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Effects of the polysaccharide fraction of Urtica fissa on castrated rat prostate hyperplasia induced by testosterone propionate.

Abstract

A crude polysaccharide fraction of Urtica fissa roots and stems (UFP) was obtained by water extraction and ultrafiltration, and its effect on castrated rat prostate hyperplasia induced by testosterone propionate was evaluated by the volume index, wet and dry weight index and histopathological tests. Results showed that the crude polysaccharide fraction significantly inhibited prostatic hyperplasia in animal models at doses of 62.5, 125, 250 mg/kg body wt. (administered orally). Treatment with UFP at 62.5 mg/kg body wt. decreased the volume index by 32%, the wet weight index by 17% and the dry weight index by 23%, respectively. In the high-dose group (UFP at 250mg/kg body wt.). the indexes of volume, wet weight and dry weight decreased further by 37%, 25% and 33%, respectively. Histopathological examination showed that proliferation of prostatic epithelial cells and fibrotic tissues were significantly inhibited.

[C] 2007 Elsevier GmbH. All rights reserved.

Keywords: Urtica fissa: Urticaceae family: Crude polysaccharide fraction: Prostate hyperplasia; Castrated rat

Introduction

Benign prostate hyperplasia (BPH) is the most common disease among aged men. 50-80% of men over 50 years of age suffer from BPH. Phytotherapeutics are very popular for the treatment of BPH, especially in Europe, where numerous plant extracts have been used. One of the most popular plants is stinging nettle (Urtica dioica, Urticaceae family). Various water extracts from Urtica dioica roots are widely used for treatment of benign prostatic hyperplasia (Safarinejad, 2005; Lopatkin et al., 2005; Bondarenko et al., 2003; Schneider and Rubben, 2004). Urtica has also been used for many years as a traditional herbal medicine in China for the treatment of eczema, rheumatism, and inflammation. Wang reported 11 species and subspecies of the genus Urtica from the Sichuan Province of China, the nettles from eight of which were commonly used for treatment of rheumatism (Wang and Wei, 2001a; Wang et al., 2002). The crude extract from Urtica fissa showed significant anti-inflammatory activities and immunological enhancement in the rat footpad swelling and pinna swelling models (Wang and Wei, 2001b). In this study we investigated the inhibitory effects of the polysaccharide fraction of Urtica fissa on castrated rat prostate hyperplasia induced by testosterone propionate.

Materials and methods

Drugs

Testosterone propionate and barbanylum were purchased from Shanghai Ninth Pharmaceutical Factory, China. Qian Lie Kang (a standard extraction product of rape pollen, 0.5 g/tablet) was purchased from Zhejiang Conba Pharmaceutical Co., LTD. China.

Plant materials

Urtica fissa was collected in July, 2005 at Anshun mountains in Guizhou Province of China, and identified by Professor Liu Cun-sheng in Beijing University of Chinese Traditional Medicine. A voucher specimen was deposited under the number 200507 in our laboratory.

Extraction of polysaccharide

Urtica fissa roots and stems (50 kg) were pre-extracted with 4001 95% ethanol to remove color materials. The plant residue was extracted with boiling water (2 x 4001) for 3 h twice. The aqueous extract was centrifuged to remove water-insoluble materials and ultrafilted to remove small molecular substances using an ultrafiltion system including a pump and a hollow fiber microporous membrane cartridge (10 cm I.D x 100 cm length) with molecular weight cutoff 5000, the concentrated fraction (41) was lyophilized to obtain the crude polysaccharide fraction (UFP, brown, 402 g).

Quantitation of total sugar and monosaccharide

Total sugar in UFP was determined spectrophotometrically with the phenol-sulfuric acid reagent at UV 490 nm (UV-2100 ultraviolet spectrophotometer, Unica, China). Monosaccharide was determined with DNS reagent at UV 540 nm. Glucose (98%, purchased from the National Institute for the Control of Pharmaceutical and Biological Products) was used as the standard in both measurements.

