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Abstract

Background

Piper chaba is a common plant in Bangladesh. It is enriched with many important biologically active compounds and popular for traditional healing.

Method

The methanol extract of the root of P. chaba was fractionated into n-hexane and ethyl acetate soluble fractions. The ethyl acetate fraction was subjected to various chromatographic techniques to isolate the pure compounds. The structures of the compounds were elucidated with the aid of UV, IR, NMR, including ¹H, ¹³C, DEPT-135, COSY, HSQC, HMBC, spectroscopy, and LC-MS. The isolated compounds were subjected to cytotoxicity assay using HeLa cell lines.

Result

β-sitosterol (1a) with trace of stigmasterol (1b), piperine (2), ergosterol peroxide (3), (E)-1-(4-chlorophenyl)-3-(4-methylphenyl)-prop-2-en-1-one (4), and (E)-1-(4-chlorophenyl)-3-(4-methoxyphenyl)-prop-2-en-1-one (5) were purified.

Conclusion

To the best of our knowledge, this is the first report of isolation of the halogenated chalcone derivatives 4 and 5 from a natural source, whereas ergosterol peroxide (3) has not been reported from this plant before. Chalcones 4 and 5 showed significant cytotoxicity on HeLa cells.

Keywords

Piperaceae piperine chalcone steroids HeLa cells cytotoxicity

Introduction

The genus Piper belongs to the Piperaceae family comprising about 1000 species and are native throughout the tropics and subtropics, with a few species in Eurasia.¹ The herb P. chaba Haunter (syn. of P. retrofractum Vahl and P. officinarum (Miq.) C. DC. locally known as Chui Jhal, a climbing, glabrous plant is available in various parts of India and Malay Islands. It grows abundantly in Bangladesh in Khulna division mainly in the Satkhira–Bagerhatt area. Its root is used as a spice in the Indian subcontinent. Different parts of this plant like stems, leaves, fruits, bark, and roots are known to be anti-flatulent, gastro-protective, appetizing, expectorant, antitussive and anti-fungal from the ancient time.^2,3 Recent research findings showed that P. chaba reveals diverse biological activities^4,5 including, anticancer^6-8 and immunomodulatory⁹ effects. Previous phytochemical investigation on this plant provided various types of promising bioactive compounds such as piperchabamides A-D, piperine, piperanine (Δ^α,β-dihydropiperine), pipernonaline, dehydropipernonaline, piperlonguminine, retrofractamide B (pipercide), guineensine, N-isobutyl-(2E,4E)-octadecadienamide, N-isobutyl-(2E,4E,14Z)-eicosatrienamide, methyl piperate, etc¹⁰

In this study with P. chava, the known ergosterol peroxide (3) and chlorinated chalcones (4 and 5) were isolated for the first time from a natural source, along with β-sitosterol (1a) with a trace of stigmasterol (1b) and piperine (2). In addition, all the compounds were subjected to cytotoxicity study using HeLa cell line.

Methods

General Experimental Procedures

In the separation process, silica gel 60 (Merck, 230-400 mesh) and F₂₅₄ (Merck) were used for column chromatography and thin layer chromatography (TLC), respectively.

Instruments

The IR spectra were recorded with IR200 spectrophotometer (Thermo Electron Corporation, Waltham, MA). The NMR spectra were acquired in CDCl₃ on an AVANCE 400 MHz instrument (Bruker Corporation, Switzerland) and the chemical shifts were referenced with the residual non-deuterated solvent signal. The ESI-MS spectra were recorded on a 6400 series triple quadrupole-time-of-flight mass spectrometer (Agilent, Palo Alto, CA).

Collection of the Plant Material

The roots of P. chaba were collected from Katalbari Upazila in the district of Kurigram, Bangladesh on May 2024 and was identified by Prof. Mohammad Zashim Uddin, Department of Botany, University of Dhaka. A voucher specimen (DUSH 10828) for this collection has been maintained at herbarium of the department. The collected roots were air dried and crushed into coarse powder and stored in airtight bottle.

