PTP1B Inhibitory Flavonoids From Orthosiphon stamineus Benth. and Their Growth Inhibition on Human Breast Cancer Cells

Abstract

In our preliminary screening study on the protein tyrosine phosphatase 1B (PTP1B) inhibitory and cytotoxic activities, an ethyl acetate soluble fraction of the aerial part of Orthosiphon stamineus Benth. was found to inhibit PTP1B activity. Thus, based on assay-guided isolation of this active fraction, ten compounds (1-10) were purified and evaluated for their inhibitory effects on PTP1B and their growth inhibition on MCF7, tamoxifen-resistant MCF7 (MCF7/TAMR), and MDA-MB-231 human breast cancer cell lines. Among the isolates, compounds 5, 6, 9, and 10 showed potencies against PTP1B with IC₅₀ values of 9.76, 10.12, 6.88, and 8.92 μM, respectively, followed by compounds 1 and 4 with IC₅₀ values of 16.92 and 22.25 μM. Kinetic study showed that the active compounds (1, 5, 9, and 10) possessed mixed-competitive inhibition, which was similar to the positive control (ursolic acid, IC₅₀ value of 3.42 μM, mixed-competitive). The others showed noncompetitive inhibition (4 and 6). In addition, all these active compounds (1, 4-6, and 9-10) displayed growth inhibition on three cancer cell lines, especially the most PTP1B inhibitory flavanones (9 and 10) exhibited comparable inhibitory effects on MCF7, MCF7/TAMR, and MDA-MB-231 cancer cells (IC₅₀ values of 11.5 and 15.4, 8.9 and 10.5, and 17.6 and 21.3 μM, respectively) with tamoxifen, the positive control used in this assay (IC₅₀ values of 11.9, 12.1, and 12.7 μM, respectively). The results suggest that these active constituents from O. stamineus might be considered as new natural compounds for the development of anticancer agents via PTP1B inhibition.

Protein tyrosine phosphatase 1B (PTP1B) is a member of nontransmembrane protein tyrosine phosphatase that plays a key role in metabolic signaling and is a promising drug target for type 2 diabetes and obesity.^1,2 Originally, this enzyme is located on the cytoplasmic face of endoplasmic reticulum, and is firstly identified from human placenta and executed as a critical regulator of multiple signaling pathways involved in many kinds of human cancers.^3,4 Accumulating evidences indicated that PTP1B is involved in leptin signal of cancer as a key negative regulator.^3,4 However, several contrasting findings suggested that it could exert both tumor-suppressing and tumor-promoting effects depending on the substrate involved and the cellular context.⁵ Among human cancers, breast cancer is the most common malignancy and accounts for the second-highest number of deaths in women cancer worldwide. Protein tyrosine phosphatase 1B had recently drawn attention as an attractive target for the treatment of breast cancer.^5,6 Protein tyrosine phosphatase 1B promoted breast cancer metastasis by regulating phosphatase and tensin homolog but not epithelial-mesenchymal transition.⁷ In addition, the level of PTP1B was higher in breast cancer tissues than the corresponding adjacent normal tissues in several patients. It was positively associated with lymph node metastasis and estrogen receptor status, and acted as an independent prognostic factor and predicted a poor prognosis in estrogen receptor-positive breast cancer patients.^7,8 These findings consequently raised the hypothesis that inhibitors of PTP1B may offer new avenues for the treatment of breast cancer. Therefore, finding new PTP1B inhibitors from natural plants was investigated. A few PTP1B inhibitors from natural products have been identified. The results showed that natural compounds found to inhibit PTP1B were diverse including pterocarpan derivatives,^9,10 diterpenoids and triterpenoids,¹¹ flavonoids,¹² sesquiterpenoids,¹² coumarins,¹² alkaloids,¹² and phenolic compounds.^13,14 These results encourage us to continue our studies in the discovery of PTP1B inhibitors from natural sources.

In our screening program for PTP1B inhibitors from medicinal plants, the ethyl acetate (EtOAc) fraction of the aerial parts of Orthosiphon stamineus Benth. (Lamiaceae) exhibited 67.8% inhibition on PTP1B at a concentration of 30 µg/mL. Orthosiphon stamineus is found mainly in Southeast Asia, Australia, and Africa.¹⁵ In Vietnam, O. stamineus is called “Rau Meo” and the aerial parts are used for the treatment of urinary lithiasis, edema, eruptive fever, influenza, hepatitis, and jaundice.¹⁶ In Myanmar, this plant is known as “Secho” or “Myit-shwe” and the leaves are reputed to be an antidiabetic drug and decoctions of the air-dried leaves are used to cure urinary tract and renal diseases.¹⁷ This plant is also known as “Neko no hige” in Japan and consumed as a healthy Java tea to facilitate body detoxification. In Indonesia, O. stamineus is known as an effective folk medicine for the treatment of diabetes, hypertension, rheumatism, tonsillitis, and menstrual disorder.¹⁸ Orthosiphon stamineus had been reported to possess various pharmacological activities such as anti-inflammatory,^19,20 antioxidant,²¹ hepatoprotective,^22,23 anticancer,^24,25 antihypertensive,^19,26 gastroprotective,²² antisebum,²⁷ hyperlipidemic,²⁸ nephroprotective,²¹ antipyretic,²⁹ anti-angiogenic,³⁰ antibacterial,³¹ antiviral,³² diuretic,³³ and antidiabetic.^34,35 Previous studies on chemical constituents of O. stamineus showed the presence of essential oils,³⁶ diterpenoids, triterpenoids, flavonoids, and phenolics.^19,21,37 Some studies have shown PTP1B inhibitory^34,35 and cytotoxic activities³⁸ of extracts and fractions from O. stamineus. However, there has been no investigation of chemical constituents and inhibitory activities of PTP1B and cytotoxicity of O. stamineus. Therefore, this paper described the isolation and structural elucidation of these compounds as well as the evaluation of their PTP1B inhibitory and cytotoxic activities on human breast cancer cells.

