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Cheng HJ et al / Act a Pharmacol Sin 2002 Nov; 23 (11): 1035-1039  2002, Act a Pharmacologica Si nica ISSN 1671-4083 Shanghai Institute of Materia Medica Chinese Academy of Sciences http:/ /www.ChinaPhar.com chronic prostatitis or infertility with chronic prostatitis CHEN Hong-Jie 1 , WANG Zhi-Ping 2 , CHEN Yi-Rong, QIN Da-Shan, FU Sheng-Jun, MA Bao-Liang 1 Now in 1st Renmin Hospital of Lanzhou, Lanzhou 730050, China. 2 Corr espondence to Prof WANG Zhi-Ping. Received 2001- 12-24 Accepted 2002-08-03 Institute of Urology, 2nd Affiliated Hospital of Lanzhou medical College, Lanzhou 730030, China KEY WORDS prostatitis; infertility; free radicals; pollen

ABSTRACT

AIM: To determine the drug action mechanism of pollen extract EA-10, P5 on the treatment of
chronic prostatitis (CP) or infertility with CP. METHODS: Malondiadehyde (MDA), super oxide
dismutase (SOD), and nitrogen monoxide (NO) were measured by biochemical assay, and zinc
content was assayed by atomical spectrophotography in the pre-treatment and post-treatment of CP or
infertility with CP. RESULTS: Compared with control group, leukocytes in expressed prostatic
secretion (LEPS), MDA, and NO were increased, and zinc content and SOD were decreased
significantly in the pre-treatment of CP. After the treatment, LEPS was improved, and MDA and NO
were reduced, while zinc content were increased apparently and the alteration of SOD was not evident
(P>0.05). In the pre-treatment of infertility with CP, LEPS, MDA, NO, sperm viability, and seminal
leukocytes were obviously higher and seminal plasma SOD, zinc content, and sperm motility were
obviously lower than those in control group. After the treatment, LEPS, sperm motility, and sperm
viability were improved, MDA, NO, and seminal leukocytes were decreased, SOD and zinc content
were increased markedly. CONCLUSION: There was inter-correlation between oxygen free radicals
(OFR) and occurrence, development, and recovery of CP; Change of OFR may be involved in the
drug action mechanism of EA-10, P5 in the treatment of CP or infertility with CP.

INTRODUCTION
Chronic prostatitis (CP) is one of t he most common diseases in andrology. Its therapeutic efficacy is
not very satisfactory. Recent studies showed that CP might defect semen quality. Thus, it is significant
to make an investigation of pathogenesis and medication of CP.
Oxygen free radical s (OFR) which causes tissue damage by lipid peroxidation (LPO)[1], includes
mainly super oxide anion (O2 ), hydrogen peroxide (H2O2 ), hydroxyl free radical (·OH), and nitrogen
monoxide (NO). LPO has yielded several types of secondary free radicals and a large number of reactive compounds (including MDA), resulting in the destruction of cellular portion. Of course, cells are equipped with various antioxidants, such as vitamin E, vitamin C, glutathione (GSH), super oxide dismutase (SOD), catalase (CAT), and so on. These can scavenge supernumerary OFR and protect organism from cytotoxic effect of OFR[2,3]. In addition, there was apparent negative correlation between semen OFR level and semen quality, but with the increasing of semen OFR level and pro-longing of contact time between OFR and sperm, sperm vital force would obviously decrease [4,5]. Studies also showed seminal MDA might be increased apparently in chronic bacterial prostatitis, resulting in the influence of sperm vitality and sperm motility [6,7]. These data indicated that OFR played an important role in pathogenesis of CP and infertility. EA-10, P5 is regarded as a satisfactory drug in the treatment of CP. At present, it is still unknown that whether OFR, antioxidase, and zinc content in semen will be regulated in t he treatment of CP or infertility with CP by EA-10, P5. Therefore, we investigated whether EA-10, P5 could inhibit LPO, and thus to obtain the primary conclusion about drug action mechanism of EA-10, P5 in our treatment.