Extraction and Isolation

The dried powder (about 568 g) of P. chaba was taken in a clean, round bottomed flask and extracted with methanol at room temperature and atmospheric pressure. The extract was concentrated to dry mass (18.74 g) using a rotary evaporator under reduced pressure. The concentrated extract was then partitioned in a separatory funnel by using n-hexane and ethyl acetate. The extracts were concentrated separately to dry mass with a rotary evaporator at 40 °C. Based on the thin layer chromatography (TLC) pattern, the ethyl acetate (5.42 g) fraction was subjected to column chromatography over silica gel and the column was eluted with n- hexane, ethyl acetate and methanol mixtures of increasing polarities (% ethyl acetate in n-hexane: 0, 1, 2, 3, 4, 5, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 and 100; % methanol in ethyl acetate: 0, 1, 2, 3, 5 and 10) to give total of 33 fractions, each 100 ml. All the column fractions were examined by TLC under UV light and by spraying with vanillin-sulphuric acid reagent. Depending on the TLC behavior, some fractions were combined in a beaker, and all were again marked as B₁-B₁₃. Among these, fraction B₄ (225 mg) (eluted with 7-8% ethyl acetate in n-hexane) showed four spots on TLC plate with mild tailing. This fraction was again subjected to column chromatography over silica gel using n- hexane and ethyl acetate mixtures of increasing polarities (% ethyl acetate in n-hexane: 0, 1, 3, 5, 7, 8, 10, 12, 15, 20, and 30) to give a total of 11 fractions, each 100 ml. Each of the fractions was monitored by TLC and the fractions of similar behaviors were combined and marked as T₁-T₆. The fractions T₃ (eluted with 7–8% ethyl acetate in n-hexane in the sub-column) and T₄ (eluted with 10–12% ethyl acetate in n-hexane in the sub-column) appeared to contain single spot on TLC plate. The isolated solids were marked as compound 4 (7 mg) and compound 5 (6 mg), respectively. Furthermore, fraction B₆ (eluted with 10–12% ethyl acetate in n-hexane) provided compound 1a with a trace of 1b as a solid crystal (8 mg). From fraction B₇ (eluted with 15% ethyl acetate in n-hexane), compound 3 (7 mg) was isolated through preparative TLC (solvent system: 12% ethyl acetate in toluene). In addition, from fraction B₈ (eluted with 20% ethyl acetate in n-hexane), compound 2 (24 mg) was purified by preparative TLC (solvent system: 10% ethyl acetate in toluene).

Cytotoxicity Assay

HeLa, a human cervical carcinoma cell line, was maintained in DMEM (1% penicillin-streptomycin (1:1) and 0.2% gentamycin) and 10% fetal bovine serum (FBS). Cells (2.0 × 10⁴/100 μl) were seeded onto 96-well plate and incubated for 24 h at 37 °C with 5% CO₂. 1 mg/ml of stock of compound in 1% DMSO in water was prepared. After incubation, 25 μl of the stock solution was added to each well. Then the plate was placed in carbon dioxide incubator at 37 °C with 5% CO₂. After 24 h, cytotoxicity was examined under an inverted light microscope. Triplicate wells were used for each sample.^11,12

Statistical Analysis

Three replicates (n = 3) of each sample were used and the values were calculated as mean ± standard deviation. Microsoft Excel 2010 software was used for this purpose.

Results

β-Sitosterol (1a) with a Trace of Stigmasterol (1b)