The methanol extract of the aerial parts of O. stamineus was partitioned into chloroform, ethyl acetate, and water-soluble fractions. In a preliminary experiment, the inhibitory effect of these fractions was tested in vitro on PTP1B enzyme assay. The result showed that the ethylacetate fraction was the most active fraction against the enzyme activity (see supplemental figures S2–S4 for details). Therefore, the ethyl acetate fraction was selected for further isolation of active components. Chromatographic purification of this fraction (see supplemental figure S3) led to the isolation of 10 compounds (1-10) (Figure 1).

Figure 1

Chemical structure of isolated compounds 1 to 10 from the aerial parts of O rthosiphon stamineus Benth.

The ¹H-nuclear magnetic resonance (NMR) spectrum of 1, 2, 3, 5, and 6 displayed characteristic signals due to 5 aromatic protons of 2 benzene rings (rings A and B), together with those of hydroxyl (C-3) and methoxy groups (Figure 1), while their ¹³C-NMR spectrum revealed the signals of a ketone (C-4), oxygen-substituted carbons, and 2 quaternary carbons (Figure 1). The ¹H- and ¹³C-NMR spectra indicated that these compounds are derivatives of kaempferol (for 1 and 2) and quercetin (for 3, 5, and 6).³⁹ Detailed analysis of the ¹H- and ¹³C-NMR spectra of compound 1 revealed that 1 possessed 1 ketone carbon, 6 oxygenated carbons, and 2 methoxy carbons at C-7 and C-4′. Similar to compound 1, compound 2 also possessed 1 ketone carbon, 6 oxygenated carbons, but showed 3 methoxy carbons at C-5, C-7, and C-4′ (Figure 1). Compound 3 possessed 1 ketone carbon, 7 oxygenated carbons, and 3 methoxy carbons at C-7, C-3′, and C-4′. Compound 5 also possessed 1 ketone carbon, 7 oxygenated carbons, and 4 methoxy carbons at C-5, C-7, C-3′, and C-4′. Similar to compound 2, compound 6 also possessed 1 ketone carbon, 3 methoxy carbons at C-5, C-7, and C-4′, but showed 7 oxygenated carbons (Figure 1). Comparison of the ¹H- and ¹³C-NMR data of these compounds with those published in the literature led to the identification structures of 1, 2, 3, 5, and 6 to be 7,4′-dimethylkaempferol,⁴⁰ 5,7,4′-trimethylkaempferol,⁴¹ 7,3′,4′-trimethylquercetin,⁴² 5,7,3′,4′-tetramethylquercetin,⁴³ and 5,7,4′-trimethylquercetin,⁴⁴ respectively.

Compound 4 was isolated as a yellow crystal. Detailed analysis of the ¹H- and ¹³C-NMR spectra of compound 4 revealed that 4 possessed 1 ketone carbon, 7 oxygenated carbons, and 4 methoxy carbons at C-3, C-5, C-3′, and C-4′ (Figure 1). The ¹H- and ¹³C-NMR spectra indicated that this compound possessed the basics of 3-methoxyflavone³⁹ and was also a derivative of quercetin. Thus, the structure of compound 4 was assigned to be 3,5,3′,4′-tetramethylquercetin.⁴⁵

Compound 7 was isolated as colorless needles. The ¹H-NMR spectrum of 7 displayed signals due to 6 methines and 4 methoxy groups at C-5, C-7, C-3′, and C-6′ (Figure 1), while its ¹³C-NMR spectrum revealed the signals of a ketone, 6 methine carbons, and 6 oxygen-substituted carbons (Figure 1). The ¹H- and ¹³C-NMR spectra indicated that 7 possessed the basics of flavone.³⁹ Analysis of these signals by the correlated spectroscopy (COSY) and heteronuclear multiple quantum correlation (HMQC) spectra led to the partial structures which were connected based on the long-range correlations observed in the heteronuclear multiple bond correlation (HMBC) spectrum. Thus, the structure of compound 7 was assigned to be 5,7,3′,6′-tetramethoxyflavone.⁴⁶ Compound 8 is a derivative compound of 7 in which the ¹H- and ¹³C-NMR spectra were similar to those of 7 except for the presence of an aromatic proton at C-6′ and a methoxy group located at C-4′ in 2 (Figure 1). Therefore, compound 8 was identified as 5,7,3′,4′-tetramethoxyflavone.⁴⁷