MATERIALS AND METHODS

Population All 68 cases of CP (group I) and 63 cases of infertility with CP (group II) were
divided into two groups, which were then subdivided into three treatment subgroups respectively
(group A: EA-10, P5 + Roxithromycin, group B: EA-10, P5 alone, and group C: Roxithromycin alone).
Twenty cases who were normal healthy donors of proven fertility were used as control group. The treatment period was four weeks. Group A received EA-10, P5 (product from Sweden Pharmacia Allergon AB, 375 mg/pill) and roxithromycin (150 mg/pill) twice daily. Group B-C received respectively EA-10, P5 and Roxithromycin twice daily. During the treatment, all 131 cases were treated with sitzing bath in hot water and controlled diet (wine and pungent diet prohibited). Semen samples and treatment Semen samples were obtained from all cases by
masturbation after 3 d of abstinence. Samples were incubated for 20 min in 37 ºC warm bath box.
Firstly, regular semen analysis and seminal MDA content were analyzed after semen has been
liquefied completely; Secondly, liquefied se-men was centrifuged at 1000×g for 10 min, and seminal
plasma was used to determine the content of NO and SOD. Finally, surplus seminal plasma was
frozen at -20 ºC until further use for zinc content assay.
Determination of seminal MDA content and SOD activity Seminal MDA
content was determined by thiobarbituric acid (TBA) method [8]. SOD activity was measured as the
inhibition of nitroblue tetrazolium reduction due to super oxide anion generation by xanthine plus
xanthine
oxidase [9].
Zinc and NO content in seminal plasma assay Zinc content was assayed by a
method based on
atomical spectrophotography [10]. The NO concentration was estimated by a method based on nitrite
salt response with sulfanilamide to from diazole, which could appear purplish red color reacting with
naphthalene ethylenediamine in the acid conditions. The absorbance of 530 nm was measured [11].
Semen parameters All semen analysis adopt with color quality analysis system of WLJY-
9000, which was devised by skill-trade Company Weili Peking. All parameters were settled down to
refer to standard of World Health Organization (WHO) [12].
Statistical Date were expressed as mean ±SD and analyzed with t-test. Value of P<0.05 was
considered to be statistically significant.


Changes in symptom and LEPS in CP or infertility with CP After the
treatment by EA-10,
P5 +Roxithromycin, EA-10, P5 alone, and roxithromyc alone in CP or infertility with CP, remissive
rate of symptom was 92 %, 66.67 %, 68.17 %, and 90 %, 61.91 %, 63.64 %, while effective rate of
LEPS was 88 %, 57. 14 %, 59.09 %, and 85 %, 52. 38 %, 54. 55 %, respectively. Therapeutic
efficacy in group A was significantly higher than that in group B or C (P<0. 01) (Tab 1, 2).
Tab 1. Changes in symptom and LEPS in different treated groups of CP. bP<0.05 vs EA-10,
P +Roxithromycin groups.

__________________________________________________________________________________ Treatment . Efficiency Percent/% Efficiency Percent/ % EA-10, P5+Roxithromycin 25 23 92 22 88 Roxithromycin 22 15 68.17b 13 59.09b
__________________________________________________________________________________
Tab 2. Changes in the symptom and LEPS in different treated groups of infertility with CP.
bP<0.05 vs
EA-10, P
+roxithromycin groups.
__________________________________________________________________________________
Treatment
Efficiency Percent/% Efficiency Percent/% Roxithromycin 22 14 63.64b 12 54.55b
__________________________________________________________________________________
Changes in LEPS, MDA, SOD, Zinc content, and NO in CP Compared with
control group, LEPS, MDA, and NO were increased, while zinc content and SOD were decreased
significantly in the pre-treatment (P<0.01). After the treatment, LEPS and zinc content were
improved, while MDA and NO were decreased apparently vs pre-treatment (P<0.01), but there was
no obvious alteration of SOD (P>0.05) (Tab 3).
Tab 3. Changes in LEPS, MDA, SOD, Zn 2+ content, and NO in different treated groups of CP.
Mean ±SD. bP<0.05, cP<0. 01 vs
control. dP>0.05, fP<0.01 vs pre-treatment at the same
group. hP<0.05 vs
EA-10, P +Roxithromycin group.
__________________________________________________________________________________ EA-10, P5 (n=21) Roxithromycin (n=22) Control (n=20) (n=25) . pre-treat post-treat pre- treat post-treat pre-treat post-treat LEPS /Hp 3.4±2.1 25±16b 5.0±2.8f 23±13b 7±4f 25±14b 7±4f MDA/μmol-L-1 4.1±1.1 8.3±1.9c 4.3±1.4f 8.3±1.7c 5.4±1.6bf h 8.4±1.8c 5.2±1.2bf h Zn2+/mmol-L-1 2.3±0.6 1.2±0.4b 1.8±0.5f 1.2±0.5b 1.6±0.5f 1.2±0.4b 1.6±0.5f SOD/kU-L-1 20±119 850±118b 851±122d 838±110b 840±113d 829±120b 831±123d NO/μmol-L-1 4.6±1.6 63±20c 39±16bf 63±20c 45±18bf 63±21c 47±18bf __________________________________________________________________________________
Changes in LEPS, MDA, SOD, Zinc content, NO, and semen parameters in
infertility

with CP In the pre-treatment, LEPS, MDA, NO, sperm viability, and seminal leukocytes were
obviously higher and SOD, zinc content, and sperm motility were obviously lower than those in
controlled group (P<0.01). After the treatment, LEPS, SOD, zinc content, sperm motility, and sperm
viability were improved and MDA, NO, and seminal leukocytes were decreased significantly
(P<0.01). Compared with the pre-treatment, MDA levels and seminal leukocytes were reduced
significantly in group A than these in group B or C in the post-treatment (P<0.01) (Tab 4).

