Royleanine A, an Antitumor Dihydro-β-agarofuran Sesquiterpene Pyridine Alkaloid from Maytenus royleanus

Abstract

Genus Maytenus (Celastraceae) has proved to be a good source of new bioactive sesquiterpene pyridine alkaloids. Maytenus royleanus is a medicinal plant used in folk medicine for different ailments in a variety of ways around the world. Investigation of the roots of M. royleanus resulted in the isolation of an undescribed antitumor sesquiterpene pyridine alkaloid: Royleanine A (1). Its structure was established by analysis of spectroscopic data and comparison with reported data. Royleanine A (1) was evaluated for its antitumor potential against several cancer cell lines and was found to be significantly active against OVC-5 cells (ovarian cancer cell line) with half maximal inhibitory concentration (IC₅₀) 28.9 µg mL^-1, cervical (Hela) IC₅₀ 0.064 µg mL^-1 and prostate (PC 3) cancer lines with 0.034 µg mL^-1.

Keywords:
Maytenus royleanus; pyridine alkaloid; Royleanine A; antitumor activity

Introduction

Since the very dawn of medicine, plant secondary metabolites have been investigated as a source of new therapeutic agents.¹ Species of Maytenus (Celastraceae) proved as promising source of new bioactive agents. Species of Maytenus have been widely used in folk medicine²,³ for different ailments like influenza, rheumatism, gastrointestinal disorders and tumors in South America and Asia.⁴

A variety of bioactive secondary metabolomes, over time, have been reported from species of Maytenus, including maytansinoids, spermidine, terpenoids, cardinolids, flavonoids and dihydro-β-agarofuran sesquiterpene pyridine alkaloids. Dihydro-β-agarofuran sesquiterpene pyridine alkaloids displayed a broad range of biological activities like antitumor,⁵ insecticidal,⁶ immunosuppressive,⁷,⁸ anti-human immunodeficiency virus (HIV),⁹ multi drugs resistant-reversing activity,¹⁰ cytotoxicity,¹¹ anti-tubercular¹² and anti-inflammatory effects.¹³

The genus Maytenus is represented in Pakistan by three species: M. willichiana, M. senegalensis and M. royleanus. M. royleanus is locally known as “soor azghee” (red thorn) in Pakistan and has been used in folk medicine of the locality. A prelude to this research work was our visit to a very popular local traditional physician (Hakeem) in district Buner of Pakistan, who used this plant in combination with other plant extracts, for the treatment of ‘Nasoor’ which is a local name for complicated diseases like cancer and up till now, many patients have been cured. The results were even confirmed by follow up test in a hospital. As part of our search to discover new antitumor agents from local medicinal plants,¹⁴-¹⁶ we investigated the roots of M. royleanus. In a previous article,¹⁶ we reported the gas chromatography mass spectrometry (GC-MS) analysis of seed oil and isolation of triterpenes from roots of M. royleanus.

Herein, we report the isolation and structural determination of a new sesquiterpene pyridine alkaloid, Royleanine A (1) and its in vitro antitumor activity against different cancer cell lines.

Experimental

General experimental procedure

All chemicals and solvents used were purchased from commercial suppliers (Sigma-Aldrich, Karachi, Pakistan). Melting point was measured with Buchi B-535 digital device (Flawil, Switzerland). Optical rotation was taken on Jasco-P2000 digital polarimeter (Easton, Maryland, USA) in MeOH at room temperature. For infrared (IR) spectral data Bruker VECTOR-22 (Billerica, Massachusetts, USA) spectrometer was used; ν_max in cm^-1. UV spectrum was recorded with the help of analytical LC-MS 996 photodiode array detector. ¹H (600 MHz, CD₃OD) and ¹³C (125 MHz, CD₃OD) nuclear magnetic resonance (NMR) data were recorded by Varian Inova 600 instrument (California, USA), values of δ in ppm and J in Hz. For chemical profiles of fractions and compounds purity, analytical Shimadzu-high performance liquid chromatography (HPLC) (Kyoto, Japan) was used (MeOH/H₂O with 0.1% formic acid), having reverse phase (RP)-C₁₈ (5 µm diameter, 15 cm × 4.6 mm length, Phenomenex Luna^®, CA, USA) column, Sedex 55 evaporative light scattering detector (ELSD), 996 photodiode array detector and electrospray ionization (ESI-MS) in + ve mode equipped with time of flight (TOF) analyzer. Both low resolution ESI and high-resolution ESI mass spectra were recorded on Mariner ESI-TOF-MS (Connecticut, USA) instrument.

