INTRODUCTION {#sec1-1}
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Plant materials have been used for treatments of diseases in the Eastern countries, especially East Asia, for thousands of years. In the specific tribes in Thailand, *Kaempferia parviflora*, a Zingiberaceae plant, has been used a folk medicine called "Krachai Dum." The rhizome and tincture of *K. parviflora* has been applied as medicine for general pain, colic gastrointestinal disorders, and male impotence. These various pharmacological activities of *K. parviflora* prompted researchers to elucidate its activities scientifically using the *in vivo* and *in vitro* assays. Among them, inhibition of P-glycoprotein function,\[[@ref1]\] anti-plasmodial, anti-fungal and anti-mycobacterial effects,\[[@ref2]\] cytotoxicity against various cancer cell lines,\[[@ref3]\] anti-cholinesterase activity,\[[@ref4]\] anti-allergic activity,\[[@ref5]\] modulation of the function of multidrug resistance associated-proteins,\[[@ref6]\] anti-gastric ulcer effect,\[[@ref7]\] anti-obese activity,\[[@ref8]\] anti-diabetes activity,\[[@ref9][@ref10]\] and inhibition of fat absorption\[[@ref11]\] have been demonstrated. These multi-functional effects of *K. parviflora* makes it an attractive potential treatment for life-style-related diseases. Thus, continuous research to discover novel effects of *K. parviflora* on life-style-related diseases has been performed in our laboratory.

Among various life-style-related diseases, we first focused on anti-hyperuricemia activity based on inhibition on xanthine oxidase. Xanthine oxidase has been recognized as a key enzyme for producing uric acid and a treatment for gout. *K. parviflora* extract showed potent inhibitory activity compared to other Zingiberaceae plants used as crude drugs and the active principles were determined as 3,5,7,3΄,4΄-pentamethoxyflavone and 5,7,3΄,4΄-tetramethoxyflavone.\[[@ref12]\] In addition, we focused on an improvement effect on blood fluidity. Stagnation of blood causes a loss of metabolism, obesity, and poor circulation. *K. parviflora* has been shown to have fibrinolysis activity and improved the blood fluidity in disseminated intravascular coagulation model rats.\[[@ref13]\] Thus, we have been demonstrated some novel *K. parviflora* function and believe that other novel functions are yet to be revealed.

Benign prostate hyperplasia (BPH) is life-style-related illnesses caused by inappropriate diet habits and the social stress leading to hormone imbalance. The incidence of BPH is approximately 50-60% in males 40-60 years old and greater than 90% in men over 80 years.\[[@ref14]\] It also causes urinary tract obstruction and infection.\[[@ref15]\] Androgen plays an important role in the growth, and maintenance of normal prostate gland, function as well as in the development of BPH. The androgens testosterone and dihydrotestosterone (DHT) contribute to the onset of BPH.\[[@ref16]\] Testosterone is converted to DHT by 5α-reductase (5αR).\[[@ref17]\] DHT is a potent androgen whose and its action is mediated through binding to the androgen receptor (AR) in the prostate, which induces protein synthesis and abnormal growth of the prostate.\[[@ref17][@ref18]\] Therefore, inhibition of 5αR could help prevent BPH. Finasteride, a potent 5αR inhibitor and flutamide, a binding inhibitor to AR are used as effective agents in BPH treatment. These drugs, however, are known to produce side-effects including impaired liver function, diarrhea, and headache. Alternatively, functional foods including natural products that show 5αR inhibitions may be useful in the prevention of BPH.

Some 5αR inhibitors from natural products have been discovered by our group, i.e., 12-methoxycarnosic acid in *Rosmarinus officinalis* leaf,\[[@ref19]\] ginsenoside Ro in the rhizome of red ginseng,\[[@ref20]\] kaikasaponin and soyasaponin in flowers of *Pueraria thomsonii*\[[@ref21]\] and fatty acids and ethinylestradiol in spores of *Lygodium japonicum*.\[[@ref22]\]

In the present study, extract from *K. parviflora* rhizome was subjected to the inhibitory assay against 5αR along with other Zingiberaceae plants. *K. parviflora* showed potent activity and the active principle was revealed. Furthermore, a result on the improvement of BPH by *K. parviflora* using *in vivo* BPH model is described.

MATERIALS AND METHODS {#sec1-2}
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Materials {#sec2-1}
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Rhizomes of *K. parviflora* were purchased from Aoba trading Co. (Tokyo, Japan). Voucher specimens were deposited at the Faculty of Pharmacy, Kinki University. Reagents used in this study were of analytical grade and were purchased from Wako Pure Chemicals Industries (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan) unless otherwise stated.