Compound 9 was isolated as yellow oil. The presence of an oxymethine group (C-2), a methylene group (C-3), and a carbonyl carbon (C-4) in the ¹H- and ¹³C-NMR spectra indicated 9 to be a flavanone (Figure 1).³⁹ The ¹H-NMR spectrum of 9 showed signals of a 1,2,3,5,6-pentasubstituted benzene ring (ring A) and a 1,3,4-trisubstituted benzene ring (ring B) characterized with an ABX spin system (Figure 1). The ¹³C-NMR spectrum exhibited 20 signals due to 12 sp ² carbons of 2 benzene rings, a ketone, and 5 methoxy carbons (Figure 1). The circular dichroism (CD) spectrum of compound 9 exhibited positive Cotton effect (∆ε ₃₃₈ (nm) +4.7770) and negative Cotton effects (∆ε ₂₉₀ (nm) –59.8223), which established the absolute configuration at C-2 to be 2S (see supplemental figure S5). Based on these evidences and in comparison with the published data, compound 9 was identified as 2S-5,6,7,3′,4′-pentamethoxyflavanone.⁴⁸

Similar to 9, compound 10 also possessed 1 ketone carbon, but showed 7 oxygenated carbons and 4 methoxy carbons at C-5, C-7, C-3′, and C-4′ (Figure 1). Analysis of these signals by the COSY, HMQC, and HMBC spectra led to the partial structures of 10. Compound 10 exhibited positive Cotton effect (∆ε ₃₃₇ (nm) +9.8024) and negative Cotton effects (∆ε ₂₈₄ (nm) –68.4875) in its CD spectrum (see supplemental figure S6). Therefore, compound 10 was identified as 2S-5′-hydroxy-5,7,3′,4′-tetramethoxyflavanone.⁴⁹

All isolates 1 to 10 were evaluated for their inhibitory activity against the PTP1B enzyme activity using in vitro assay, and the results are presented in Table 1 as IC₅₀ values. Among these isolates, compounds 5, 6, 9, and 10 displayed the most potency with IC₅₀ values of 9.76 ± 0.67, 10.12 ± 1.09, 6.88 ± 0.76, and 8.92 ± 0.72 µM, respectively, followed by compounds 1 and 4 with IC₅₀ values of 16.92 ± 2.12 and 22.25 ± 1.70 µM. The remaining compounds were less active with IC₅₀ values > 30 µM (Table 1). Among these methoxylated flavonoids, the flavanones (9 and 10) possessed the strongest activity (IC₅₀ < 10 µM), the flavonols (1-6) possessed significant activity (IC₅₀ ranging from 9.82 to 22.25 µM), and the flavones (7 and 8) were inactive (IC₅₀ >30 µM). Ursolic acid was used as a positive control in this assay and displayed an IC₅₀ value of 3.42 ± 0.45 µM and mixed-competitive inhibition mode. It showed 2 times stronger PTP1B inhibition than compound 9, 3 times than compounds 5, 6, and 10 as well as 5 and 7 times stronger PTP1B inhibition than compounds 1 and 4, respectively (Table 1).

Table 1

Protein tyrosine phosphatase 1B Inhibitory Activity of Isolated Compounds (1-10) From Orthosiphon stamineus Benth.

Compounds	IC₅₀, µM^a	K _i values, µM	Inhibition type
1	16.92 ± 2.12	75.62 ± 1.38	Mixed-competitive
2	>30	- b	-
3	>30	-	-
4	22.25 ± 1.70	76.86 ± 2.25	Noncompetitive
5	9.76 ± 0.67	55.26 ± 0.96	Mixed-competitive
6	10.12 ± 1.09	58.48 ± 1.25	Noncompetitive
7	>30	-	-
8	>30	-	-
9	6.88 ± 0.76	42.27 ± 0.78	Mixed-competitive
10	8.92 ± 0.72	51.38 ± 2.13	Mixed-competitive
Ursolic acid c	3.42 ± 0.45	6.46 ± 0.23	Mixed-competitive

^aResults are expressed as IC₅₀ values (µM), determined by regression analysis and expressed as the means ± SD of 3 replicates.

^bData not determined.

^cPositive control.

The Lineweaver-Burk plot and Dixon plot experiments were performed with the presence and absence of the inhibitors with various concentrations of the substrate para-Nitrophenylphosphate (p-NPP) to determine the inhibition type of the active compounds (1, 4- 6, and 9- 10). The double reciprocal Lineweaver-Burk plot (Figure 2) and the Dixon plot (Figure 3) data were analyzed by using the Sigma plot program (SPCC Inc., Chicago, IL, United States). Table 1 listed the K _i values of the active compounds which were determined by Dixon plot experiment analysis (Figure 3). The inhibition type of the active compounds (1, 4- 6, and 9- 10) was listed in Table 1 as determined by the Lineweaver-Burk plot experiment (Figure 2). Of these active flavonoids, compounds 1, 5, 9, and 10, as well as the positive control (ursolic acid), exhibited mixed-competitive inhibition (the lines intersected at a value upper to the y-axis and under zero on the x-axis), while compounds 4 and 6 exhibited noncompetitive inhibition types because the lines intersected at a value of 1/[S] under zero on the x-axis (1/(intensity/min) =0) (Figure 2).