White crystalline solid; IR (KBr) υ_max 3238, 2958, 2933, 2864, 1454 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃): δ 5.35 (1H, br. s, 6-H), 5.33 (1H, dd, 23-H of 1b), 5.00 (1H, dd, 22-H of 1b), 3.5 (1H, m, 3-H), 1.01 (3H, s, 19-H), 0.90 (2H, d, 22-H of 1a), 0.899 (3H, d, 26-H), 0.831 (3H, d, 27-H), 0.79 (2H, d, 23-H of 1a), 0.66 (3H, s, 18-H); ¹³C-NMR (100 MHz, CDCl₃): δ 140.81 (C-5), 138.37 (C-22), 129.69 (C-23), 121.69 (C-6), 71.81 (C-3), 56.91 (C-14), 56.02 (C-17), 51.29 (C-24), 50.2 (C-9), 42.55 (C-4), 42.35 (C-13), 40.54 (C-20), 39.77 (C-12), 37.31 (C-1), 36.56 (C-10), 31.94 (C-7), 31.94 (C-8), 31.69 (C-2), 31.49 (C-25), 28.96 (C-16), 25.44 (C-28), 24.39 (C-15), 21.26 (C-26), 21.11 (C-11), 21.11 (C-21), 19.42 (C-19), 19.02 (C-27), 12.29 (C-29), 12.07 (C-18).

Piperine (2)

White crystalline solid; IR (KBr) υ_max 2993, 2855, 1636, 1549, 1193, 922 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃): δ 7.36 (1H, d, H-3), 6.95 (1H, d, H-2’), 6.86 (1H, d, H-6’), 5.95 (2H, s, -OCH₂O-), 3.56 (2H, br. s, H-6”), 1.54 (2H, m, H-3”), 1.64 (2H, m, H-5”), 1.57 (2H, m, H-4”); ¹³C-NMR (100 MHz, CDCl₃): 165.4 (C-1), 119.7 (C-2), 142.8 (C-3), 125.3 (C-4), 138.3 (C-5), 131.0 (C-1’), 105.6 (C-2’), 148.2 (C-3’), 148.1 (C-4’), 108.4 (C-5’), 122.5 (C-6’), 101.2 -OCH2O-), 45.3 (C-2”), 26.1 (C-3”), 75.3 (C-4”), 25.3 (C-5”), 46.9 (C-6”).

Ergosterol peroxide (3)

White crystalline solid; IR (KBr) υ_max. 3178, 3032, 1711, 1490, 1247 cm⁻¹; ¹H-NMR (400 MHz, CDCl₃): δ 6.49 (1H, d, H-7), 6.31 (1H, d, H-6), 5.21 (1H, dd, H-22), 5.13 (1H, dd, H-23), 3.96 (1H, m, H-3), 0.99 (3H, d, H-21), 0.911 (3H, d, H-28), 0.87 (3H, s, H-19), 0.83 (3H, s, H-18), 0.80 (3H, d, H-27), 0.79 (3H, d, H-26); ¹³C-NMR (100 MHz, CDCl₃): 135.41 (C-7), 135.21 (C-22), 132.31 (C-23), 130.76 (C-6), 82.17 (C-8), 79.44 (C-5), 66.49 (C-3), 56.19 (C-17), 51.68 (C-9), 51.07 (C-14), 44.56 (C-13), 42.77 (C-24), 39.75 (C-20), 39.34 (C-12), 37.96 (C-4), 36.92 (C-10), 34.69 (C-1), 33.07 (C-25), 30.11 (C-2), 28.66 (C-16), 23.4 (C-15), 20.88 (C-11), 20.63 (C-21), 19.96 (C-27), 19.65 (C-26), 18.19 (C-19), 17.57 (C-28), 12.88 (C-18).

(E)-1-(4-chlorophenyl)-3-(4-methylphenyl)-prop-2-en-1-one (4)

Pale yellow solid; m.p. 156–158 °C (lit.¹³ 155-157 °C) IR (KBr) υ_max 3100–3000, 1658, 1375–1331, 1275–1222, 738, 705–667 cm⁻¹. ¹H and ¹³C NMR data ( Table 1 ); ESI-MS m/z 257 [M + H]⁺.

Table 1.

¹H NMR (400 MHz) and ¹³C NMR (100 MHz) Spectral Data of Compounds 4 and 5 in CDCl₃.