Quantitation of amino acid

100.0 mg UFP was hydrolyzed with 2 ml of 6 M hydrochloric acid in a screw-cap vial at 110[degrees]C for 24 h. Amino acid was determined spectrophotometrically with 2% ninhydrin reagent at UV 570 nm (UV-2100 ultraviolet spectrophotometer, Unica, China). Glutamic acid (99.0%, purchased from the National Institute for the Control of Pharmaceutical and Biological Products) was used as the standard.

Quantitation of N content

N content in UFP was determined using a Finsons 1108 Element analyzer (Finsons, Italy).

TLC analysis of sugar composition

UFP (12 mg) was hydrolyzed with 5 ml of 2 M TFA in a screw-cap vial at 110[degrees]C for 6h. After cooling, the solution was neutralized with 2 ml MeOH and concentrated for TLC analysis. Baker-flex microcrystalline cellulose-precoated flexible plates were obtained from J.T. Baker Chemical Co. (Phillipsburg, NJ, USA). TLC analysis was developed twice using butanol-acetic ether-pyridine-water (6:1:5:4, v/v) as the mobile phase. The spots on chromatograms were detected with aniline-o-phthalic acid after heating at 105[degrees]C for 10 min.

HPGPC of molecular weight (MW)

Molecular weight of UFP was determined by HPGPC, using a P230 GPCHPLC system (Elite Co, Dalian, China) and a WellChrom K-2301 Refractive Index Detector (Knauer, Berlin, Germany). 0.7% [Na.sub.2][SO.sub.4] was used as the mobile phase with a flow rate of 0.5 ml/min at 40[degrees]C. A calibration curve was prepared from known MW Dextran standards (D-133800, D-41100, D-10000, D-2500, purchased from the National Institute for the Control of Pharmaceutical and Biological Products).

Animals and castration procedure

Male Wistar rats, 8 weeks of age and weighing 180[+ or -]20g each at the beginning of the experiments were provided by the Animal Center of the Academy of Military Medicines and maintained under standard environmental conditions (air-conditioned room and 12 h/day controlled lighting, and fed with rodent diet and water ad libitum). Animal care and surgery protocols were approved by the Animal Care Committees of the Academy of Military Medicines. All animals were treated in accordance with recognized scientific ethics.

The castration was performed 1 week before the beginning of the experiments. Barbanylum was used for anesthesia. Castration was performed through the scrotum sack, taking out the testicles and the epididymal fat according to the procedure described by Coppenolle et al. (2000). In the sham castration, an incision was made and sutured afterwards in the same area. Both sets of operated animals were treated with penicillin to prevent infection.

Induction and treatment of prostatic hyperplasia

Testosterone propionate (0.5 ml/0.1 ml olive oil per rat) was injected subcutaneously in castrated rats to induce prostatic hyperplasia. Castrated rats were divided randomly into five groups: (A) Control sham castrated group, to which saline solution was given orally and olive oil injected subcutaneously; (B) model group, to which saline solution was given orally and testosterone (0.5 ml/0.1 ml oil per rat) was injected subcutaneously; (C) three UFP groups, to which 250, 125, 62.5 mg/kg body wt. were administrated orally, respectively, and testosterone propionate (0.5 ml/0.1 ml oil per rat) injected subcutaneously; (D) positive control group, to which Qian Lie Kang (QLK, 1000 mg/kg body wt., orally) was given and testosterone propionate (0.5 ml/0.1 ml oil per rat) injected subcutaneously. All rats were treated once a day for 3 weeks.

Twenty-four hours after the last administration, rats were killed and prostates were removed. Each prostate was dissected into two parts: the ventral lobe and the dorsalateral lobe. Each lobe was weighed separately, and the volume was measured using a highly graduated cylinder. Half of the gland tissue from each rat was fixed in a 10% formalin solution for histopathological examination. The other half of the gland was baked in an oven for 24 h to calculate the dry weight index.