Figure 2

Lineweaver-Burk plots for the inhibition of compounds (5, 6, 9, and 10) on the protein tyrosine phosphatase 1B-catalyzed hydrolysis of p-NPP. Data are expressed as the mean reciprocal of initial velocity for n = 3 replicates at each substrate concentration.

Figure 3

Dixon plots for isolated compounds (5, 6, 9, and 10) used for determining the inhibition constant K _i. K _i values were determined from the negative x-axis value at the point of the intersection of 3 lines. Data are expressed as the mean reciprocal of initial velocity for n = 3 replicates at each substrate concentration.

For diabetic and cancer objects, the PTP1B enzyme displayed an important role as an attractive target in the treatment, especially for breast cancer. Many studies have shown that in human breast cancer PTP1B was overexpressed, and that the inhibition of PTP1B delayed erbB2-induced mammary tumorigenesis and protected from lung metastasis.^6,50 Thus, the isolated flavonoids were further investigated for their growth inhibition on MCF7, MCF7/TAMR, and MDA-MB-231 breast cancer cell lines (Table 2). Among the isolates, compounds 5, 6, 9, and 10 which potentially inhibited PTP1B (Table 1) also displayed potency against these 3 cancer cell lines. Especially, compound 9 which was the most PTP1B inhibitor (IC₅₀ value of 6.88 ± 0.76 µM) also showed the most cytotoxicity against MCF7 and MCF7/TAMR (IC₅₀ values of 11.5 ± 0.7 and 8.9 ± 1.0 µM, respectively) when compared with positive control (tamoxifen, IC₅₀ values of 11.9 ± 0.4 and 12.1 ± 0.7 µM, respectively). In the same manner, flavanones (9 and 10) were the most active compounds on MCF7/TAMR and MDA-MB-231 cell lines, followed by flavonols 1, 5, and 6, and then the worst compounds 2 and 3 and flavones 7 and 8 (see also Figure 1 and Table 2 for more details). However, this rule was irregular for the cytotoxic activity of these isolates on MCF7 human cancer cell lines. Interestingly, in this study, we found that flavanones (9 and 10) bearing the more methoxy moiety in the structure exhibited the stronger inhibitory effect on both PTP1B and human breast cancer cells. From our research data, we might suggest that the polymethoxylated flavanones 9 and 10 isolated from Vietnamese cat’s whisker (O. stamineus Benth.) can be considered as new natural compounds for the development of anticancer agents via PTP1B inhibition.

Table 2

Cytotoxic Activities of Isolated Compounds (1-10) Against Human Breast Cancer Cell Lines.

Compounds	Cell lines/IC₅₀, µM^a
Compounds	MCF7	MCF/TAMR	MDA-MB-231
1	>30	14.3 ± 1.1	27.3 ± 0.8
2	>30	>30	>30
3	>30	>30	>30
4	15.7 ± 1.9	>30	>30
5	28.4 ± 1.6	9.7 ± 0.4	15.8 ± 0.2
6	22.9 ± 0.6	10.8 ± 0.2	>30
7	25.6 ± 1.3	>30	>30
8	14.8 ± 0.9	>30	>30
9	11.5 ± 0.7	8.9 ± 1.0	17.6 ± 1.2
10	15.4 ± 1.6	10.5 ± 0.7	21.3 ± 0.9
Tamoxifen^b	11.9 ± 0.4	12.1 ± 0.7	12.7 ± 0.6

^aResults are expressed as IC₅₀ values (µM), determined by regression analysis and expressed as the means ± SD of 3 replicates.

^bPositive control.

Experimental

General Experimental Procedures

¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) were measured on a Varian Unity Inova 400 MHz spectrometer. Electron spray ionization mass spectroscopy (ESI-MS) was obtained from a Varian FT-MS spectrometer and MicroQ-TOF III (Bruker Daltonics, Ettlingen Germany). Infrared (IR) spectroscopy was fulfilled on Nicolet Impact 410 spectrometer. UV was performed in spectroscopic V-630 UV-VIS instrument. Column chromatography was carried out on silica gel (Si 60 F₂₅₄, 40-63 mesh, Merck, St. Louis, MO, United States). All solvents were redistilled before use. Precoated thin layer chromatography (TLC) plates (Si 60 F₂₅₄) were used for analytical purposes. Compounds were visualized under UV radiation (254-365 nm) and by spraying plates with 10% H₂SO₄ followed by heating with a heat gun. The high performance liquid chromatography (HPLC) runs were carried out using a Gilson System LC-321 pump with an UV/Vis-155 UV detectors, and an Optima Pak C18 column (10 × 250 mm, 10 µm particle size), J. sphere ODS-H80 (20 × 150 mm, 4 µm particle size) RS Tech, Korea, and Shodex-C18M-10E (10 × 250 mm, 10 µm particle size), and/or YMC-Pak ODS-AM (6.0 × 150 mm, 5 µm particle size) for semipreparative runs.

Plant Materials

The aerial parts of O. stamineus Benth. were collected in January 2017 at Ngu Hiep, Thanh Tri, Ha Noi. The sample was identified by Dr Nguyen Quoc Binh (Viet Nam National Museum of Nature, VAST). A voucher specimen (SH-164) was deposited at the Institute of Natural Products Chemistry (INPC), VAST (see supplemental figure S1 and supplemental references S1–S2 for details).