Preparations of extracts and methoxyflavones {#sec2-2}
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Preparations of extracts from rhizomes of *K. parviflora*, *Curcuma zedoaria* and *Zingiber officinale*, and methoxyflavones were performed according to the methods previously reported.\[[@ref12]\]

Inhibitory assay for 5αR {#sec2-3}
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The enzyme 5αR was prepared according to the method reported\[[@ref23]\] with modifications. Rats (Wistar, 9 weeks, 270-290 g) were purchased from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan), and were maintained at a constant temperature and humidity under a 12 h light/dark cycle for 11 days. Water and pellet chow (Labo MR stock, Nosan Corporation, Tokyo, Japan) were freely available. The epididymis was removed from 100 rats and homogenized with a blender in cooled physiological saline containing 0.25 M sucrose and protease inhibitor cocktail. Homogenates were filtered and centrifuged at 300 ×g for 10 min, and the supernatants were centrifuged again under the same conditions to obtain crude enzyme the protein assay method (Bio-Rad Laboratories, Inc., Hercules, CA) and crude enzyme solution was diluted to 10 mg/ml and stored at −85°C until use.

The 5αR inhibition assay was performed as reported previously\[[@ref24][@ref25]\] with minor modifications. The reaction solution containing 50% ethanol (50 μl) with various concentrations of test compounds, 600 μl of 100 mM Mcllvaine buffer (pH 5.0), 20 μl of testosterone (0.4 mM in propylene glycol \[PG\]-citrate/phosphate buffer, pH 5.0 \[1:1 v/v\]) and 110 μl of enzyme solution.\[[@ref26]\] Reactions were initiated by the addition of 20 μl of 34 mM nicotinamide adenine dinucleotide phosphate reduced form. The mixture was incubated at 37°C for 30 min. After addition of 1.0 ml of dichloromethane and 20 μl of *p*-hydroxybenzoate *n*-hexyl ester as an internal standard (IS), the organic layer was obtained by centrifugation (3000 × *g* for 3 min), and transferred to another tube and air dried. The residue was dissolved in 0.2 ml of methanol, and an aliquot of 30 μl was injected into an high performance liquid chromatography (HPLC) system under the following conditions: Column, YMC-Park ODS-AM302 (4.6 i.d. × 150 mm); column temperature, 40°C; mobile phase, methanol/water (65:35, v/v); flow rate, 1.0 ml/min; detection, UV at 254 nm, *t*~R~; testosterone, 7 min, *t*~R~; IS, 14 min. The control-0-min tube received 1.0 ml of dichloromethane before addition of the enzyme solution, whereas, the control-30-min tube received 50 μl of methanol instead of the test sample. A similar procedure to that described previously was carried out for these control tubes. The 5αR inhibitory activity was determined from the following equation using the peak-area ratios (*r* = testosterone/IS). Finasteride (Tokyo Chemical, Tokyo, Japan) was used as a reference drug.

Inhibition (%) = 100 × (C~sample~/C~control~)

C: Conversion rate (%) of testosterone to DHT

C~sample~ (C for sample groups) = *r* of the test sample -- *r* of control-30 min

C~control~ (C for control groups) = *r* of control-0 min -- *r* of control-30 min.

Animals {#sec2-4}
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Male Crj: CD Sprague-Dawley (SD) rats, which castrated at 6 weeks age and normal (not castrated) SD rats (7 weeks age) were purchased from Shimizu Laboratory Supplies (Kyoto, Japan). The rats were acclimatized for 1 week in an animal room, which is maintained by air-conditioning with lighting from 7 a.m. to 7 p.m. The temperature and humidity were controlled automatically. Laboratory pellet chow (Labo MR stock, Nihon Nosan Kogyo, Tokyo, Tokyo, Japan) and water were freely available. All experimental protocols using animals were approved by the Committee for the Care and Use of Laboratory Animals at Kinki University.

Animal treatments {#sec2-5}
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The growth suppression assay of prostates and seminal vesicles in castrated rats was performed based on the Hershberger\'s method.\[[@ref27][@ref28]\] The castrated rats were randomized to four groups of 6 rats: (1) A negative control group receiving intraperitoneal administration (i.p.) of olive oil and oral administration (p.o.) of 100 μl of 50% PG, (2) testosterone propionate (TP) (2 mg/kg, i.p.) and 100 μl of PG (p.o.), (3) TP (2 mg/kg, i.p.) and *K. parviflora* ext in PG (200 mg/kg, p.o.) and (4) TP (2 mg/kg, i.p.) and finasteride in PG (2 mg/kg, p.o.). Non-treatment group (not castrated rats) was also prepared. The administrations were performed once a daily for 14 days.

Sacrifice and evaluation of prostates and seminal vesicles weights {#sec2-6}
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The day after the last treatment (at the day 15 from the starting of administration), rats were sacrificed under anesthesia with pentobarbital (35 mg/kg). Their prostates and seminal vesicles were removed and weighted.

Statistical analysis {#sec2-7}
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Data were evaluated statistically by Bonferroni/Dunn multiple range tests using a Microsoft Excel add-in software Statcel (Ver. 3, OMS publishing, Saitama, Japan). Significant differences were detected at *P* \< 0.05 and 0.01.

RESULTS AND METHODS {#sec1-3}
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Effects of *K. parviflora*, *C. zedoaria* and *Z. officinale* extracts on 5αR inhibition {#sec2-8}
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Firstly, the inhibitory activity (conversion rate of testosterone to DHT) of the *K. parviflora* extract was studied compared with those of *C. zedoaria* and *Z. officinale* extracts on 5αR. The results are shown in [Table 1](#T1){ref-type="table"}. *K. parviflora* extract showed enzyme inhibition of 75.8% at 500 μg/ml while the values for the *C. zedoaria* and *Z. officinale* extracts were 28.2% and 21.1%, respectively. This result suggested that *K. parviflora* has the most potent activity and may be effective in the treatment of BPH.