Plant materials

M. royleanus roots were collected in northern area (Buner) of Pakistan in May 2018. The selected specie was identified by Prof Ambara Khan and Dr Fazle Rahim. A voucher specimen (HBKU-825) was deposited in the Herbarium of Department of Botany, Bacha Khan University Charsadda, Khyber Pukhtoonkhwa, Pakistan. Fresh plant material was washed with distilled water to remove dust particles and was shade dried. The plant material was grinded to fine particles and extracted with methanol.

Extraction and isolation

Dried roots (13 kg) of M. royleanus were extracted with commercial MeOH at 25 °C after 24 h and the process was repeated three times. The filtered extract after concentration at 40 °C resulted into brownish thick methanolic extract (100 g) which was stored in refrigerator at 4 °C. According to our reported fractionation procedure, crude methanolic extract was partitioned on the basis of increasing solvent polarity, into seven fractions (FH1, FH2, FD1, FD2, FD3, FM1 and FM2) (Supplementary Information (SI) section Figure S2).

The liquid chromatography mass spectometry (LC MS) profiles of all crude fractions were developed and all showed complex peaks cluster. Dichloromethane fraction (FD2, 25 g) was partitioned into five subfractions (FD2A-E) by column chromatography, the LCMS chemical profile of all five fractions (FD2A-E) were developed. In chromatogram of FD2B peak at m/z 909 become prelude for future study, after dereplication of known data. The selected peak was isolated with preparative HPLC, using reverse phase C₁₈ column (5 µm, 15 cm × 4.6 mm), gradient solvent system MeCN/H₂O (flow rate of 10-100 mL in 30 min) 0.1% acidic (formic acid), resulted into pure compound 1 (14 mg).

Royleanine A (1)

[α]_D^27.9 -115; UV (MeOH) λ / nm 240; IR (KBr) ν / cm^-1 2922, 1731, 1592; ¹H NMR (600 MHz, CD₃OD) δ 5.62 (d, J 3.6 Hz, H-1), 5.20 (dd, J 3.6, 1.8 Hz, H-2), 4.65 (d, J 1.8 Hz, H-3), 7.00 (brd s, H-5), 2.25 (d, J 4.2 Hz, H-6), 5.54 (dd, J 6.0, 4.2 Hz, H-7), 5.40 (d, J 6.0 Hz, H-8), 5.22 (d, J 13.4 Hz, H-11a), 4.41 (d, J 13.4 Hz, H-11b), 1.51 (s, H-12), 1.61 (s, H-14), 5.91 (d, J 11.4 Hz, H-15a), 3.93 (d, J 3.6 Hz, H-15b), 7.42 (d, J 7.1 Hz, H-3’), 8.11 (dd, J 7.1, 1.8 Hz, H-4’), 8.68 (d, J 1.8 Hz, H-6’), 4.65 (q, J 7.0 Hz, H-7’), 2.54 (q, J 7.1 Hz, H-8’), 1.42 (d, J 7.0 Hz, H-9’), 1.61 (d, J 7.1 Hz, H-10’), 2.91 (s, Ac-1), 2.61 (s, Ac-2), 1.84 (s, Ac-5), 2.16 (s, Ac-7), 1.98 (s, Ac-8), 2.13 (s, Ac-11); ¹³C NMR (125 MHz, CD₃OD) δ 74.8 (C-1), 69.8 (C-2), 77.6 (C-3), 71.9 (C-4), 75.4 (C-5), 51.6 (C-6), 70.2 (C 7), 72.1 (C-8), 53.5 (C-9), 95.0 (C-10), 61.0 (C-11), 23.6 (C 12), 85.8 (C-13), 18.5 (C-14), 71.3 (C-15), 165.8 (C 2’), 123.0 (C-3’), 139.0 (C-4’), 127.0 (C-5’), 152.5 (C 6’), 36.6 (C 7’), 46.4 (C-8’), 12.1 (C-9’), 9.9 (C-10’), 175.6 (C-11’), 170.2 (C-12’), 20.9 (Ac-1), 20.8 (Ac-2), 21.2 (Ac 5), 20.8 (Ac-7), 20.9 (Ac-8), 21.8 (Ac-11), 171.8 (CO-1), 170.5 (CO-2), 171.0 (CO-5), 171.4 (CO-7), 171.2 (CO-8), 172.0 (CO-11); HR-ESI-MS m/z, calcd. for C₄₅H₅₁NO₁₉ [M]⁺: 909.3171, found: 909.3078.