Positions	4		5
Positions	δ_H mult., (J in Hz)	δ_C, type	δ_H mult., (J in Hz)	δ_C, type
1	-	189.3, C = O	-	189.2, C = O
2	7.44, d, 15.6	120.5, CH	7.36, d, 15.6	119.1, CH
3	7.79, d, 15.6	145.5, CH	7.78, d, 15.6	145.2, CH
1’	-	131.9, C	-	127.4, C
2’/6’	7.54, d, 8	128.6, CH	7.60, d, 8.8	130.3, CH
3’/5’	7.23, d, 8	128.9, CH	6.93, d, 8.8	114.5, CH
4’	-	141.4, C	-	161.8, C
1’’	-	136.6, C	-	136.8, C
2’’/6”	7.95, d, 8.4	129.9, CH	7.95, d, 8.8	129.8, CH
3’’/5”	7.47, d, 8.4	129.8, CH	7.46, d, 8.8	128.9, CH
4”	-	139.1, C	-	138.8, C
4’-OMe	-	-	3.85, s	55.5, CH₃
4’-Me	2.39, s	21.6, CH₃	-	-

(E)-1-(4-chlorophenyl)-3-(4-methoxyphenyl)-prop-2-en-1-one (5)

Pale yellow solid; m.p. 117–119 °C (lit.¹³ 116-118 °C); IR (KBr) υ_max 3110–3000, 1711, 1400–1350, 1300–1217, 740 cm⁻¹. ¹H and ¹³C-NMR data ( Table 1 ). ESI-MS m/z 273 [M + H]⁺; m/z 296 [M + Na]⁺.

Cytotoxicity Assay

Table 2 represents the cytotoxicity of the isolated compounds (1a-5) on HeLa cells.

Table 2.

Cytotoxicity of the Isolated Compounds (1a-5) on HeLa Cells.

Sample	Survival of HeLa cells	Cytotoxicity status
Vehicle	100%	Not cytotoxic
1a with trace 1b	>95%	Not cytotoxic
2	>95%	Not cytotoxic
3	>95%	Not cytotoxic
4	10%–20%	Moderate cytotoxic
5	<5%	Cytotoxic

Discussion

β-sitosterol (1a) with a trace (13.5%) of stigmasterol (1b) and piperine (2), two previously reported (Figure 1) compounds from P. chaba were readily characterized by comparing their NMR spectral data (Supplementary figures S1 –S7) with literature.^14-18 The ¹³C NMR spectrum (Supplementary figure S9) of compound 3 displayed 28 carbon resonances while the ¹H NMR spectrum (Supplementary figure S8) showed olefinic proton signals at δ 6.49, 6.31, 5.21 & 5.13 and an oximethine proton shift at δ 3.96. In addition, it revealed six methyl group signals. Compound 3 was identified as ergosterol peroxide (Figure 1) by comparing its spectroscopic data with the published values.^19,20 This is the first report of its occurrence from P. chaba.

Figure 1.

Structures of Compounds Isolated from P. chaba Roots.

Compound 4 was isolated as a pale yellow solid having R_f value of 0.51 (5% ethyl acetate in dichloromethane). The ESI-MS spectrum showed a protonated molecular ion peak at m/z 257 [M + H]⁺ corresponding to the molecular formula C₁₆H₁₃OCl (Figure 1, Supplementary figure S15). The peaks at m/z 257 and 259 with an approximate intensity of 3:1 confirmed the presence of Cl atom in the parent compound. Its IR spectrum (Supplementary figure S16) displayed absorption bands at 3100–3000 cm⁻¹ for aromatic and vinyl C-H stretching, at 1658 cm⁻¹ due to C-O stretching for carbonyl group, at 1375–1331 cm⁻¹ due to methyl asymmetric C-H bending and 1275–1222 cm⁻¹ for methyl symmetric C-H bending. The band at 738 cm⁻¹ and a loop between 705 and 667 cm⁻¹ substantiated the presence of C-Cl group¹³ in compound 4.

