Effects of Pueraria Mirifica and Miroestrol on The

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Research Paper Journal of Pharmacy And Pharmacology

Effects of Pueraria miriﬁca and miroestrol on the antioxidation-related enzymes in ovariectomized mice Waranya Chatuphonpraserta,c, Latiporn Udomsukd, Orawan Monthakantiratb, Yaowared Churikhitb, Waraporn Putaluna,b and Kanokwan Jarukamjorna,b aResearch Group for Pharmaceutical Activities of Natural Products using Pharmaceutical Biotechnology (PANPB), and bAcademic Ofﬁce for Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, cFaculty of Medicine, Mahasarakham University, Mahasarakham, and dCollege of Medicine and Public Health, Ubon Ratchathani University, Ubon Ratchathani, Thailand

Keywords Abstract catalase; glutathione peroxidase; miroestrol; Pueraria mirifica; superoxide dismutase Objectives The influences of Pueraria candollei var. mirifica (PM), a Thai medicinal plant with long tradition of medicinal consumption among menopausal Correspondence women for rejuvenation and estrogen hormone replacement, on oxidative status Kanokwan Jarukamjorn, Research Group for in ovariectomized (OVX) mice were determined. Pharmaceutical Activities of Natural Products Methods The crude extract of PM and its active phytoestrogen, miroestrol (MR), using Pharmaceutical Biotechnology (PANPB), Faculty of Pharmaceutical Sciences, Khon were given to OVX mice. The effect of them on antioxidation enzymes and glu- Kaen University, Khon Kaen 40002, Thailand. tathione (GSH) levels in livers and uteri were examined in OVX mice and com- E-mail: [email protected] pared with the synthetic estradiol hormone. Key findings Ovariectomy significantly decreased total GSH content, reduced Received August 7, 2012 GSH content, and the ratio of GSH to oxidized glutathione (GSSG) in both the Accepted October 16, 2012 livers and the uteri of mice. Moreover, an ovariectomy reduced the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT). doi: 10.1111/jphp.12003 The crude extract of PM as well as MR significantly increased levels of GSH, levels of reduced GSH, and the ratio of GSH/GSSG in both the livers and the uteri, while estradiol did not. In addition, the potential of PM and MR to return the activities of GPx, SOD, and CAT to normal levels was noted. Conclusions These observations support using PM and MR as promising alternative medicine candidates for hormone replacement therapy of estradiol because of their ability to improve GSH levels and the activities of antioxidative enzymes, especially in OVX mice.

Introduction Pueraria mirifica (synonym: Pueraria candollei Wall. Ex defence systems are composed of both the enzyme system – Benth var mirifica (Airy Shaw & Suvat.) Nyomdham), of the with enzymes glutathione peroxidase (GPx), catalase, and family Leguminosae, is a Thai medicinal plant with a long superoxide dismutase (SOD) – and a nonenzyme system tradition of medicinal consumption among menopausal of reduced glutathione (GSH).[6,7] Levels of GPx, catalase, women for rejuvenation and oestrogen replacement.[1,2] The SOD, and GSH were decreased in ovariectomized ani- tuberous roots of P. mirifica contain several phytoestrogens, mals.[8,9] Several studies have demonstrated the antioxidant including miroestrol (Figure 1), deoxymiroestrol, and iso- activity of P. mirifica and miroestrol. For example, the root- flavonoids.[3,4] Antioxidant properties are one of the most cultured extract of P. candollei inhibited lipid peroxida- important claims for food ingredients, dietary supplements, tion in mouse brain.[10] P. mirifica tuberous extracts have cosmetics, and natural anticancer products. Phytoestrogen- shown antioxidant activity by DPPH assay, compared with rich plants have an established antioxidant activity.[5] Reac- a-tocopherol, and by the b-carotene bleaching method.[11,12] tive oxygen species (ROS) cause damage to cells and organs Oestrogen (estradiol) is considered the first-line drug for when the rate of ROS generation exceeds the rate of their the prevention and treatment of multiple conditions affect- decomposition by antioxidant defence systems. These ing women’s health. It has been widely recommended for

H OH 21 H OH H H H 21 H H3C 12 12 H C 13 OH H3C 11 13 17 OH 3 11 18 17 18 20 20 H H 19 19 H H 1 H H 1 14 16 14 16 10 H 10 H 2 9 H 2 9 H 8 15 8 15 OH H 3 O 3 O 7 5 7 HO 5 O HO 4 O 6 4 6

