Accepted Manuscript

A -3-O-β-D-glucoside, Intervention Effect of Astragalin on Estra- diol Metabolism

Xin Meng, Aihua Zhang, Xijun Wang, Hui Sun

PII: S0039-128X(19)30097-2 DOI: https://doi.org/10.1016/j.steroids.2019.05.005 Reference: STE 8413

To appear in: Steroids

Received Date: 12 December 2018 Revised Date: 30 January 2019 Accepted Date: 21 May 2019

Please cite this article as: Meng, X., Zhang, A., Wang, X., Sun, H., A Kaempferol-3-O-β-D-glucoside, Intervention Effect of Astragalin on Estradiol Metabolism, Steroids (2019), doi: https://doi.org/10.1016/j.steroids.2019.05.005

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A Kaempferol-3-O-β-D-glucoside, Intervention Effect of Astragalin on Estradiol Metabolism

Xin Meng, Aihua Zhang, Xijun Wang, Hui Sun*

National Chinmedomics Research Center, Sino-America Chinmedomics Technology

Collaboration Center, National TCM Key Laboratory of Serum Pharmacochemistry,

Laboratory of Metabolomics, Department of Pharmaceutical Analysis, Heilongjiang

University of Chinese Medicine

NO.24 Heping Road, Harbin 150040, P. R. China

Corresponding author：Hui Sun

Email: [email protected]

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Abstract

Kaempherol-3-O-β-D-glucoside, known as astragalin, is one of flavonoids found in a variety of plants including Cuscuta australis R.Br. In recent studies, astragalin possess many biological functions.

Although astragalin is formed by linking glucose to kaempherol, its biological activity is not the same as kaempferol. In vivo, 17 β-estradiol (E2) is hydroxylated by cytochrome P450 (CYP) 1B1 to form

4-hydroxy-E2 (4-OH-E2). This metabolite 4-OH-E2 is highly expressed in tumor tissues and has a strong tumorigenic effect. In this paper, the inhibition of astragalin and kaempferol on the activity of cytochrome 1B1 catalyzing estradiol to form 4-hydroxy-estradiol was studied, and the structure-activity relationship between astragalin and kaempferol due to their structural differences was discussed. This study showed that astragalin could inhibit the activity of CYP1B1. The inhibitory effect of astragalin (IC50 5.36±1.13μM) was weaker than kaempferol (IC50 0.45±0.11μM). For astragalin, Ki and Vmax values were 4.061±0.737μM and 1.457pmol/μg protein/min, while for kaempferol, Ki and

Vmax values were 2.631±0.381 μM protein/min and 1.023±0.231pmol/μg. By kinetic analysis, astragalin and kaempferol were all mixed inhibition, indicating that although astragalin is formed by linking glucose to kaempherol, its inhibitory mechanism on CYP1B1 remained unchanged, and still belonged to a mixed inhibition. The data indicated that astragalin has been able to inhibit the metabolism of estradiol into the carcinogenic metabolite 4-hydroxyl-estradiol in vivo and illustrated an anti-tumor mechanism of astragalin. This study helps to reveal the structure-activity relationship between CYP1B1 activity and its inhibitors.

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Keywords: Hormones, estradiol, 4-hydroxy-estradiol, astragalin, kaempherol, anti-tumor

Chemical compounds studied in this article:

Astragalin (PubChem CID: 5282102); 17β-Estradiol (PubChem CID: 5757); Kaempherol (PubChem

CID: 5280863)

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1. Introduction

Astragalin, known as kaempferol-3-O-β-D-glucoside (Fig.1), is one of flavonoids found in a variety of plants including Cuscuta australis R.Br[1, 2]. In recent studies, astragalin possess many biological functions, including anti-inflammatory, anti-allergic, antioxidant, anti HIV and so on[3-5]. It has also been reported that astragalin could inhibit the production of prostaglandin and angiotensin converting enzyme activity, inhibit apoptosis of ovarian granulosa cells in senile rats during menopause, increase endogenous estrogen and progesterone, and reduce inflammation induced by lipopolysaccharide by inhibiting the signal pathway of NF- кB[6-8].