Extraction and Isolation

The aerial parts of O. stamineus (1.1 kg) were extracted with methanol (5 L × 3 times) at room temperature using sonication for 5 hours. After removing the solvent under reduced pressure, the methanol extract (121 g) was suspended in hot water (500 mL) and partitioned with n-hexane (1 L × 3), chloroform (CHCl₃, 1 L × 3), and ethylacetate (EtOAc, 1 L × 3), successively. The resulting fractions were concentrated in vacuo to give the hexane, CHCl₃, and EtOAc-soluble fractions, respectively. By the guided-fractionation activity, the EtOAc-soluble fraction (43.2 g) was directly chromatographed on an open silica gel column (15 × 60 cm; 63-200 mm particle size, Merck) using a stepwise gradient of n-hexane/EtOAc (from 20:1 to 1:0) to give 5 fractions (OA-1-OA-5) according to their TLC profiles. Fraction OA-1 (3.2 g) was then subjected onto an open silica gel column chromatography (4.0 × 60 cm; 40-63 mm particle size), eluted with a stepwise gradient solvent system of n-hexane/acetone (from 20:1 to 1:1), resulted in the isolation of compound 4 (20.4 mg), and 4 subfractions (OA-1.1-OA-1.4). Further purification of subfraction OA-1.1 (80 mg) by semipreparative Gilson HPLC (using an isocratic solvent system of 50% acetonitrile [ACN] in H₂O + 0.1% formic acid [flow rate = 2 mL/min], over 25 minutes; UV detections at 205 and 254 nm; RS Tech Optima Pak C₁₈ column [10 × 250 mm, 10 µm particle size]), resulted in the isolation of compounds 1 (15.8 mg; t _R = 14.9 minutes) and 5 (3.2 mg; t _R = 18.3 minutes).

Fraction 3 (OA-3) (650 mg) was also subjected onto an open silica gel column chromatography (4.0 × 60 cm; SiO₂, 40-63 mm particle size), eluted with a stepwise gradient solvent system of n-hexane/acetone (from 10:1 to 1:0) to afford 6 subfractions (OA-3.1-OA-3.6). Compounds 7 (4.9 mg; t _R = 10.1 minutes), 2 (5.8 mg; t _R = 12.5 minutes), and 8 (4.4 mg; t _R = 14.5 minutes) were isolated from subfraction OA-3.2 (69 mg) by Gilson HPLC system, using an isocratic solvent system of 50% ACN in H₂O + 0.1% formic acid, flow rate was set as 2 mL/min, over 20 minutes; UV detections at 205 and 254 nm; RS Tech Optima Pak C₁₈ column (10 × 250 mm, 10 µm particle size). Further purification of subfraction OA-3.2 (35 mg) by semipreparative Gilson HPLC system with a RS Tech Optima Pak C₁₈ column (10 × 250 mm, 10 µm particle size), eluted with an isocratic solvent system of 50% MeCN in H₂O + 0.1% formic acid (flow rate = 2 mL/min), over 40 minutes; UV detections at 205 and 254 nm resulted in the isolation of compound 6 (5.3 mg; t _R = 36.3 minutes). The subfraction OA-3.4 (55 mg) was also purified by semipreparative Gilson HPLC using same column and flow rate with 60% ACN in H₂O + 0.1% formic acid over 25 minutes to give compounds 9 (4.0 mg; t _R = 16.6 minutes), 10 (3.5 mg; t _R = 18.6 minutes), and 3 (1.5 mg; t _R = 21.6 minutes), respectively.

Protein Tyrosine Phosphatase 1B Inhibition Assay

Protein tyrosine phosphatase 1B (human recombinant) was purchased from Biomol International LP, Plymouth Meeting, PA, United States, and the inhibitory activities of the tested samples were evaluated using the method as described.⁵¹ Briefly, 0.05 to 0.1 µg of PTP1B (BIOMOL International L.P., Plymouth Meeting, PA, United States) and 4 mM p-NPP in a buffer containing 1 mM dithiothreitol, 0.1 M NaCl, 1 mM EDTA, and 50 mM citrate (pH 6.0), with or without test compounds, were added as 100 µL of a final volume to each of the 96 well. After the reaction mixture was incubated at 37°C for 30 minutes, 10 M NaOH was added to quench the reaction. Protein tyrosine phosphatase 1B enzyme activity was determined by the amount of produced p-nitrophenol at 405 nm. The nonenzymatic hydrolysis of the substrate was corrected by measuring the control, which did not contain PTP1B enzyme.

Determination of the Inhibition Mode of Active Compounds

The Lineweaver-Burk plot and Dixon plot experiments were carried out in the presence and absence of the inhibitors with various concentrations of p-NPP as the substrate. The inhibition modes of the tested compounds were assessed on the basis of their inhibitory effects on K _m (dissociation constant) and V _max (maximum reaction velocity) of the enzyme, which were determined by Lineweaver-Burk plot experiment. The Lineweaver-Burk plot is the double reciprocal plot of the enzyme reaction velocity (V) vs the substrate (p-NPP) concentration (1/V vs 1/[p-NPP]).