Anti-proliferative disk diffusion assay

In vitro anti-proliferative disk diffusion assay was performed in Josephine Ford Cancer Centre (JFCC), Henry Ford Hospital, Detroit (USA).¹⁷ Royleanine A (1) was screened for determination of its potency and selectivity against twelve cancer cell lines. Measure of the differential inhibition of solid tumor against normal or leukemia cell line is considered as selectivity, while positive activity is greater than 250 zone units (200 zone units = 6.5 mm) inhibition. The sample was first dissolved in dimethyl sulfoxide (DMSO), then transferred onto a paper disk, applied to an agar plate which was then seeded with a specific tumor cell line. For cell growth, the agar plates were incubated and the activity of the sample was measured from the size of zone of inhibition in zone units or mm on agar plate. In order to determined half maximal inhibitory concentration (IC₅₀) value for the sample, human tumor cells were cultured in T25 tissue culture flasks in concentration of 5 × 10⁴ cells, then 5 mL of media Roswell Park Memorial Institute (RPMI) 1640 provided with 15% bovine calf serum, 5% penicillin and 5% glutamine. After 72 h of incubation, sample was transferred to culture flasks in order to achieve concentrations from 10¹ 10 ⁵ µg mL^-1. After three days incubated of flasks, the cells were cleaned, trypsinized, rotated and the number of both viable and dead cells were counted by using 0.08% trypan blue stain. The number of living cells was plotted as function of concentration and the IC₅₀ value determined by interpolation. Each point was performed in triplicate and then standard deviation was calculated.

Anti-proliferative assays

For compound 1, the antiproliferative potential was determined by the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyltetrazolium bromide) assay on Hela (cervical) and PC-3 (prostate) cancer lines. Cells have grown in DME (Dulbecco’s Modified Eagle) medium for PC-3 and ME (minimal essential) medium for Hela, containing 10% fetal bovine serum (FBS) and 2% penicillin/streptomycin and maintained at 37 °C with 5% CO₂ level for 24 h in flask. Cells (1 × 10⁵ cells mL^-1) were placed in a 96 well plates for 24 h incubation, to allow cell attachment. Various concentrations of sample compound varying from 100 1 µM are added into the well and incubated for 48 h. The IC₅₀ values were calculated and at least three independent experiments were carried out for the sample. Doxorubicin was used as positive control in this assay for both PC-3 and Hela.¹⁸

Results and Discussion

After dereplication of known data by using available chemical database,¹⁹ no hits were observed for peak at m/z 909 in LC-MS profile of fraction FD1B (see Experimental section). FD1B was subjected to reversed-phase high-performance liquid chromatography (RP HPLC), which resulted in the isolation of an undescribed dihydro-β agarofuran sesquiterpene pyridine alkaloid, Royleanine A (1), Figure 1.

Figure 1
Chemical structure of compound 1.