Statistical analysis

Data were expressed as mean [+ or -] S.D. and compared using the double-tail Student's test; p < 0.05 was taken as statistically significant.

Results

Fifty kg of roots and stems of Urtica fissa were extracted with hot water and subjected to ultrafiltration to obtain 402 g of crude polysaccharide fraction. The percentage of total sugar in crude fraction was determined to be 61.44% by phenol-sulfuric acid method; the reducing saccharide (including oligo or monosaccharides) content was determined to be 8.04% by the DNS method; and the percentage of polysaccharide (including glycans) in the crude fraction was calculated to be 53% based on percentage of total sugar minus that of reducing sugars. The percentages of amino acid and N content in UFP were determined to be 9.1% and 1.23%, respectively. These results showed that polysaccharide was the main component in crude fraction.

Following total acid hydrolysis of the polysaccharide fraction with TFA, the sugar components were analyzed using TLC and the resultant chromatogram indicated the presence of six monosaccharides, including glucose, galactose, arabinose, xylose, rhamnose and glucuronic acid, according to the spots matching with corresponding monosaccharide standards. Based on the color intensities of these spots, glucose, galactose and arabinose were determined to be the main sugar components. The molecular weight of the polysaccharide fraction was analyzed by HPGPC, and three peaks corresponding to molecular weights of 2.3 x [10.sup.6], 7.5 x [10.sup.4], and 5.9 x [10.sup.3] were found. This result showed that the crude polysaccharide fraction was not homogenous from molecular weight 2.3 x [10.sup.6] to 5.9 x [10.sup.3].

UFP was tolerated by all animals and no side-effects were observed. As shown in Table 1, after inducing by subcutaneous injection of testosterone for 30 days, the ratios of prostate volume-to-body weight (V/W) and prostate weight-to-body weight (W/W) were twice the values among control group rats (p < 0.001). These higher ratios indicated that significant prostatic hyperplasia was induced by testosterone. However, in the UFP and Qian Lie Kang treated groups, the volume and weight index of prostate gland were reduced significantly, as compared with those of the model group. Treatment with UFP at 62.5 mg/kg body wt. notably decreased the volume index by 32%, the wet weight index by 17% and the dry weight index by 23%, respectively. In the high-dose group (UFP at 250 mg/kg body wt.), the indexes of volume, wet weight and dry weight decreased further by 37%, 25% and 33%, respectively (Figs. 1 and 2). These results demonstrated that oral administration of UFP could inhibit prostatic hyperplasia induced by testosterone in terms of total volume and weight.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]
Table 1. Effect of UFP on volume and weight index of castrated rat
prostate gland

Groups Dose B.W (g) Prostate Wet weight Dry weight
 (mg/kg) volume index index
 (ml) (mg/g (mg/g
 B.W) B.W)

Control -- 311.9 0.55 0.186 0.044
 [+ or -] [+ or -] [+ or -] [+ or -]
 12.6 0.07 0.030 0.009

Model -- 275.0 1.47 0.507 0.122
 [+ or -] [+ or -] [+ or -] [+ or -]
 16.5 0.29 0.105 0.030
 ### ### ### ###

QLK 1000 275.8 0.99 0.410 0.090
 [+ or -] [+ or -] [+ or -] [+ or -]
 12.4 0.11 *** 0.081 * 0.022 *

UFP 62.5 274.3 1.00 0.423 0.086
 [+ or -] [+ or -] [+ or -] [+ or -]
 18.9 0.14 *** 0.059 * 0.020 *

UFP 125 275.1 0.98 0.392 0.077
 [+ or -] [+ or -] [+ or -] [+ or -]
 16.0 0.11 *** 0.067 ** 0.013 **

UFP 250 270.3 0.92 0.382 0.075
 [+ or -] [+ or -] [+ or -] [+ or -]
 13.2 0.09 *** 0.074 ** 0.007 ***


Rat prostate consists of two parts: a ventral lobe and a dorsalateral lobe. Each lobe was dissected and weighed separately. As shown in Figs. 1 and 2, wet or dry weights of the lobes of model group rats were increased due to castration and administration of testosterone, and after treatment with UFP and QLK, wet or dry weights of both the ventral and dorsalateral lobes decreased significantly, as compared with model rats.