Cytotoxic Activity Assay

The cancer cell lines were maintained in Roswell Park Memorial Institute (RPMI) 1640, which included l-glutamine with 10% fetal bovine serum (FBS) and 2% penicillin-streptomycin. Cells were cultured at 37°C in a 5% CO₂ incubator. Cytotoxic activity was measured using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.⁵² Viable cells were seeded in the growth medium (100 µL) into 96-well microtiter plates (1 × 10⁴ cells per well) and incubated at 37°C in a 5% CO₂ incubator. The test sample was dissolved in dimethyl sulfoxide (DMSO) and adjusted to final sample concentrations ranging from 5.0 to 150 µM by diluting with the growth medium. Each sample was prepared in triplicate. The final DMSO concentration was adjusted to <0.1%. After standing for 24 hours, 10 µL of the test sample was added to each well. The same volume of DMSO was added to the control wells. On removing medium after 48 hours of the test sample treatment, MTT (5 mg/mL, 10 µL) was also added to the each well. After 4 hours incubation, the plates were removed and the resulting formazan crystals were dissolved in DMSO (150 µL). The OD was measured at 570 nm. The IC₅₀ value was defined as the concentration of sample that reduced absorbance by 50% relative to the vehicle-treated control.

Statistical Analysis

Data are represented as means ± SD of at least 3 independent experiments performed in triplicated assays and determined by regression analysis. For statistical analysis of the data for single comparison, the significance between means was determined by Student’s t test. Sigma Plot program version 11.0 was used for analysis of the kinetic data.

Supplemental Material

Supplementary material - Supplemental material for PTP1B Inhibitory Flavonoids From Orthosiphon stamineus Benth. and Their Growth Inhibition on Human Breast Cancer Cells

Supplemental material, Supplementary material, for PTP1B Inhibitory Flavonoids From Orthosiphon stamineus Benth. and Their Growth Inhibition on Human Breast Cancer Cells by Dao-Cuong To, Duc-Thuan Hoang, Manh-Hung Tran, Minh-Quan Pham, Nhu-Tuan Huynh and Phi-Hung Nguyen in Natural Product Communications

Footnotes

Acknowledgment

The authors wish to thank the Center for Applied Spectroscopy, Institute of Chemistry, VAST, for the spectroscopic measurements.

Declaration of Conflicting Interests

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

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by a Grant from Vietnam Academy of Science and Technology (VAST) (Project code number: VAST.ĐLT.06/17-18).

ORCID ID

Minh-Quan Pham

Phi-Hung Nguyen

References

Zhang

. PTP1B as a drug target: recent developments in PTP1B inhibitor discovery. Drug Discov Today. 2007;12(9-10):373-381.doi:10.1016/j.drudis.2007.03.011

Montalibet

Kennedy

. Therapeutic strategies for targeting PTP1B in diabetes. Drug Discov Today. 2005;2(2):129-135.doi:10.1016/j.ddstr.2005.05.002

Yip

S-C.

Saha

Chernoff

. PTP1B: a double agent in metabolism and oncogenesis. Trends Biochem Sci. 2010;35(8):442-449.doi:10.1016/j.tibs.2010.03.004

Bakke

Haj

. Protein-Tyrosine phosphatase 1B substrates and metabolic regulation. Semin Cell Dev Biol. 2015;37(0):58-65.doi:10.1016/j.semcdb.2014.09.020

Wiener

JR.

Kerns

B-JM.

Harvey

et al. Overexpression of the protien tyrosine phosphatase PTP1B in human breast cancer association with p185 ^c-erbB-2 protein expression. J Natl Cancer Inst. 1994;86(5):372-378.doi:10.1093/jnci/86.5.372

Tonks

NK.

Muthuswamy

. A brake becomes an accelerator: PTP1B—A new therapeutic target for breast cancer. Cancer Cell. 2007;11(3):214-216.doi:10.1016/j.ccr.2007.02.022

Bollu

LR.

Mazumdar

Savage

MI.

Brown

et al. Molecular pathways: targeting protein tyrosine phosphatases in cancer. Clin Cancer Res. 2017;23(9):2136-2142.doi:10.1158/1078-0432.CCR-16-0934

Liu

Chen

X-G

et al. Ptp1B promotes aggressiveness of breast cancer cells by regulating PTEN but not EMT. Tumor Biol. 2016;37(10):13479-13487.doi:10.1007/s13277-016-5245-1

Nguyen

PH.

TVT.

Thuong

et al. Cytotoxic and PTP1B inhibitory activities from Erythrina abyssinica . Bioorg Med Chem Lett. 2009;19(23):6745-6749.doi:10.1016/j.bmcl.2009.09.108

10.

Dao

TT.

Nguyen

PH.

Thuong

et al. Pterocarpans with inhibitory effects on protein tyrosine phosphatase 1B from Erythrina lysistemon Hutch. Phytochemistry. 2009;70(17-18):2053-2057.doi:10.1016/j.phytochem.2009.09.012

11.

J-P.

Kim

Cho

et al. Protein Tyrosine Phosphatase 1B Inhibitors from the Stems of Akebia quinata . Molecules. 2016;21(8):1091.doi:10.3390/molecules21081091

12.