Miroestrol Deoxymiroestrol

Figure 1 Chemical structures of miroestrol and deoxymiroestrol. the prevention of osteoporosis, reduction of the risk of Pharmaceutical Sciences, Chulalongkorn University, mortality from cardiovascular disease, maintaining lipid Bangkok, Thailand. Estradiol benzoate (E2), reduced profiles, improvement of the signs and symptoms of meno- glutathione (GSH), oxidized glutathione (GSSG), super- pause, and possible protection against the development of oxide dismutase (SOD), xanthine, xanthine oxidase, Alzheimer’s disease.[13,14] However, the current issue of nitroblue tetrazolium (NBT), ammonium molybdate, debate is whether oestrogen therapy might increase the risk 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and 4- of carcinogenesis, especially breast cancer and endometrial vinylpyridine were products of Sigma (St Louis, MO, USA). adenocarcinoma in the uterus.[15] Therefore, the search for All other laboratory chemicals were of the highest purity natural products possessing oestrogenic activity with ben- available from commercial suppliers. efits on cancer prevention continues. Butea monosperma, a herb of the family Popilionaceae Plant materials containing miroestrol in the stem bark, showed a significant recovery in the level of GSH, and elevated levels of hepatic The tuberous roots of P. mirifica werecollectedinUbon GPx and SOD in thioacetamide-treated rats.[16] Daidzin is a Ratchathani, Thailand, in March 2010. Plant materials were major isoflavone from Puerariae radix, a plant in the same identified by Dr Thaweesak Juengwatanatrakul, Faculty of family as P. mirifica, that increased the level of hepatic Pharmaceutical Sciences, Ubon Ratchathani University, reduced GSH in mice with lipopolysaccharide-induced Ubon Ratchathani, Thailand. Reference specimens (NI- hepatic failure.[17] Therefore, P. mirifica extract or miroestrol PSKKU 007–010) were deposited at the Herbarium of the might possess the potency to modify the level or activity of Faculty of Pharmaceutical Sciences, Khon Kaen University, GSH, GPx, SOD, and catalase. However, the effects of P. Thailand. mirifica or miroestrol on the levels of GSH and on the activity of GPx, SOD, and catalase in ovariectomized mice have Preparation of the P. mirifica crude extract undergone less research.[18] Therefore, our study has deter- and miroestrol mined the influences of P. mirifica extract and miroestrol Dried tuberous root bark of P. mirifica was powdered and on antioxidation systems, including the levels of GSH, GPx, extracted three times with hexane, and the maceration was SOD, and catalase, in the liver and uterus of ovariectomized extracted three times with ethyl acetate, following the mice. protocol of Yusakul et al.[19] The ethyl acetate crude extracts were combined, evaporated, and fractionated by column Materials and Methods chromatography (Silica gel 60 with hexane: ethyl acetate 3 : 1, 3 : 2, 1 : 1, and 0 : 1). The miroestrol fraction Chemicals (compared with the referenced standard) continued to be Miroestrol was isolated from the tuberous roots of P. miri- purified using a Sephadex LH-20 column (ethyl acetate : fica plants as described previously.[4] NMR identification methanol, 7 : 3). Miroestrol was recrystallized and iden- was performed, and the results were compared with authen- tified using high-performance liquid chromatography tic standards from Dr Chaiyo Chaichantipyuth, Faculty of (HPLC) (Figure 2) and 1H NMR spectrum. The ethyl