Compound 1 was purified as white amorphous powder, its molecular formula (C₄₅H₅₁NO₁₉) was assigned on the basis of NMR data and HR-ESI-MS which showed molecular ion peak at m/z 910.3128 [M + H]⁺. The UV spectrum showed maximum absorption at 240 and 274 nm indicating the presence of aromatic moiety in compound 1. In the IR spectrum absorption bands at 1735 and 1592 cm^-1 indicated ester carbonyl and C=C of aromatic group. The ¹³C NMR spectra of 1 indicated total 45 carbon atoms, including nine CH₃, four CH₂, sixteen CH and sixteen non-hydrogenated carbons. The NMR data further suggested the presence of nine esters including six acetate esters, one benzoate and one evoninate ester.

In the ¹³C NMR (125 MHz, CD₃OD) spectra ester carbonyls appeared at δ 171.5, 170.0, 171.0, 171.9, 171.2, 171.7, 172.6, 168 and 172.8, being assigned to C-1, 2, 5, 7, 8, 11, 11’, 12’ and 7’’, respectively, by heteronuclear single quantum coherence spectroscopy (HSQC) correlations (Table 1). The six acetate protons in ¹H NMR (600 MHz, CD₃OD) spectra resonated at δ 2.22 (s, OAc-1), 1.85 (s, OAc-2), 1.74 (s, OAc-5), 2.06 (s, OAc-7), 2.01 (s, OAc-8), 2.28 (s, OAc-11) were assigned to respective positions by 2D NMR data. The ¹H NMR spectra of 1 further showed the presence of three sec-methyls at δ 1.38 (d, J 7.8 Hz, H-12), 1.63 (s, H-14), 1.97 (s, H-10’), two methine protons at δ 2.76 (d, J 7.8 Hz, H-4), 2.76 (d, J 3.6 Hz, H-6) and six oxygenated methine protons at δ 5.79 (d, J 3.6 Hz, H-1), 5.47 (dd, J 2.4, 3.6 Hz, H-2), 4.80 (d, J 2.4 Hz, H-3), 6.41 (brd s, H-5), 5.71 (dd, J 9.6, 3.6 Hz, H-7) and 5.73 (d, J 9.6 Hz, H-8) which were characteristic peaks of dihydroagarofuran polyester sesquiterpene skeleton in compound 1.²⁰ In ¹H NMR spectrum downfield signals at δ 8.87 (brd s, H-3’), 7.50 (d, J 4.8 Hz, H-5’), 8.61 (d, J 4.8 Hz, H-6’) were assigned to 2’,4’-disubstituted pyridine moiety, present in majority of Maytenus alkaloids,²⁰ while protons at δ 8.07 (dd, J 1.2, 7.8 Hz, H-2’’, 6’’), 7.54 (t, J 7.2 Hz, H-3’’, 5’’) and 7.67 (t, J 7.8 Hz, H-4’’) were assigned to benzoate moiety. The presence of pyridine and benzoate moieties were supported by ¹³C NMR spectra, the pyridine carbons resonated at δ 153.6 (C-2’), 152.0 (C-3’), 128.7 (C-4’), 127.5 (C-5’), 153.0 (C-6’) and six phenyl carbons at δ 166.6 (C-1’’), 131.1 (C-2’’, 6’’) and 130.1 (C-3’’, 5’’). Analysis of 2D NMR data confirmed that pyridine ring was 2,4-disubstituted and benzoate group was attached at position C-9’ (Figures 2 and S1, SI section). The heteronuclear multiple bond correlation (HMBC) spectrum of 1 exhibited long range correlations between H-1/C-11, COO-1; H-2/C-9, COO-2; H-3/C-10, 12, 11’; H-4/C-2, 5; H-5/C-7; H-6/C-10; H-7/C-9, 13; H-11/C-1, 8, COO-11; and H-15/C-13, COO-12’, which confirmed the presence of dihydroagarofuran sesquiterpene skeleton. The relative stereochemistry of 1 was confirmed by nuclear overhauser effect spectroscopy (NOESY) spectrum and comparison with spectral data of related reported compounds.²⁰,²¹

Figure 2
Selected COSY, HMBC and NOESY correlations for compound 1.