Jiang

C-S.

Liang

L-fu.

Guo

Y-wei

. Natural products possessing protein tyrosine phosphatase 1B (PTP1B) inhibitory activity found in the last decades. Acta Pharmacol Sin. 2012;33(10):1217-1245.doi:10.1038/aps.2012.90

13.

Kostrzewa

Przychodzen

Gorska-Ponikowska

et al. Curcumin and cinnamaldehyde as PTP1B inhibitors with antidiabetic and anticancer potential. Anticancer Res. 2019;39(2):745-749.doi:10.21873/anticanres.13171

14.

Przychodzen

Kuban-Jankowska

Wyszkowska

et al. PTP1B phosphatase as a novel target of oleuropein activity in MCF-7 breast cancer model. Toxicology in Vitro. 2019;61:104624.doi:10.1016/j.tiv.2019.104624

15.

Hsu

C-L.

Hong

B-H.

Y-S.

Yen

G-C

. Antioxidant and anti-inflammatory effects of Orthosiphon aristatus and its bioactive compounds. J Agric Food Chem. 2010;58(4):2150-2156.doi:10.1021/jf903557c

16.

Tran

. Medicinal Plants in Viet Nam. WHO Regional Office for the Western Pacific Manila and Institute of Material Medica Hanoi, Hanoi, Vietnam: Science and Technology Publishing House; 1990.

17.

Bwin

DM.

Gwan

. Burmese Indigenous Medicinal Plants: 1. Plants with reputed Hypoglycemic Action, Health and Myanmar Traditional Medicine. Yangon: Burma Medical Research Institute, Ministry of Health; 1967.

18.

Eisai Indonesia PT . Medicinal Herb Index in Indonesia. 2nd ed. Indonesia: PT Eisai Indonesia; 1995.

19.

Ameer

OZ.

Salman

IM.

Asmawi

MZ.

Ibraheem

ZO.

Yam

. Orthosiphon stamineus: traditional uses, phytochemistry, pharmacology, and toxicology. J Med Food. 2012;15(8):678-690.doi:10.1089/jmf.2011.1973

20.

Yam

MF.

Lim

Salman

et al. HPLC and anti-inflammatory studies of the flavonoid rich chloroform extract fraction of Orthosiphon stamineus leaves. Molecules. 2010;15(6):4452-4466.doi:10.3390/molecules15064452

21.

Adnyana

IK.

Setiawan

Insanu

. From ethnopharmacology to clinical study of Orthosiphon stamineus Benth. Int J Pharm Pharmaceut Sci. 2013;5(3):66-73.

22.

Yam

MF.

Ang

LF.

Salman

et al. Orthosiphon stamineus Leaf Extract Protects Against Ethanol-Induced Gastropathy in Rats. J Med Food. 2009;12(5):1089-1097.doi:10.1089/jmf.2008.0005

23.

Maheswari

Maryammal

Venkatanarayanan

. Hepatoprotective activity of Orthosiphon stamineus on liver damage caused by paracetamol in rats. Jordan J Biol Sci. 2008;1(3):105-108.

24.

Pauzi

Mohd

KS.

Abdul Halim

NH.

Ismail

Abdul

. Orthosiphon stamineus extracts inhibits proliferation and induces apoptosis in uterine fibroid cells. Asian Pac J Cancer Prev. 2018;19(10):2737-2744.doi:10.22034/APJCP.2018.19.10.2737

25.

Chitra

Karthikeyan

. In-vitro cytotoxicity of java tea mediated selenium nanoballs against L6 cell lines. JDDT. 2018;8(6):195-200.

26.

Koay

YC.

Amir

. A survey of the chemical constituents and biological activities of Orthosiphon stamineus . Sci Int. 2012;24(2):133-138.

27.

Vogelgesang

Abdul-Malak

Reymermier

Altobelli

Saget

et al. On the effects of a plant extract of Orthosiphon stamineus on sebum-related skin imperfections. Int J Cosmet Sci. 2011;33(1):44-52.doi:10.1111/j.1468-2494.2010.00581.x

28.

Mariam

Asmawi

MZ.

Sadikun

. Hypoglycaemic activity of the aqueous extract of Orthosiphon stamineus . Fitoterapia. 1996;67(5):465-468.

29.

Yam

MF.

Asmawi

MZ.

Basir

. An investigation of the anti-inflammatory and analgesic effects of Orthosiphon stamineus leaf extract. J Med Food. 2008;11(2):362-368.doi:10.1089/jmf.2006.065

30.

Majid

ASA.

Ahamed

et al. Extract of Orthosiphon stamineus, formulations, and uses thereof. PCT Int Appl. 2019;WO 2019087084 A1 20190509.

31.

Norefrina

Mohd

et al. Antibacterial activity of Orthosiphon stamineus towards Staphylococcus aureus and methicilin resistant Staphylococcus aureus (MRSA. J Chem Pharm Sci. 2018;11(1):1-5.

32.

Wahab

NZA.

Ibrahim

Kamarudin

MKA

et al. In vitro antiviral activity of Orthosiphon stamineus extract against dengue virus type 2. J Fundam Appl Sci. 2018;10(1S):541-551.