[mV] using a PerkinElmer Series 200 LC pump coupled with (a) 20 miro a PerkinElmer 785A UV/vis detector. A RP-18 column (LiChroCART, 125 mm ¥ 4 mm, 5 mm particle-size, Merck, 15 Germany) was employed. A series of standard solutions was d-miro prepared by mixing standards of miroestrol and deoxymi- 10 roestrol to obtain the solutions with final concentrations of 5 25.00, 12.50, 6.25, 3.13, 1.56 and 0.78 mg/ml for each compound. Triplicate injections were made for each of the six 0 concentrations. Calibration curves for miroestrol and deoxymiroestrol were constructed. The linearity of each stand- [mV] 0105 15 20 ard curve was confirmed by plotting the peak area vs the 20 Time (min) (b) concentration. 15 Animal design and treatments 10 miro Seven-week-old female ICR mice were supplied by the 5 National Laboratory Animal Center (Mahidol University, d-miro Nakhon Pathom, Thailand) and were housed in the Labo- 0 ratory Animal Unit of the Faculty of Pharmaceutical Sci- ences (Khon Kaen University, Khon Kaen, Thailand) under [mV] 01015205 Time (min) the supervision of the Animal Ethics Committee for Use 20 (c) and Care, Khon Kaen University, Khon Kaen, Thailand 15 (Approval No. AEKKU 01/2555). The mice were intraperitoneally anaesthetized with pentobarbital sodium 10 (100 mg/kg; Nembutal, Ceva Sante Anamale, France) and underwent a bilateral ovariectomy via dorsolateral inci- 5 sion. The ovaries were excised and the oviducts replaced; the incisions were closed. Three days after the operation, 0 mice (n = 5) received intraperitoneal injections of either 01015205 E2 (1 mg/kg per day) in oil vehicle, or the crude extract Time (min) of P. mirifica (2.5 or 25 mg/kg per day) in corn oil, or miroestrol (0.1 or 1.0 mg/kg per day) in 15% Tween 20 Figure 2 HPLC chromatogram of (a) standards of miroestrol (miro) daily for two months. The sham control was simply left and deoxymiroestrol (d-miro), (b) the crude extract of Pueraria mirifica, untreated to exhibit the constitutive levels of the enzymes. and (c) the pure compound of miroestrol. The control groups were intraperitoneally injected with corn oil (0.2 ml per mouse per day) or 15% Tween 20 in acetate crude extract (the P. mirifica crude extract) was sus- distilled water (0.2 ml per mouse/day) daily for the same pended in corn oil, while miroestrol was dissolved in 15% time. The animals were killed 24 h after the last treatment, Tween 20 for the animal treatments. and the livers and uteri were immediately excised for further analysis. Determination of miroestrol and deoxymiroestrol by high-performance Determination of protein content liquid chromatography analysis The livers and uteri were homogenized in cold 1.15% Dried powder of the P. mirifica crude extract (1 g) was KCl (1 g sample per 3 ml 1.15% KCl) with a hand- washed with 5 ml hexane for 1 h with sonication and then homogenizer in an ice bath. The protein content in the extracted four times with 5 ml ethyl acetate-chloroform homogenized samples was determined according to (3 : 1, v/v) with sonication for 1 h. The extracts were com- the Bradford method.[20] As per this method, 50 ml of the bined and evaporated at 60°C. The residual solid was dis- diluted sample was mixed with 200 ml Bio-Rad Protein solved in 1 ml ethanol for HPLC analysis. The mobile phase Assay reagent (Bio-Rad, Hercules, CA, USA). The forma- for HPLC consisted of 20% acetonitrile containing 1.5% tion of protein-dye complexes was measured at a wave- acetic acid at a flow rate of 1.0 ml/min. The detection length of 595 nm and compared with the standard, bovine wavelength was set at 254 nm. HPLC was performed serum albumin.

Measurement of glutathione/oxidized added to each well, followed by the xanthine oxidase solu- glutathione content tion (1.5 mU/well). The reaction was incubated at 25°C for 20 min and stopped by adding 25 ml 0.8 mm copper chlo- GSH/GSSG content in liver and uterus was determined ride (CuCl ). The production of formazan was measured using the glutathione assay kit, according to the manufac- 2 at a wavelength of 550 nm. The percentage of formazan turer’s instructions (Sigma, St Louis, MO, USA and Trevi- inhibition was determined and compared with the standard gen, Gaithersburg, MD, USA). Liver or uterus homogenates curve of bovine Cu-Zn SOD.[23] were extracted in 5% sulfosalicylic acid. After centrifugation at 10 000g at 4°C for 10 min, the supernatant was collected Determination of catalase activity and diluted for GSH/GSSG content analysis using DTNB. To measure GSSG content, the supernatant was treated with Catalase activity was determined by Goth’s colorimetric 4-vinylpyridine and incubated for 1 h at room temperature method[25] with some modifications. Sample was incubated before performing the assay. The formation of thiol anions inaH2O2 substrate, and the enzymatic reaction was termi- was measured at a wavelength of 405 nm at 1-min intervals nated by the addition of ammonium molybdate. Specifically, over a 5-min period. The GSH or GSSG contents were 15 ml homogenate sample was incubated in 50 ml 130 nm determined as mmol/mg protein or nmol/mg protein, by H2O2 substrate at 37°C for 1 min. The reaction was stopped comparison with the slope of the GSH or GSSG standard using 65 ml 32.4 mm ammonium molybdate, and the yellow [21] curve, respectively. complex of molybdate and H2O2 was measured at a wavelength of 405 nm.[23] The percentage of inhibition of the Determination of glutathione molybdate-H2O2 complex was collected and compared with peroxidase activity the standard curve of the hepatic bovine catalase. The activity of GPx in liver and uterus was determined Statistical analysis according to the methods of Pinto and Bartley[22], with some modifications. The assay mixture sequentially contained The data was presented as the mean Ϯ SD and analysed 3.0 ml of a homogenate of either liver or uterus, 0.5 ml by one-way analysis of variance followed by the Tukey 0.25 m phosphate buffer at pH 7.4, 0.1 ml 25 mm ethylenedi- post-hoc test (SPSS ver. 17.0). P < 0.05 was considered to aminetetraacetic acid (EDTA), 0.1 ml 0.4 m sodium azide be statistically significant.