###### 

Inhibitory effects of crude Zingiberaceae extracts on 5αR

![](PR-5-309-g001)

Effects of methoxyflavones from *K. parviflora* on 5αR {#sec2-9}
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Secondly, the inhibitory activities of methoxyflavones from *K. parviflora* were examined since methoxyflavones are known to be multi-potential active compounds. The results are shown in [Table 2](#T2){ref-type="table"}. Among the compounds tested, 3, 5, 7, 3΄, 4΄-pentamethoxyflavone and 5, 7, 3΄,4΄-tetramethoxyflavone showed the most potent inhibitory activities of 55.9% at 50 μM ([Figure 1](#F1){ref-type="fig"}). Their IC~50~ values were 46.6 and 48.7 μM for 3, 5, 7, 3΄, 4΄-pentamethoxyflavone and 5, 7, 3΄, 4΄-tetramethoxyflavone, respectively. These results indicated that these two methoxyflavones were the most potent among the nine methoxyflavones.

###### 

Inhibitory effects of methoxyflavones on 5αR

![](PR-5-309-g002)

![Chemical structures of 3,5,7,3',4'-pentamethoxyflavone and 5,7,3',4'-tetramethoxyflavone](PR-5-309-g003){#F1}

Effects of *K. parviflora* extract on rat prostate and seminal vesicle weights {#sec2-10}
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The effects of *K. parviflora* extract on the growth of rat prostates and seminal vesicles were studied. The results are shown in [Figure 2](#F2){ref-type="fig"}. The mean prostate weight/body weight of the group, which received PG (control group) was 145.0 ± 30.7 mg/kg \[[Figure 2a](#F2){ref-type="fig"}\]. The mean prostate weight/body weight of the group with BPH induced by TP injection was 1898.0 ± 325.2 mg/kg, which was significantly higher than that of the control group (*P* \< 0.01). The mean prostate weight/body weight of the rats that received the *K. parviflora* extract (200 mg/kg) was 986.1 ± 124.6 mg/kg. Compared with the BPH group, *K. parviflora* showed a significant reduction in prostate weight (*P* \< 0.05). The mean prostate weight/body weight of the finasteride-administered (2 mg/kg) rats was 742.3 ± 84.0 mg/kg, which was significantly lower than the BPH group (*P* \< 0.01). The mean prostate weight/body weight of the non-castrated and treated rats was 397.0 ± 61.9 mg/kg \[[Figure 2a](#F2){ref-type="fig"}\].

![Suppression of testosterone propionate-induced hyperplasia of prostates (a) and seminal vesicles (b) in castrated rats by *Kaempferia parviflora* extract. Values represent mean ± SE of *n* = 6. Significantly different from control group at \#*P* \< 0.01. Significantly different from negative control group at \*\**P* \< 0.01](PR-5-309-g004){#F2}

The mean seminal vesicles weight/body weight of the group, which received PG (control group) was 78.7 ± 16.7 mg/kg \[[Figure 2b](#F2){ref-type="fig"}\]. The mean seminal vesicles weight/body weight of the group with BPH induced by TP injection was 1099.1 ± 73.9 mg/kg, which was significantly higher than that of the control group (*P* \< 0.01). The mean seminal vesicles weight/body weight of the rats that received *K. parviflora* extract (200 mg/kg) was 737.2 ± 85.4 mg/kg. Compared with the BPH group, *K. parviflora* showed a significant reduction in seminal vesicle weight (*P* \< 0.05). The mean seminal vesicles weight/body weight of the finasteride (2 mg/kg)-administered rats was 415.2 ± 36.0 mg/kg, which was significantly lower than that of the BPH group (*P* \< 0.01). The mean seminal vesicles weight/body weight of the non-castrated and treated rats was 477.7 ± 28.3 mg/kg \[[Figure 2b](#F2){ref-type="fig"}\].

In the present study, *K. parviflora* extract treatment resulted in a significant decrease in the weights of prostates and seminal vesicles in TP injected-castrated rats. These effects can be attributed to the inhibitory activity against 5αR exhibited by *K. parviflora* extract as shown in this manuscript.

Search for effective plant resources for the improvement of BPH has been performed extensively. Among them, *Serenoa repens*\[[@ref29][@ref30]\] *Ganoderma lucidum*,\[[@ref31]\] *Lepidium meyenii* (Red Maca),\[[@ref32]\] banana peel,\[[@ref33]\] and *Echinacea purpurea*\[[@ref34]\] showed significant improvements of BPH. The results obtained in this study could have successfully presented that *K. parviflora* is a novel entry from Zigiberaceae plant and *K. parviflora* would be a promising candidate. Further, studies on the mode of action of *K. parviflora* extract are now underway.

**Source of Support:** Nil

**Conflict of Interest:** None declared.