It is well known that long-term exposure to estrogen can induce estrogen-related tumors such as breast cancer, ovarian and endometrial cancer[9-11]. The pathogenesis of estrogen related tumors is due to the metabolism of estrogen to produce carcinogenic metabolites.

17β-estradiol is hydroxylated by cytochrome P450 (CYP) 1B1 (CYP1B1) to form in vivo

4-hydroxy-E2 (4-OH-E2), as a major estrogen metabolite [12]. It can cause DNA damage, for example

DNA strand breaks, oxidized DNA bases, and covalent adducts to DNA [13]. At the same time, the expression of CYP1B1 is different in various tissues of human body [14-16]. The expression of

CYP1B1 is higher in various tumors and inflammatory tissues, but is usually very low or even undetectable in normal extrahepatic tissues [17-20]. Therefore, inhibition on CYP1B1 is important for the development and progression of tumor and inflammation.

However, it is not known whether astragalin is able to inhibit the activity of CYP1B1. Compared with kaempferol (Fig.1) in this study, the inhibition of astragalin on the activity of CYP1B1 catalyzing estradiol to form 4-hydroxy-estradiol and the inhibition kinetics was studied. Although astragalin is obtained by linking kaempferol to a glucose, its biological activity may be not the same as kaempferol.

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This study reveals an anti-tumor mechanism of astragalin, which is helpful to illustrate the structure-activity relationship between CYP1B1 and its inhibitors, and lay a foundation for the treatment and prevention of estrogen related tumors and the discovery of new drugs.

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2. Experimental

2.1 Materials

HPLC grade astragalin and kaempferol were obtained from China Food and Drug Testing Institute

(BeiJing, China). Recombinant human CYP1B1 supersomes (Gentest Corporation via BD Biosciences,

San Jose, CA), and E2, 6-estrone (6-E1), 4-hydroxyl-estradiol, 2-hydroxyl-estradiol, glucose-6-phosphate dehydrogenase, glucose-6-phosphate and β-NADP+ were purchased from Sigma

Chemical Co. (St. Louis, MO). All the other chemicals and reagents were obtained from Xilong

Scientific (Shantou, China).

2.2 Catalytic effect of CYP1B1 enzyme on E2 metabolism in vitro

The reaction rate of 4-OH-E2 catalyzed by CYP1B1 with different concentrations of E2 (0, 0.5, 1.0,

2.0, 5.0, 10.0, 20.0, 40.0μM) was analyzed. Firstly, the reaction solution, ascorbic acid (0.5 mM),

+ Tis-HCl buffer (37◦C, pH 7.4) (50 mM), β-NADP (0.5 mM), MgCl2 (5 mM), EDTA (50 μM), G6PDH

(1U ml-1), G6P (5 mM) were prepared. The mixed solution was incubated for 3 minutes at 37◦C in an oscillating water bath. After incubation, recombinant microsome CYP1B1 (10 pM) was added to make the reaction start. Then, it should be incubated for 30 minutes in an oscillating water bath[21]. The termination reaction needs to be heated for 2 minutes in 95◦C water bath to inactivate the enzyme, and then cool down in ice water immediately.

2.3 Inhibition of astragalin on carcinogenic metabolites of E2

Briefly, as above reaction mixture was incubated at 37◦C for 30 minutes with astragalin (0, 0.005,

0.05, 0.5, 5, 50μM) or kaempferol (0, 0.005, 0.05, 0.5, 5, 50μM). The concentration of E2 was 20μM, which had 10pM recombinant microsomal CYP1B1. E2 was catalyzed by CYP1B1 to produce

4-hydroxyl-estradiol, as mentioned above.