(NaN3), 0.3 ml 50 mm GSH, and 0.1 ml 50 mm hydrogen peroxide (H2O2). The reaction was stopped using 1.0 ml 5% Results sulfosalicylic acid 10 s after the addition of the H2O2.After centrifugation at 10 000g at 4°C for 10 min, the supernatant Identification and quantitative was taken for GSSG measurement as described above. The determination of miroestrol amount of GSSG contained in this reaction was an accumu- Miroestrol was isolated as a white powder: 1H NMR lation of GSSG that was created in this reaction as well as (CD3OD, 400 MHz): 6.99 (d, H-1), 6.50 (dd, H-2), 6.30 GSSG that was endogenous to the sample, in addition to the (d, H-4), 6.29 (s, H-7), 3.31 (s, H-9), 2.74 (m, H-12), 2.43 GSSG that was contained in the GSH solution. One unit of (d, H-13), 2.58 (dd, H-16a), 2.89 (bd, H-16b), 3.70 GPx activity was calculated from 1 mmol GSSG formed/min (s, H-18), 1.87 (dd, H-19a), 1.99 (1H, br t, H-19b), 0.56 in the reaction at 30°C and pH 7.4. (s, H-20), 1.23 (s, H21), according to a previous report of Chansakaow et al.[4] The contents of miroestrol and deox- Determination of superoxide ymiroestrol in the crude extract of P. mirifica were quanti- dismutase activity fied as previously described using the HPLC method of Yusakul et al.[19] The P. mirifica crude extract contained For the sample’s preparation, 0.5 ml of liver or uterus 69.37 Ϯ 1.91 mg/g dry weight miroestrol and 15.41 Ϯ homogenate was extracted using 0.3 ml chloroform and 0.71 mg/g dry weight deoxymiroestrol (Figure 2b). The 0.5 ml ethanol, vigorously vortex-mixed for 1 min, and sub- purity of miroestrol employed in this study was > 99% as jected to centrifugation at 13 000g at 4°C for 30 min.[23] The shown in the HPLC chromatogram (Figure 2c). supernatants were used for the SOD assay. SOD activity was determined by the inhibition of NBT reduction due to the Glutathione content in liver and uterus of O2- generated by the xanthine/xanthine oxidase system.[24] ovariectomized mice Either 12.5 ml supernatant or bovine Cu-Zn SOD standard (10–100 mg/ml) was added to a 96-well microplate. Then, Total GSH, reduced GSH, and oxidized GSH (GSSG) levels 107.5 ml reagent mixture (1.2 mm xanthine, 140 mm EDTA, were measured in the livers (Table 1) of ovariectomized

140 mm NBT, 56 mm Na2CO3, and 70 mg/ml BSA) was mice. The hepatic total GSH content, the reduced GSH

Table 1 Effect of the Pueraria miriﬁca crude extract and miroestrol on total glutathione, reduced glutathione, and oxidized glutathione content in the liver of the ovariectomized mouse

Total GSH content Reduced GSH content GSSG content Ratio of GSH/ Treatment group (mmol/mg protein) (mmol/mg protein) (mmol/mg protein) GSSG