Table 1
¹H (600 MHz) and ¹³C NMR (125 MHz) data of Royleanine A (1) in CD₃OD

C No.	δC / ppm	C-type	δH (multi, integral, J) / ppm	HMBC
C-1	74.8	CH	5.62 (d, J 3.6 Hz)
C-2	69.8	CH	5.20 (dd, J 3.6, 1.8 Hz)	C4, 11, COO-2
C-3	77.6	CH	4.65 (d, J 1.8 Hz)	C1, 4, 10
C-4	71.9	C	-	-
C-5	75.4	CH	7.0 (brd. s)	-
C-6	51.6	CH	2.52 (d, J 4.2 Hz)	C7, 8, 10
C-7	70.2	CH	5.54 (dd, J 6.0, 4.2 Hz)	C9, COO-7
C-8	72.1	CH	5.40 (d, J 6.0 Hz)	C1, 7, 11
C-9	53.5	C	-	-
C-10	95.0	C	-	-
C-11	61.2	CH2	5.22 (d, J 13.4 Hz, Ha)	C8, 9, COO-11
			4.41 (d, J 13.4 Hz, Hb)	C8, 9, 10
C-12	23.6	CH3	1.51 (s)	C3, 4, 10
C-13	85.8	C	-	-
C-14	18.5	CH3	1.61 (s)	-
C-15	71.3	CH2	5.91 (d, J 11.4 Hz, Ha)	C12’
			3.93 (d, J 11.4 Hz, Hb)	C12’, 13, 14
C-2’	165.8	C	-	-
C-3’	123.0	CH	7.42 (d, J 7.1 Hz)	C7,5’
C-4’	139.0	CH	8.11 (dd, J 7.1,1.8 Hz)	C2’,5’,12’
C-5’	127.0	C	-	-
C-6’	152.5	CH	8.68 (d, J 1.8 Hz)	C2’,4’,12’
C-7’	36.6	CH	4.65 (q, J 7.0 Hz)	C2’, 9’, 11’
C-8’	46.4	CH	2.54 (q, J 7.1 Hz)	C9’, 10’
C-9’	12.1	CH3	1.42 (d, J 7.0 Hz)	C2’, 8’
C-10’	9.9	CH3	1.16 (d, J 7.1 Hz)	C7’, 8’, 11’
C-11’	175.6	C	-	-
C-12’	170.2	C	-	-
1-Ac	20.9	CH3	2.19 (s)	COO-1
2-Ac	20.8	CH3	2.16 (s)	COO-2
5-Ac	21.2	CH3	1.84 (s)	COO-3
7-Ac	20.8	CH3	2.16 (s)	COO-4
8-Ac	20.9	CH3	1.98 (s)	COO-5
11-Ac	21.8	CH3	2.13 (s)	COO-6
1-CO	171.8	-	-	-
2-CO	170.5	-	-	-
5-CO	171.0	-	-	-
7-CO	171.4	-	-	-
8-CO	171.2	-	-	-
11-CO	172	-	-	-

HMBC: heteronuclear multiple bond correlation.

The relative stereochemistry of compound 1 was determined on the basis of NOESY correlations, chemical shift (δ) and coupling constant (J) values. Acetyl groups at C-1 and C-2 were assigned as axial configuration on the basis of J value for coupling of H_eq-1 with H_eq-2 (J 3.6 Hz) and H_eq-2 with H_eq-3 (J 1.8 Hz). This was further confirmed by NOESY correlations between H-1/H-2 and H-2/H-3. Proton at C-3 also showed NOESY correlations with equatorial methyl at C-12 and with OBz protons at C-9’ which were assigned α configuration. Acetyl groups at C-5, 7 and 8 were assigned axial configuration on the similar way with the help of chemical shift, coupling constant values and NOESY correlations. All spectral data analysis and comparison with related reported compounds were in agreement that the structure of compound 1 was 1β,2β,5α,7α,8α,11-hexaacetoxy-3α,15-[2’-methyl-2’α-benzoate-4’(4’’-carboxy-2’’-pyridyl)-butanoic acid] dicarbolactone-dihydroagarofuran, which was given a trivial name Royleanine A.