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2.4 CYP1B1 enzyme kinetics study

The concentration of astragalin and kaempferol in the study of enzyme inhibition kinetics was set at

IC50. Enzyme kinetic assay was carried out at different E2 concentrations (0, 0.5, 1.0, 2.0, 5.0, 10.0,

20.0, 40.0μM). E2 was catalyzed by CYP1B1 to produce 4-hydroxyl-estradiol, as mentioned above.

Then HPLC-ECD was used to analyze the samples. The Km and Vmax values were determined by using GraphPad Prism 4 (GraphPad Software, Inc., San Diego, CA) software Michaelis-Menten equation non-linear regression curve fitting.

2.5 Extraction and analysis of samples

The 500pM internal standard 6-E1 was added to the sample. Samples were extracted with 5ml and centrifuged for 5 minutes with 13000rpm. It should be extracted three times and the supernatant was incorporated into a glass centrifugal tube. Added ascorbic acid (25nmol) to the supernatant and dried in nitrogen streams at 37◦C. The sample was dissolved in methanol at 50μl.

HPLC-ECD condition: Injection volume 5μl, chromatographic column Mightisil RP-18 GP 250-3.0

(5μm) (Kanto Chemical Co.), mobile phase 45% NH4H2PO4 buffer (pH=3, 37◦C) and 55% methanol, flow rate 0.5 ml min-1, Guard cell (ESA 5020) at a potential of +500 mV, ESA 5010 analytical cell (E1

= +300 mV, E2 = +900 mV) (ESA Coulochem II, ESA, Inc., Chelmsford, MA).

2.6 Statistical analysis of data

The results were expressed as mean value ± standard deviation; four times parallel to each experiment. Dunnett's multiple comparison test was used to evaluate the statistical significance of the difference using double tail paired ANOVA.

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3 Results and discussion

The kinetic parameters of the catalyzed E2 hydroxylation by CYP1B1 determining the rate of

4-OH-E2 formation. The apparent kinetics parameters Km and Vmax of CYP1B1 enzymatic reaction were 0.942 μM and 2.217±0.165 pmol/min/pmol of enzyme. The inhibitory effects of astragalin and kaempferol on the production of 4-hydroxy-estradiol by CYP1B1 catalysts were studied. The percentage of CYP1B1 activity was plotted with the concentration of astragalin or kaempferol, and

IC50 was estimated by S-shaped dose-response curve (Fig.2). As shown in Fig.2, kaempferol has a strong inhibitory effect on the catalytic activity of CYP1B1, and IC50 is 0.45 ± 0.11μM. Compared with kaempferol, IC50 of kaempferol-3-O-β-D-glucoside (astragalin) was 5.36±1.13μM, and its inhibitory activity on CYP1B1 was slightly weaker.

Michaelis–Menten enzyme kinetic model was used to analyze the kinetic parameters of 4-OH-E2 formation catalyzed by recombinant CYP1B1 at IC50 concentration of astragalin (5μM) and kaempferol (0.5μM) (Fig.3). For astragalin, Ki and Vmax values were 4.061±0.737 μM and

1.457pmol/μg protein/min, while for kaempferol, Ki and Vmax values were 2.631±0.381 μM and

1.023±0.231pmol/μg protein/min. By analyzing the Lineweaver-Burk curve (Fig.4), it was confirmed that both astragalin and kaempferol were mixed inhibitors because the results showed lines intersecting the primitive enzyme-substrate diagram in the second quadrant.

Metabolic inactivation of estradiol includes conversion to low-activity estrogens, such as estriol, which can be excreted from urine [22]. Estradiol also forms estradiol glucuronide and estradiol sulfate in the liver, which is excreted through the kidney. One of the most important ways of stimulating metabolism is to metabolize catechol estrogens through hydroxylation. CYP1B1 acts as an important enzyme involved in the metabolism of estradiol. The steroid 4-OH-E2 produced by the enzyme has a

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strong tumorigenic effect.

We used estradiol as substrate to study the inhibitory effect of astragalin and kaempherol on

CYP1B1 in vitro. Because CYP1B1 formed hydrogen bonds with estradiol, it had strong catalytic activity in NADPH-dependent oxidative metabolism of E2 and produced estrogen metabolite 4-OH-E2.