Sham 8.64 Ϯ 0.56 6.55 Ϯ 0.46 2.36 Ϯ 0.20 2.78 Ϯ 0.03 Ovariectomized-corn oil 5.55 Ϯ 0.80A 2.87 Ϯ 0.36A 3.03 Ϯ 0.15 0.95 Ϯ 0.18A Ovariectomized-Tween 3.39 Ϯ 0.07A 1.21 Ϯ 0.30A 2.19 Ϯ 0.60 0.55 Ϯ 0.05A Ovariectomized-E2 4.88 Ϯ 0.47A 2.55 Ϯ 0.32A 2.33 Ϯ 0.16 1.09 Ϯ 0.06A Ovariectomized-PM2.5 5.41 Ϯ 0.90A 4.19 Ϯ 0.97A,B 1.27 Ϯ 0.19A,B 3.30 Ϯ 0.04A,B Ovariectomized-PM25 6.11 Ϯ 0.49A,B 4.41 Ϯ 0.72A,B 1.70 Ϯ 0.12A,B 2.59 Ϯ 0.02B Ovariectomized-MR0.1 6.97 Ϯ 0.71C 4.24 Ϯ 0.61A,C 2.73 Ϯ 1.20 1.55 Ϯ 0.07A,C Ovariectomized-MR1 8.72 Ϯ 0.36C 6.52 Ϯ 0.93C 2.20 Ϯ 1.40 2.96 Ϯ 0.10C

E2, estradiol benzoate 1 mg/kg per day; GSH, glutathione; GSSG, oxidized glutathione; MR, miroestrol (mg/kg per day); PM, P. miriﬁca crude extract (mg/kg per day); Sham, sham operated mice. AP < 0.05 vs sham; BP < 0.05 vs ovariectomized-corn oil; CP < 0.05 vs ovariectomized-Tween.

Table 2 Effect of the Pueraria miriﬁca crude extract and miroestrol on total glutathione, reduced glutathione, and oxidized glutathione content in the uterus of the ovariectomized mouse

Total GSH content Reduced GSH content GSSG content Ratio of GSH/ Treatment groups (nmol/mg protein) (nmol/mg protein) (nmol/mg protein) GSSG

Sham 68.89 Ϯ 12.65 66.16 Ϯ 11.49 3.78 Ϯ 0.69 22.75 Ϯ 5.31 Ovariectomized-corn oil 14.17 Ϯ 4.85A 11.77 Ϯ 9.64A 2.62 Ϯ 1.25A 5.77 Ϯ 0.53A Ovariectomized-Tween 12.41 Ϯ 2.34A 9.22 Ϯ 4.01A 3.36 Ϯ 2.20 3.37 Ϯ 1.97A Ovariectomized-E2 12.24 Ϯ 1.42A 10.51 Ϯ 0.96A 2.30 Ϯ 0.33A 5.84 Ϯ 1.79A Ovariectomized-PM2.5 23.75 Ϯ 2.59A,B 22.12 Ϯ 2.56A,B 1.63 Ϯ 0.23A,B 13.74 Ϯ 2.36A,B Ovariectomized-PM25 17.13 Ϯ 3.07A 16.66 Ϯ 4.11A 1.47 Ϯ 0.43A,B 19.21 Ϯ 5.96A,B Ovariectomized-MR0.1 24.77 Ϯ 3.61A,C 23.63 Ϯ 3.92A,C 1.26 Ϯ 0.59A,C 16.15 Ϯ 3.19A,C Ovariectomized-MR1 15.25 Ϯ 3.50A 14.82 Ϯ 3.61A 1.12 Ϯ 0.41A,C 18.68 Ϯ 6.26A,C

E2, estradiol benzoate 1 mg/kg per day; GSH, glutathione; GSSG, oxidized glutathione; MR, miroestrol (mg/kg per day); ovariectomized, ovariectomized mice; PM, P. miriﬁca crude extract (mg/kg per day); Sham, sham operated mice. AP < 0.05 vs sham; BP < 0.05 vs ovariectomized-corn oil; CP < 0.05 vs ovariectomized-Tween.