Royleanine A (1) was tested in Josephine Ford Cancer Centre, Henry Ford Hospital, Detroit, Michigan (USA), against twelve cancer cell lines (Table 2). Compound 1 was found active against OVC-5 cells (ovarian cancer cell line), with IC₅₀ values of 28.9 µg mL^-1, but did not show any significant activity against other cell lines.

Table 2
Anti-proliferative disk diffusion assay of Royleanine A (1)

Tumor cell line	IC50 / (µg mL-1)
Murine lyphocytic leukemia (L1210)	> 100
Murine granulocyte macrophage colony formy unit (CFU-GM)	> 100
Murine colon adrenocarcinoma (Colon38)	> 100
Human lung adrenocarcinoma (H125)	> 100
Human colon adrenocarcinoma (H116)	> 100
Androgen sensitive prostate cancer (LNCaP)	> 100
Hormone responsive breast cancer (MCF-7)	> 100
Melanoma (MDA)	> 100
Ovarian cancer (OVC-5)	28.9
Glioblastoma (U251N)	> 100
Murine pancreatic solid tumor (PANC-1)	> 100
Humane leukemic lymphoid (CEM)	> 100

₅₀

Compound 1 (Royleanine A) was also tested against two available cancer cell lines; Hela (cervical cancer cells) and PC-3 (prostate cancer cells) for its anti-proliferative activity (Table 3) at International Center for Chemical and Biological Sciences (ICCBS), university of Karachi, Pakistan. Compound 1 showed excellent activity against both cell lines even more than the standard used, with IC₅₀ values of 0.064 and 0.034 µg mL^-1 for Hela and PC-3, respectively.

Table 3
Antiproliferative activity of Royleanine A (1) against Hela and PC-3

Compound	IC50 ± SD / (µmol L-1)
	Hela	PC-3
Royleanine A	1.616 ± 0.04 (0.064 µg mL-1)	0.845 ± 0.01 (0.034 µg mL-1)
Doxorubicin	3.10 ± 0.20	0.91 ± 0.12

₅₀1^-1-1₅₀

The selectivity of compound 1 for ovarian, cervical and prostate cancer cell lines were very surprising and interesting results.

Conclusions

The phytochemical study of Maytenus royleanus roots resulted into a new compound 1: 1β,2β,5α,7α,8α,11 hexaacetoxy-3α,15-[2’-methyl-2’α-benzoate-4’(4’’-carboxy-2’’-pyridyl)-butanoic acid] dicarbolactone dihydroagarofuran (Royleanine A). Royleanine A (1) was tested for their anti-proliferative potential against fourteen cancer cell lines. The excellent cytotoxic selectivity against ovarian cancer cell line was in harmony with ethnomedicinal uses of this plant. Royleanine A (1) is a good candidate for anti-tumor drug development after extensive further work on it. M. royleanus not only will have greater use in folk medicine but is a promising source of new bioactive secondary metabolomes for future research work.

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br as PDF file.

https://minio.scielo.br/documentstore/1678-4790/spbvcxjFKRMhZpGqXpzpMXw/817682664bf4b11cc0719d1d79d80f6ad55944b1.pdf

Acknowledgments

The authors acknowledged Higher Education Commission (HEC) of Pakistan for financial assistance under IRSIP, University of California Santa Cruz for providing technical facilities and Frederick A. Valeriote. We are thankful to Prof Ambara Khan (Govt. Degree College Daggar, Pakistan), for his support in the identification and collection of plant.