Histopathological examination (Fig. 3) showed no histopathological change in the prostate glands of control group rats. But in the model group, prostatic epithelial cell proliferation occurred and more fibrotic tissues were found in the interstices of the rat prostate. Some papilla had protruded into cavities. The state of rat prostate glands in the UFP group (250mg/kg body wt.) was improved significantly, as compared with the model group; only a minor proliferation of epithelial cells was found and there was nearly no fibrotic tissue in the interstices of the prostate in the UFP-treated group.

[FIGURE 3 OMITTED]

Discussion

The main active compounds of Urtica remain undetermined, although many compounds were reported in Urtica water or aqueous alcohol extracts, including: fatty acids, sterols, flavonoids, proteins, polysaccharides and lectins (Akbay et al., 2003; Hirano et al., 1994; Schottner et al., 1997; Budzianowski, 1991; Wagner et al., 1989; Chaurasia and Wichtl, 1987; Ganzera et al., 2005). Some macromolecular materials, such as polysaccharide and glycoprotein, are considered to be the pharmacological components. Lichius et al. reported that the polysaccharide fraction of the 20% methanolic extract of Urtica dioica showed an inhibitory effect on the growth of prostatic lymph node carcinoma cells and BPH-model induced by implanting an urogenital sinus (UGS) into the ventral prostate gland of an adult mouse (Lichius et al., 1999a, b). Urtica dioica agglutinin (UDA), a lectin from stinging nettle roots, was reported to directly inhibit cell proliferation of Hela cells and block the binding of EGF to its receptor, and was regarded to be responsible for inhibiting effects in BPH treatment (Wagner et al., 1994).

In this study, the water extract of Urtica fissa was subjected to ultrafiltration in order to remove the monosaccharides or oligosaccharides, which were decreased to 8% in UFP. Amino acid and N contents in UFP only reached 9.1% and 1.23%, respectively, and polysaccharide is considered to be the main active component in UFP. Since protein in Urtica dioica was involved in the inhibition of prostatic cell proliferation (Konrad et al., 2000; Wagner et al., 1994) and fibrotic tissues (Durak et al., 2004). Proteins or glycoproteins in UFP might be responsible for the synergy effects in inhibiting BPH.

Thus far, only the root extracts of Urtica dioica have been reported to show antiprostatic activities and are used as medicine in Europe. Extracts from leaves of Urtica dioica are used as antiinflammatory remedies in rheumatoid arthritis (Schulze-Tanzil et al., 2002; Riehemann et al., 1999). This is the first report about biological effects of the crude polysaccharide fraction from Urtica fissa roots and stems on BPH.

In conclusion, a 3-week treatment with testosterone can induce rat prostatic hyperplasia by increasing the indexes of prostatic volume and weight, and enlarging both the ventral and dorsalateral prostatic lobes. These changes could be inhibited by oral administration of the crude polysaccharide fraction of Urtica fissa. Further study might be required for exploring the structure of active polysaccharides and their molecular mechanisms. This study might provide a foundation for phytotherapy using Urtica fissa for benign prostate hyperplasia.

Acknowledgment

We thank Prof. Liu Cun-sheng of Beijing University of Chinese Traditional Medicine for plant identification.

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Q. Zhang *, L. Li, L. Liu, Y. Li, L. Yuan, L. Song, Z. Wu

Beijing Institute of Radiation Medicine, Taiping Road 27, Beijing 100850, China

* Corresponding author. Tel./fax: + 86 1066932270.

E-mail address: qinglz@yahoo.com (Q. Zhang).
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Author:Zhang, Q.; Li, L.; Liu, L.; Li, Y.; Yuan, L.; Song, L.; Wu, Z.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Sep 1, 2008
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