content, and the ratio of GSH/GSSG were markedly Glutathione peroxidase activity in liver and decreased in both groups of control ovariectomized mice. uterus of ovariectomized mice E2 did not improve any of the levels of the various GSH measures in the liver. The crude extract of P. mirifica signifi- Liver and uterus glutathione peroxidase activity (GPx) was cantly increased the levels of hepatic total GSH and the suppressed after ovariectomy and treatment with corn oil or reduced GSH, while decreasing the GSSG content in ova- Tween 20 (Figure 3). E2 numerically increased, but not sig- riectomized mice treated with corn oil. However, only a nificantly, the GPx activity in liver, and caused a further high dose of P. mirifica (25 mg/kg per day) restored GSH/ reduction of GPx activity in uterus, compared with the ova- GSSG to a sham control level. All miroestrol injections riectomized control groups. The crude extract of P. mirifica restored the hepatic total GSH and GSSG to sham control increased hepatic GPx activity in a dose-dependent pattern levels, whereas the reduced GSH content and the ratio of (Figure 3a). Miroestrol showed a dose-dependent effect of GSH/GSSG reached their normal values only at the high increasing hepatic GPx activity, though the activity was still dose of miroestrol (1 mg/kg per day). On the other hand, less than the sham control level (Figure 3b). In the uteri of in uteri (Table 2), the total GSH content, the reduced post-ovariectomy mice, P. mirifica significantly increased GSH content, the GSSG content, and GSH/GSSG were all the activity of GPx (Figure 3c). Miroestrol restored the strongly reduced post-ovariectomy. E2 did not improve any activity of GPx, but not dose-dependently, whilst the higher of the levels of the various GSH measures in uterus. Admin- dose exhibited the lower increase to the sham control istration of either P. mirifica or miroestrol significantly (Figure 3d). The dose-dependent pattern of P. mirifica and increased the total GSH, the reduced GSH, and the ratio of miroestrol on liver GPx level was not observed in their GSH/GSSG, while the amount of GSSG was suppressed. effect on uterus.

3.5 2.5 (a) ** (b) 3.0 ** 2.0 2.5 1.5 * 2.0 ** **

1.5 1.0

(U/mg protein) 1.0 (U/mg protein) * ** Hepatic GPx activity ** Hepatic GPx activity 0.5 ** 0.5 ** ** 0.0 0.0 Corn oil E2 PM 2.5 PM 25 Tween E2 MR 0.1 MR 1 Sham Sham Ovariectomized Ovariectomized

0.08 0.08 (c) (d) 0.07 0.07 0.06 0.06 0.05 * * 0.05 0.04 0.04 0.03 * ** 0.03 (U/mg protein) (U/mg protein) 0.02 ** 0.02 ** Uterus GPx activity Uterus GPx activity ** 0.01 0.01 ** ** ** ** 0.00 0.00 Corn oil E2 PM 2.5 PM 25 Tween E2 MR 0.1 MR 1 Sham Sham Ovariectomized Ovariectomized

Figure 3 Glutathione peroxidase (GPx) activity in the liver and uterus of ovariectomized mice. Ovariectomized mice were intraperitoneally injected with estradiol benzoate (E2, 1 mg/kg per day), the crude extract of Pueraria miriﬁca (PM; 2.5 or 25 mg/kg per day), or miroestrol (MR; 0.1 or 1.0 mg/kg per day) daily for two months. The ovariectomized control groups were intraperitoneally injected with either corn oil or 15% Tween 20 daily for two months. The sham control mice were left untreated. The data are presented as the mean Ϯ SD from duplicate (n = 4–5) independent experiments. A signiﬁcant difference was determined by one-way analysis of variance followed by a Tukey post-hoc test. *P < 0.01, **P < 0.001 compared with sham group or ovariectomized control.

Superoxide dismutase activity in liver and uterus (Figure 4d), again with no dose-dependent pattern uterus of ovariectomized mice observed. Superoxide dismutase (SOD) activity was measured in liver Catalase activity in liver and uterus of and uterus (Figure 4). Ovariectomy significantly reduced ovariectomized mice the SOD activity in both organs (Figure 4). E2 numerically increased the activity of SOD in liver and uterus compared Catalase activity was significantly decreased in both liver with the corn oil ovariectomized group, but the increase and uterus of mice post-ovariectomy (Figure 5). E2 could was significant compared with the Tween 20 ovariectomized not recover the catalase activity in either the liver or uterus group (Figure 4b and d). The crude extract of P. mirifica of these ovariectomized mice, where it tended to suppress improved the SOD activity in the liver of ovariectomized the hepatic catalase activity (Figure 5). Interestingly, the mice, following a dose-dependent pattern (Figure 4a). The crude extract of P. mirifica increased the catalase activity hepatic SOD activity in ovariectomized mice was signi- in liver and uterus of ovariectomized mice up to the levels ficantly elevated by miroestrol, but no dose-dependent of the sham group, but the P. mirifica crude extract at effect was observed (Figure 4b). The crude extract of P. 25 mg/kg per day exhibited less potential than at 2.5 mg/kg mirifica also increased the uterus SOD activity (Figure 4c). per day (Figure 5a and c). Miroestrol showed the poten- Miroestrol extensively increased the activity of SOD in tial to enhance the activity of catalase in only uterus