References

• ¹
  Kuete, V.; Ango, P. Y.; Yeboah, S. O.; Mbaveng, A. T.; Mapitse, R.; Kapche, G. D.; Ngadjui, B. T.; Efferth, T.; BMC Complementary Altern. Med. 2014, 14, 340. [Crossref]
• ²
  Gonzalez, J. G.; Delle-Monache, G.; Delle-Monache, F.; Marini-Bettolo, G.; J. Ethnopharmacol. 1982, 5, 73. [Crossref]
• ³
  González, A.; Bazzocchi, I.; Moujir, L.; Jiménez, I.; Stud. Nat. Prod. Chem. 2000, 23, 649. [Crossref]
• ⁴
  Itokawa, H.; Shirota, O.; Ikuta, H.; Morita, H.; Takiya, K.; Itaka, Y.; Phytochemistry 1991, 30, 3713. [Crossref]
• ⁵
  Wu, W.; Wang, M.; Zhu, J.; Zhou, W.; Hu, Z.; Ji, Z.; J. Nat. Prod. 2001, 64, 364. [Crossref]
• ⁶
  Jiménez, I. A.; Bazzocchi, I. L.; Núñez, M. J.; Mukainaka, T.; Tokuda, H.; Nishino, H.; Konoshima, T.; Ravelo, A. G.; J. Nat. Prod. 2003, 8, 1047. [Crossref]
• ⁷
  Wang, X.; Gao, W.; Yao, Z.; Zhang, S.; Zhang, Y.; Takaishi, Y.; Duan, H.; Chem. Pharm. Bull. 2005, 6, 607. [Crossref]
• ⁸
  Duan, H.; Takaishi, Y.; Momota, H.; Ohmoto, Y.; Taki, T.; Jia, Y.; Li, D.; J. Nat. Prod. 2001, 5, 582. [Crossref]
• ⁹
  Gutiérrez-Nicolás, F.; Oberti, J. C.; Ariza-Espinar, L.; Ravelo, Á. G.; Estévez-Braun, A.; Planta Med. 2011, 15, 1718. [Crossref]
• ¹⁰
  Delgado-Méndez, P.; Herrera, N.; Chávez, H.; Estévez-Braun, A.; Ravelo, Á. G.; Cortes, F.; Castanys, S.; Gamarro, F.; Bioorg. Med. Chem. 2008, 3, 1425. [Crossref]
• ¹¹
  Zhu, Y.; Miao, Z.; Ding, J.; Zhao, W.; J. Nat. Prod. 2008, 6, 1005. [Crossref]
• ¹²
  Chen, J. J.; Chou, T. H.; Peng, C. F.; Chen, I. S.; Yang, S. Z.; J. Nat. Prod. 2007, 2, 202. [Crossref]
• ¹³
  Jin, H. Z.; Hwang, B. Y.; Kim, H. S.; Lee, J. H.; Kim, Y. H.; Lee, J. J.; J. Nat. Prod. 2002, 65, 89. [Crossref]
• ¹⁴
  Din, A. U.; Uddin, G.; Hussain, N.; Khan, A.; Khan, I.; Shad, A.; Choudhary, M. I.; J. Cancer Sci. Ther. 2013, 5, 404. [Crossref]
• ¹⁵
  Uddin, G.; Din, A. U.; Khurshid, A.; Valeriote, F. A.; Choudhary, M. I.; J. Chin. Chem. Soc. 2013, 60, 92. [Crossref]
• ¹⁶
  Din, A. U.; Uddin, G.; Hussain, N.; Choudary, M. I.; J. Braz. Chem. Soc. 2013, 24, 663. [Crossref]
• ¹⁷
  Gao, S.; Xu, Y. M.; Valeriote, F. A.; Gunatilaka, A. L.; J. Nat. Prod. 2011, 74, 852. [Crossref]
• ¹⁸
  Forguson, L. R.; Mutat. Res. 1994, 1, 395. [Crossref]
• ¹⁹
  Brüning, R.; Wagner, H.; Phytochemistry 1978, 17, 1821. [Crossref]
• ²⁰
  Corsino, J.; Bolzani, V. S.; Pereira, A. M. S.; França, S. C.; Furlan, M.; Phytochemistry 1998, 49, 2181. [Crossref]
• ²¹
  Han, B. H.; Ryu, J. H.; Han, Y. N.; Park, M. K.; Park, J. H.; Naoki, H.; J. Nat. Prod. 1990, 53, 909. [Crossref]

Edited by

Editor handled this article: Paulo Cezar Vieira

Publication Dates

• Publication in this collection
  28 Nov 2022
• Date of issue
  2022

History

• Received
  16 Nov 2021
• Accepted
  06 May 2022