The ¹H NMR spectrum (Supplementary figure S10) of the compound 4 exhibited four aromatic proton signals each of two proton intensity at δ_H 7.95 (d, 8.4 Hz), 7.54 (d, 8.0 Hz), 7.47 (d, 8.4 Hz), and 7.23 (d, 8.0 Hz) for two sets of para-disubstituted aromatic rings in the compound. It also revealed trans- coupled (J = 15.6 Hz) protons at δ_H 7.79 and 7.74, and a methyl protons at δ_H 2.39 (3H, s) (Table 1). The ¹³C NMR spectrum (Supplementary figure S11) exhibited 12 carbon signals out of 16 carbons due to local symmetry of benzene rings, including a carbonyl participation at δ_C 189.3, whereas the DEPT (Supplementary figure S12) and HSQC (Supplementary figures S13) experiment indicated that 11 out of the 12 carbons had attached protons. The HMBC correlation (Supplementary figures S14) from H-2 (δ_H 7.36) to C-1’ (δ_C 131.9), from H-3 (δ_H 7.79) to C-1 (δ_C 189.3)/C-2’/6’ (δ_C 128.6), from H-2’/6’ (δ_H 7.60) to C-3 (δ_C 145.5)/C-4’(δ_C 141.4), from H-3’/5’ (δ_H 7.23) to C-1’ (δ_C 131.9)/4’-Me (δ_C 21.6), from H-2”/6” (δ_H 7.95) to C-1 (δ_C 189.3)/ C-3”/5” (δ_C 129.8)/ C-4” (δ_C 139.1), and from H-3”/5” (δ_H 7.47) to C-1” (δ_C 136.6)/ C-4” (δ_C 139.1) revealed the direct connection of C-1 with C-2, C-3 with C-1’, C-1” with C-1, and C-3”/5” with C-4”. HMBC correlations from the methyl group signal at δ_H 2.39 (3H, s) to C-4’ (δ_C 161.8)/C-3’/5’ (δ_C 128.9) confirmed that the methyl group was linked to C-4’, as shown in Figure 2. Thus, the chlorine atom was assumed to attach at C-4”. On the basis of above features, compound 4 was characterized (E)-1-(4-chlorophenyl)-3-(4-methylphenyl)-prop-2-en-1-one. Although it has previously been obtained by synthesis,¹³ this is the first report of its isolation from a natural source.

Figure 2.

¹H-¹H COSY and HMBC Correlations of Compounds 4 and 5.

Compound 5 was obtained as a pale yellow solid. It turned purple upon spraying the developed TLC plate with vanillin-sulfuric acid mixture followed by heating at 105 ^oC for few minutes. Its molecular formula was deduced as C₁₆H₁₃O₂Cl based on a pseudomolecular ion peak at m/z 273 [M + H]⁺ and a sodium adduct at m/z 296 [M + Na]⁺ in the ESI-MS spectrum (Figure 1, Supplementary figure S23).

The ¹H NMR spectrum (Supplementary figures S17) of the compound 5 exhibited four aromatic proton signals each of two proton intensity each at δ_H 7.95 (d, 8.8 Hz), 7.60 (d, 8.8 Hz), 7.46 (d, 8.8 Hz), and 6.93 (d, 8.8 Hz) for two sets of para-disubstituted aromatic rings in the compound. It also revealed trans- coupled (J = 15.6 Hz) protons at δ_H 7.36 and 7.78, and a methoxyl protons at δ_H 3.85 (3H, s) (Table 1). The ¹³C NMR spectrum (Supplementary figures S18) exhibited 12 carbon signals out of 16 carbons due to local symmetry of benzene rings, including a carbonyl participation at δ_C 189.2, whereas the DEPT (Supplementary figure S19) and HSQC (Supplementary figures S20) experiments indicated that 11 of the 12 carbon signals had attached protons.

The ¹H and ¹³C NMR spectral data of compound 5 was almost identical to those observed for compound 4, thus suggesting a close structural similarity between these two compounds. However, the methyl group signal observed in the NMR spectrum at δ_H 2.39 (δ_C 21.6) for compound 4 was replaced by a methoxyl group resonance δ_H 3.85 (δ_C 55.5). In addition, the signal for H-3’/H-5’ and C-3’/C-5’ in compound 5 were shielded to δ_H 6.93 and δ_C 114.5 as compared to δ_H 7.23 and δ_C 128.9 in compound 4. On the basis of the above structural features and comparing with previously published data,^13,21 compound 5 was identified as (E)-1-(4-chlorophenyl)-3-(4-methoxyphenyl)-prop-2-en-1-one, which also appears to be a new natural product.