18 16 (a) (b) 16 ** 14 * 14 * 12 * * ** 12 * 10 * 10 ** 8 * 8 6 6 (U/mg protein) (U/mg protein) 4 4 Hepatic SOD activity Hepatic SOD activity 2 2 0 0 Corn oil E2 PM 2.5 PM 25 Tween E2 MR 0.1 MR 1 Sham Sham Ovariectomized Ovariectomized

25 25 (c) (d)

20 20

15 * 15

10 * * 10 * *

(U/mg protein) * Uterus SOD activity (U/mg protein) 5 5 Uterus SOD activity ** ** 0 0 Corn oil E2 PM 2.5 PM 25 Tween E2 MR 0.1 MR 1 Sham Sham Ovariectomized Ovariectomized

Figure 4 Superoxide dismutase (SOD) activity in liver and uterus of ovariectomized mice. Ovariectomized mice were injected with estradiol benzoate (E2, 1 mg/kg per day), the crude extract of Pueraria mirifica (PM; 2.5 or 25 mg/kg per day), or miroestrol (MR; 0.1 or 1.0 mg/kg per day) daily for two months. The ovariectomized control groups were injected with vehicle for two months. The sham control mice were left untreated. The data are presented as the mean Ϯ SD from duplicate (n = 4–5) independent experiments. A significant difference was determined by one-way analysis of variance followed by a Tukey post-hoc test. *P < 0.01, **P < 0.001 compared with the sham group or ovariectomized control. ovariectomized mice, without dose-dependency (Figure 5b via the catecholestrogen pathway.[28] Some studies have and d). focused on the involvement of ovarian steroid hormones, especially oestrogens, in the phenomena of oxidative [28,29] Discussion stress. The oxidative stress in uterus was increased by E2, resulting in a significant increase in malondialde- The level of oestrogen in ovariectomized mice is greatly hyde.[30] The oestrogenic potential of P. mirifica has been decreased, leading to body weight gain, abdominal fat demonstrated by both its crude extract and the potent stores, increased liver fat, and osteoporosis.[13,14] Therefore, phytoestrogen, miroestrol, in several experimental models, ovariectomized mice were used as a model to reflect the including immature mice, female mice, ovariectomized pathological changes in perimenopausal and postmeno- rats, female monkeys, and human clinical trials.[31–36] pausal women.[26,27] Oestrogen (estradiol) is considered as In this study, E2 did not vary any levels of the various the first-line drug for the prevention and treatment of GSH measured in either liver or uterus, while P. mirificai multiple conditions affecting menopausal women.[13,14] extract and miroestrol improved the GSH level. These However, the current issue of debate is whether oestrogen observations suggested that P. mirifica extract and therapy might increase the risk of carcinogenesis, espe- miroestrol were more effective than E2 for restoring the cially breast cancer and endometrial adenocarcinoma in GSH status in both liver and uterus of ovariectomized mice. the uterus.[15] Mechanisms of hormone therapy perform- This is the first report revealing the effects of the P. mirifica ing on the carcinogenesis process are various such as crude extract and miroestrol on the antioxidation-related DNA damage induction, enhancement of DNA adduct in enzymes and the GSH content in the liver and uterus of the chronic oestrogen exposure, and free radical generation ovariectomized mouse.

300 250 (a) (b) 250 * 200 200 * 150 * 150 ** ** 100 ** 100 **

(U/mg protein) ** (U/mg protein)

Hepatic CAT activity Hepatic CAT 50 activity Hepatic CAT 50

0 0 Corn oil E2 PM 2.5 PM 25 Tween E2 MR 0.1 MR 1 Sham Sham Ovariectomized Ovariectomized

25 25 (c) (d) 20 20

15 15 ** 10 10

** * (U/mg protein) Uterus CAT activity Uterus CAT (U/mg protein) 5 5 Uterus CAT activity Uterus CAT ** ** ** ** 0 0 Corn oil E2 PM 2.5 PM 25 Tween E2 MR 0.1 MR 1 Sham Sham Ovariectomized Ovariectomized