Then, this compound would to form estradiol-3,4-semi-quinone (E2-3,4-SQ) and estradiol-3,4-quinone

(E2-3,4-Q). The purine reaction between E2-3,4-Q and DNA formed a purine adduct, which produces depurine sites, causing carcinogenic mutations.

Both astragalin and kaempferol were able to inhibit the activity of recombinant human CYP1B1.

The IC50 values of astragalin and kaempferol were 5.36±1.13μM and 0.45 ± 0.11μM, respectively, indicating that the inhibitory effect of astragalin was weaker than that of kaempferol. Astragalin is a glycoside formed by kaempferol linking a glucose. This study demonstrated that the inhibitory effect of flavonoids on CYP1B1 was weaker than that of flavonoids themselves. Because of the connection of glucose, it maybe affects the inhibition of CYP1B1. This structure may affect the binding of the compound to the active site pocket of CYP1B1, thereby affecting the inhibitory activity. In recent studies, 4'-OCH3 on B ring or 5,7-dihydroxy on A ring of flavonoids played an important role in inhibiting EROD activity catalyzed by CYP1B1. By kinetic analysis, astragalin and kaempferol were all mixed inhibition. Therefore, inhibitory effect of astragalin and kaempferol was due to its binding with CYP1B1 enzymes, whether CYP1B1 already binds E2 or not, but it had greater affinity for one state or another. The results showed that although kaempherol is linked to a glucose in astragalin, the mechanism of this flavonoid glucoside on CYPiB1 remained unchanged, and still belonged to mixed inhibition.

In conclusion, this study examined the inhibition of astragalin on the formation of

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4-hydroxy-estradiol catalyzed by CYP1B1 in vitro. The results indicated that that the inhibition of astragalin on CYP1B1 was weaker than that of kaempferol. By kinetic analysis, both astragalin and kaempferol showed mixed inhibition. Data suggested that astragalin possessed CYP1B1 inhibitory activity, which may be partly attributed to its cancer chemoprevention potential. Meanwhile, this study further revealed the relationship between the structure of CYP1B1 inhibitor and its inhibitory activity.

In order to prove that the inhibitory effect of flavonoid glycosides is weaker than that of flavonoids, more studies on the relationship between structure and activity will be carried out.

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Acknowledgments

This work was supported by the Science Foundation of Heilongjiang Administration of Traditional

Chinese Medicine (No.ZHY18-021).

Conflict of interest

The authors declare that they have no conflict of interests.

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Footnotes

Abbreviations

E2, 17β-Estradiol; 2-OH-E2, 2-Hydroxy-17β-estradiol; 4-OH-E2, 4-Hydroxy-17β-estradiol; CYP1B1, cytochrome P450 (CYP) 1B1; 6-E1, 6-estrone; IC50, 50% inhibition

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Figure legends

Fig.1 Structures of estradiol, astragalin and kaempferol

Fig.2 Dose–response curves of astragalin and kaempferol inhibition of CYP1B1–E2 activity. Error bars represent mean ± SD for four independent experiments

Fig.3 Michaelis–Menten kinetics of CYP1B1-catalyzed E2 activity. Human recombinant CYP1B1 was incubated with E2 in the absence or presence of astragalin and kaempferol. Error bars represent mean ±

SD for four independent experiments

Fig.4 Lineweaver–Burk plots showing inhibition kinetics of CYP1B1-catalyzed E2 activity in the presence of astragalin and kaempferol. Human recombinant CYP1B1 was incubated with E2 in the absence or presence of astragalin and kaempferol. Error bars represent mean ± SD for four independent experiments

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Figures

Fig.1

Fig.2

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Fig.3

Fig.4

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Astragalin inhibits the formation of a carcinogenic estrogen metabolite.

One of the astragalin anti-cancer mechanism has been revealed.

A relationship between the structure of flavonoids and the inhibition CYP1B1 was found.

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