The survival rate of HeLa Cells was less than 5% for the 5 indicating its highest cytotoxic potential (Table 2). Moderate cytotoxicity was seen in case of the compound 4. The presence of a methoxy of group in C-4’ in the chalcone 5 increased the cytotoxicity compared to the methyl group in C-4’ of chalcone 4. Remaining compounds showed no significant cytotoxicity on HeLa cell lines. This study was unable to perform molecular cell biological analyses to uncover the mechanism underlying the cytotoxic effects of compounds 4 and 5. Investigating the effects of these isolated compounds on different cancer cell lines could further clarify their impact on various types of cancer.

Conclusions

In the present study with the root of P. chava, the well-known ergosterol peroxide (3) and chlorinated chalcones (4 and 5) were isolated for the first time from a natural source, together with β-sitosterol (1a) with a trace of stigmasterol (1b) and piperine (2). Additionally, the HeLa cell line was used to test the cytotoxicity of each compound, and compound 5 had the highest level of cytotoxicity. Further studies are required to explore the biological mechanisms of the cytotoxic compounds.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251334718 - Supplemental material for Chlorinated Chalcones, Steroids and Piperine from the Roots of Piper chaba H. Growing in Bangladesh and Their Cytotoxicity on HeLa Cell Lines

Supplemental material, sj-docx-1-npx-10.1177_1934578X251334718 for Chlorinated Chalcones, Steroids and Piperine from the Roots of Piper chaba H. Growing in Bangladesh and Their Cytotoxicity on HeLa Cell Lines by Anath Chandra Roy, Md. Hasanur Rahman, Mohammad Rashedul Haque, Mohammad Sharifur Rahman, Tofail Ahmad Chowdhury and Mohammad Abdur Rashid in Natural Product Communications

Footnotes

Acknowledgments

The authors thank the members of the Division of Natural Products at Honam National Institute of Biological Resources for their preparations.

ORCID iDs

Md. Hasanur Rahman

Mohammad Sharifur Rahman

Ethical Considerations

Ethical Approval is not applicable for this article.

Author Contributions/CRediT

ACR performed the experiments, MHR provided methodology and supervision, MRH reviewed the manuscript, MSR conducted writing and data analysis, TAC designed and supervised the study, MAR conducted supervision, data analysis and reviewing. All authors have read and agreed to the published version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Conflicting Interests

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

Data Availability

All data either generated or analyzed during this study are included in this published article or available from the corresponding author upon reasonable request.

Statement of Informed Consent

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

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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Chlorinated Chalcones,Steroids and Piperine from the Roots of Piper chaba H. Growing in Bangladesh and Their Cytotoxicity on HeLa Cell Lines

Abstract

Background

Method

Result

Conclusion

Keywords

Introduction

Methods

General Experimental Procedures

Instruments

Collection of the Plant Material

Extraction and Isolation

Cytotoxicity Assay

Statistical Analysis

Results

β-Sitosterol (1a) with a Trace of Stigmasterol (1b)

Piperine (2)

Ergosterol peroxide (3)

(E)-1-(4-chlorophenyl)-3-(4-methylphenyl)-prop-2-en-1-one (4)

(E)-1-(4-chlorophenyl)-3-(4-methoxyphenyl)-prop-2-en-1-one (5)

Cytotoxicity Assay

Discussion

Conclusions

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251334718 - Supplemental material for Chlorinated Chalcones, Steroids and Piperine from the Roots of Piper chaba H. Growing in Bangladesh and Their Cytotoxicity on HeLa Cell Lines

Footnotes

Acknowledgments

ORCID iDs

Ethical Considerations

Author Contributions/CRediT

Funding

Conflicting Interests

Data Availability

Statement of Informed Consent

Supplemental Material

References

Supplementary Material