Figure 5 Catalase (CAT) activity in the liver and uterus of ovariectomized mice. Ovariectomized mice were injected with estradiol benzoate (E2, 1 mg/kg per day), the crude extract of Pueraria miriﬁca (PM; 2.5 or 25 mg/kg per day), or miroestrol (MR; 0.1 or 1.0 mg/kg per day) daily for two months. The ovariectomized control groups were injected with vehicle for two months. The sham control mice were left untreated. The data are presented as the mean Ϯ SD from duplicate (n = 4–5) independent experiments. A signiﬁcant difference was determined by one-way analysis of variance followed by a Tukey post-hoc test. *P < 0.01, **P < 0.001 compared with the sham group or ovariectomized control.

From the HPLC chromatogram (Figure 2), we showed and Zn in its catalytic centre and is localized in intracellular that the crude extract of P. mirifica consisted of both cytoplasm compartments (CuZn-SOD or SOD1). Another miroestrol and deoxymiroestrol (in which deoxymiroestrol isoform of SOD has manganese (Mn) as a co-factor and has was usually metabolized to miroestrol). Moreover, P. miri- been localized in the mitochondria of aerobic cells (Mn-SOD fica actually contains other compounds such as chromenes, or SOD2).[38] GPx exists in the cytosol as a homotetramer, flavonoids, and isoflavones, while miroestrol is the pure with each subunit containing a selenium atom incorporated compound of miroestrol.[37] Hence, a difference between the within a catalytically active selenocysteine residue.[40] This P. mirifica crude extract and miroestrol might be observed. amino acid is sterically exposed on a flat lipophilic segment In addition, the liver is the most abundant organ of antioxi- of the protein, allowing it to be oxidized by an approaching dative enzymes, while the uterus contains less of these hydroperoxide.[41] GSH has long been used as a defensive enzymes than the liver (< 10-fold) (Figures 3–5). Therefore, agent against the action of toxic xenobiotics, including drugs, the constitutive levels of enzymes might reflect the dose pollutants, and carcinogens. As a prototypical antioxidant, response of the P. mirifica crude extract or miroestrol, GSH has been involved in protecting cells from the noxious namely the dose-dependent pattern was observed in the effect of excessive oxidative stress.[42] Hence, the decrease in liver, but not in the uterus. levels of these defensive enzymes including GSH might affect The free radical tissue-defence mechanisms including the defence mechanisms of organs, allowing any abnormality SOD, GPx, and GSH, are the important antioxidative or disease to progress. enzymes against ROS and, particularly, against superoxide SOD and catalase activity were significantly decreased in anion radicals in mammals.[38,39] The isoform of SOD has Cu ovariectomized rats.[8] Moreover, the activity of GPx and

SOD were decreased in the femur of ovariectomized albino Conclusion rats.[43] Ovariectomy reduced the GSH levels in the bone marrow of rats.[44] We correspondingly found a decrease in These findings support the view that P. mirifica crude the total GSH content, the reduced GSH content, and the extract or miroestrol could be candidates as alternative ratio GSH/GSSG, as well as decreased activity of GPx, SOD, medicine in oestrogen replacement therapy, based on their and catalase enzymes in both the liver and the uterus of oestrogenic activity and ability to improve menopausal syn- mice after undergoing ovariectomy. A previous study by dromes. Moreover, their potential to improve the level of Tiwari et al.[45] reported that Butea monosperma, a plant oxidative stress-related enzymes and to maintain the level of containing miroestrol, increased the levels of GSH in GSH content in ovariectomized mice over estradiol should carbon tetrachloride-induced hepatotoxicity in rats.[46] The be promoted. effects of either P. mirifica or miroestrol on GPx, SOD, and catalase activity have not been reported previously. Declarations In this study, the crude extract of P. mirifica and the isolated miroestrol showed their potential to return the levels of Conflict of interest total GSH and GSSG to nearly those of the sham control The Author(s) declare(s) that they have no conflicts of mice. Therefore, the potential of the P. mirifica crude extract interest to disclose. and miroestrol to improve the activity of the GPx, SOD, and catalase enzymes to those of the normal levels might help Funding counterbalance the endogenous ROS and oxidative stress induced by the oestrogen deficient state, and halt the progres- This research received no specific grant from any funding sion of diseases related to oxidative stress and menopause. agency in the public, commercial, or not-for-profit sectors.

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