁻¹,⁵⁸ the FTIR spectrum presented a typical acetyl C=O stretching vibration at 1732 cm⁻¹ and a typical acetyl C–O stretching vibration at 1248 cm⁻¹. A peak at 1029 cm⁻¹ was assigned as a polysaccharide-characteristic band due to asymmetric stretching of C-O-C tensile vibrations and C–O–H linkages.^41,42 The monosaccharide adopts four different configurations in solution including two types of sugar ring (furan and pyran). Once a glycosidic bond is formed, the configuration of anomeric carbon is fixed and two different anomeric carbon configurations (α or β) are formed in a monosaccharide solution.³⁸ As pyranose rings were represented by three absorption peaks in the region of 1000-1200 cm⁻¹, three typical vibrations at 1013 cm⁻¹, 1027 cm⁻¹, and 1030 cm⁻¹ indicated the pyranose forms of the polysaccharides in the P. micrantha rhizomes.^38,39 In addition, IR bands at 814 cm⁻¹, 827 cm⁻¹, and 922 cm⁻¹ confirmed the existence of α-anomeric carbon configurations of the polysaccharide.^37,39,40 Spectra absorbance at 2918 cm⁻¹ was assigned to the stretching vibration of aliphatic C-H absorption and asymmetric stretching vibration of the C–H bond in the alkyl groups of the polysaccharides.^37,47 A small peak occurred at 1414 cm⁻¹ which is assigned to O–H/C–H deformation and C–H/C–H₂ stretching vibrations in the 1200-1500 cm⁻¹ region.⁴³ In addition, a small peak at 3282 cm⁻¹, assigned to O-H symmetric and asymmetric stretching vibration of the polysaccharides.⁴²

Cytotoxic Activity of P. micrantha Rhizome Ethanolic Extract

The cytotoxic activity of alcohol extract from P. micrantha rhizomes was evaluated against A549, Hela, HepG2, HT-29, and MCF-7 cells using MTT assay. At the same extract dilution concentration of 200 µg/mL, the inhibition rates decreased from 78.70, 71.03, 40.32, 31.49, and 30.45 against HT29, Hela, HepG2, A549, and MCF7, respectively. This sequence was also observed for the extract concentration of 100 µg/mL. Surprisingly, the extract did not show the highest cytotoxicity against HT29 cells compared to other cancer cells in other tested concentrations. It showed the highest cytotoxicity against HepG2 cells in the low range between 0.8-20 µg/mL. Meanwhile, it always presented the lowest cytotoxicity against A549 cells in the tested range. The IC50 value of PM-R-ET against HT29 cells was 96.14 ± 6.12 µg/mL. The study established the IC50 against the rested cancer cell lines higher than 100 µg/mL but did not yet determine their values. Ellipticine was used as reference, yielding IC50 values of 0.40 ± 0.03, 0.34 ± 0.03, 0.32 ± 0.02, 0.36 ± 0.03, and 0.39 ± 0.02 µg/mL for A549, Hela, HepG2, HT-29, and MCF-7, respectively. The results showed that the PM-R-ET was much less cytotoxic against all the tested cancer cells compared to the reference. In this work, we have isolated pumilaside A (2) and β-sitosterol (4) from the alcohol extract from P. micrantha rhizomes. As the constituents of the PM-R-ET, they could contribute their own cytotoxic activities from the extract against the tested cancer lines. Hanh et al⁵⁹ isolated pumilaside A from the aerial parts of Artemisia vulgaris and showed that the compound did not show any significant cytotoxicity on HepG2 and MCF7 cell lines by the SRB assay. However, Xu et al⁶⁰ also isolated pumilaside A from Litchi semen and it exhibited significant in vitro antitumor activity against A549, Hela, Hep-G2, and LAC cell lines with IC50 values of 0.012-6.29 μM by MTT assay. Pumilaside A from Litchi semen has been proven to induce apoptosis in human gastric cancer BGC823 cells.⁶¹ From the literature, among five tested cancer lines, we could not yet find any cytotoxic activity of pumilaside A against HT-29 cells. β-sitosterol showed promising anticancer activities for all the tested A549, HeLa, HepG2, HT-29, MCF-7, and several other cancer lines.^62–67

Limitations of the Study

The results of this research provided information about the first chemical investigation of the Vietnamese plant P. micrantha rhizome ethanolic extract. Additionally, the study dealt with its ATR-FTIR spectroscopy characteristics and cytotoxic activities. The significance of this study is the first presentation of the chemical compositions of isolated compounds, volatile compounds, and IR assignments of the functional groups of the constituents in its rhizomes, and anticancer activity. However, the chemical, spectroscopic, and cytotoxic investigations were carried out only for the alcoholic extract. Therefore, the main limitations can be considered that: 1) only a few constituents could be isolated, not yet quantified and their roles in the anticancer activity would not be verified, and 2) there might be different phytochemical profiles of P. micrantha rhizome alcoholic, hydroalcoholic, and aqueous extracts which could present different biological activities. It is necessary to expand research using different extraction solvents, and then to further isolate the phytoconstituents from both P. micrantha rhizomes and leaves to reduce limitations and provide a more complete contribution to the chemical literature of Vietnamese natural flora. Further studies are needed for in-depth biological activities of P. micrantha rhizomes.

Conclusion

This research studied the presence of chemical compounds in P. micrantha rhizomes including eleven constituents in the essential oil, and two isolated constituents in the ethanolic extract. ATR-FTIR spectra of P. micrantha rhizome powder presented several functional groups that could come from the presently and previously identified chemical compounds. Its ethanolic extract showed IC50 cytotoxicity of 96.14 ± 6.12 µg/mL against HT29 cells. The findings would encourage further in vitro and in vivo tests to verify the pharmaceutical activities of P. micrantha rhizomes.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X261418394 - Supplemental material for Chemico-Physical Characterization and Cytotoxicity of the Peliosanthes micrantha Rhizomes Collected in Highland Province Dak Nong of Vietnam

Supplemental material, sj-docx-1-npx-10.1177_1934578X261418394 for Chemico-Physical Characterization and Cytotoxicity of the Peliosanthes micrantha Rhizomes Collected in Highland Province Dak Nong of Vietnam by Le Ngoc Hung, Do Ngoc Thuy, Nguyen Thi Huong, Pham Thi Nhung, Phung Van Trung and Do Thi Nguyet Que in Natural Product Communications

Footnotes

ORCID iD

Le Ngoc Hung

Ethical Approval

Ethical approval is not applicable for this article.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

Funding

The author disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Vietnam Academy of Science and Technology [grant number UDNDP.04/2022-2023].

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Supplemental Material

Supplemental material for this article is available online.

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