33.

Adam

Somchit

MN.

Sulaiman

et al. Diuretic properties of Orthosiphon stamineus Benth. J Ethnopharmacol. 2009;124(1):154-158.doi:10.1016/j.jep.2009.04.014

34.

Sriplang

Adisakwattana

Rungsipipat

Yibchok-anun

et al. Effects of Orthosiphon stamineus aqueous extract on plasma glucose concentration and lipid profile in normal and streptozotocin-induced diabetic rats. J Ethnopharmacol. 2007;109(3):510-514.doi:10.1016/j.jep.2006.08.027

35.

Mohamed

EAH.

Mohamed

AJ.

Asmawi

et al. Antihyperglycemic effect of Orthosiphon stamineus Benth leaves extract and its bioassay-guided fractions. Molecules. 2011;16(5):3787-3801.doi:10.3390/molecules16053787

36.

Hossain

MA.

Ismail

Rahman

Kang

et al. Chemical composition and anti-fungal properties of the essential oils and crude extracts of Orthosiphon stamineus Benth. Ind Crops Prod. 2008;27(3):328-334.doi:10.1016/j.indcrop.2007.11.008

37.

Guerin

J-C.

Reveillere

H-P.

Ducrey

Toupet

et al. Orthosiphon stamineus as a potent source of Methylripariochromene a. J Nat Prod. 1989;52(1):171-173.doi:10.1021/np50061a023

38.

Sahib

HB.

Ismail

Othman

et al. Orthosiphon stamineus Benth. Methanolic extract enhances the anti-proliferative effects of tamoxifen on human hormone dependent breast cancer. Int J Pharmacol. 2009;5(4):273-276.

39.

Agrawal

. Carbon-13 NMR of Flavonoids. Amsterdam-Oxford-NewYork-Tokyo: Studies in Organic Chemistry 39; 1989.

40.

Mitscher

LA.

Gollapudi

SR.

Drake

Oburn

et al. Amorphastilbol, an antimicrobial agent from Amorpha nana . Phytochemistry. 1985;24(7):1481-1483.doi:10.1016/S0031-9422(00)81048-1

41.

Ibrahim

Ambreen

Hussain

et al. Phytochemical investigation on Eucalyptus globulus labill. Asian J Chem. 2014;26(4):1011-1014.doi:10.14233/ajchem.2014.15765

42.

Valesi

AG.

Rodriguez

Vander Velde

Mabry

et al. Methylated flavonols in Larrea cuneifolia . Phytochemistry. 1972;11(9):2821-2826.doi:10.1016/S0031-9422(00)86519-X

43.

Ishikawa

Takagi

Kanou

KAWAI

OHASHI

et al. Radical-capturing reaction of 5,7,3',4'-Tetramethylquercetin with the AIBN radical initiator. Biosci Biotechnol Biochem. 2000;64(1):173-177.doi:10.1271/bbb.64.173

44.

Nugroho

BW.

Edrada

RA.

Wray

et al. An insecticidal rocaglamide derivatives and related compounds from Aglaia odorata (Meliaceae). Phytochemistry. 1999;51(3):367-376.doi:10.1016/S0031-9422(98)00751-1

45.

Happi

EN.

Mpondo

. Two Polymethoxylated flavones from Distemonanthus benthamianus . J Nat Prod. 1994;57(2):291-293.doi:10.1021/np50104a015

46.

Reddy

NP.

Reddy

BAK.

Gunasekar

et al. Flavonoids from Limnophila indica . Phytochemistry. 2007;68(5):636-639.doi:10.1016/j.phytochem.2006.12.018

47.

Rajudin

Ahmad

Sirat

HM.

Arbain

Aboul-Enein

. Chemical constituents from tiger's betel, Piper porphyrophyllum N.E.Br. (Fam. Piperaceae. Nat Prod Res. 2010;24(4):387-390.doi:10.1080/14786410903421826

48.

Iwase

Takahashi

Takemura

et al. Isolation and identification of two new flavanones and a chalcone from citrus kinokuni. Chem Pharm Bull. 2001;49(10):1356-1358.doi:10.1248/cpb.49.1356

49.

Endo

Taguchi

Yosioka

. The glycosides of Plantago major var. japonica Nakai. A new flavanone glycoside,plantagoside. Chem Pharm Bull. 1981;29(4):1000-1004.doi:10.1248/cpb.29.1000

50.

Julien

SG.

Dubé

Read

et al. Protein tyrosine phosphatase 1B deficiency or inhibition delays ErbB2-induced mammary tumorigenesis and protects from lung metastasis. Nat Genet. 2007;39(3):338-346.doi:10.1038/ng1963

51.

Nguyen

P-H.

Yang

J-L.

Uddin

et al. Protein Tyrosine Phosphatase 1B (PTP1B) Inhibitors from Morinda citrifolia (Noni) and Their Insulin Mimetic Activity. J Nat Prod. 2013;76(11):2080-2087.doi:10.1021/np400533h

52.

Van

LTK.

Hung

TM.

Thuong

et al. Oleanane-type triterpenoids from Aceriphyllum rossii and their cytotoxic activity. J Nat Prod. 2009;72(8):1419-1423.doi:10.1021/np900273z

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