Tab 4. Changes in LEPS, MDA, SOD, Zinc content, NO, and Semen parameters in different
treated groups of infertility with CP. Mean±SD. aP>0.05, bP<0.05, cP<0.01 vs
control.
dP>0.05, eP<0.05, fP<0.01 vs
pre- treatment at the same group. hP<0. 05 vs EA-10,
P +Roxithromycin groups.

____________________________________________________________________
____________
Control
(n=20) (n=25) pre-treat post-treat pre-treat post-treat pre-treat post-treat LEPS/Hp 3.4±2.1 23±13c 6±4f 23±12c 7±5f 23±12c 6±4f MDA/μmol-L-1 4.1±1.1 9.2±1.6c 5.5±2.1f 9.1±1.9c 7.5±2.4beh 9.1±1.7c 7.2±2.5 bch Zn2+/mmol-L-1 2.3±0.6 1.1±0.4c 1.6±0.4bf 1.1±0.4c 1.5±0.4bf 1.1±0.3c 1.4±0.4bf SOD/kU-L-1 920±119 653±115c 736±125bf 663±91c 727±104bf 660±97c 722±109b f NO/μmol-L-1 4.6±1.6 78±20c 55±18bf 76±27c 63±27bf 77±25c 61±21bf
10-9 x Sperm density/L-1 76±24 82±49a 79±46ad 79±42a 77±41ad 80±41a 79±40ad
Sperm motility/% 75±12 37±14c 46±14bf 38±17c 43±19bf 37±16c 43±18bf
Sperm viability/% 14±8 36±14c 24±10bf 34±14c 28±11bf 34±13c 28±11bf
10-9x Seminal leukocytes/L-1 0.5±0.3 1.6±0.9c 0.7±0.4af 1.6±0.8c 0.9±0.4bf 1.6±0.8c 0.9±0.5bf

DISCUSSION

In this test, we have used EA-10, P5 and roxithromycin to treat CP and infertility with CP.
Roxithromycin has a good effect to chlamydia besides much of Gram-negative bacteria [13]. Therapeutic efficacy was lower in our works than that in literature. But our therapeutic efficacy was still satisfactory. We considered that the reason may be as follows: (1) Chronic bacterial prostatitis may be selected in all the chosen cases, which might influence therapeutic efficacy of EA-10, P5. (2) The treatment period was shorter compared with that illustrated in literature. In addition, we have found that therapeutic efficacy in group A was better than in group B or C. This indicated that EA-10, P5 should be used together with effective antibiotic in the treatment of CP. Some studies have proved that OFR was related to occurrence and development of CP[3- 4,14 ]. In our studies, MDA was higher and SOD was lower significantly in the pre-treatment of CP than those in the control group, which suggested that there be an increase of OFR, a decrease of antioxidation, and reinforce a of LPO. But MDA was decreased after the treatment, indicated that OFR was scavenged massively and LPO was obviously inhibited. Similarly, MDA was higher and SOD was lower significantly in pre-treatment of infertility with CP than those in the control group, which suggested that oxidation be increased and antioxidation be decreased in semen. At the same time, we discovered that sperm motility was declined and sperm viability was raised significantly. But after the treatment, MDA was decreased and SOD was increased significantly than those in the pre-treatment (P<0.01), accompanying with improvement of sperm motility and sperm viability apparently. This indicated that LPO was inhibited and antioxidation was reinforced. From the result above, we believed that EA-10, P5 could reduce LPO and enhance antioxidation in the treatment of CP or infertility with CP. In our treatment, antibiotic and EA-10, P5 were used not only to cure CP but also to improve semen quality. We found that EA-10, P5 had an effect on weakening oxidative stress and increasing antioxidation in prostatic secret ion and semen. This suggested that change of OFR may be involved in the drug action mechanism of EA-10, P5 in the treatment of CP or infertility with CP. At present, it is known that ferulic acid was an antioxidant containing phenolic hydroxyl [15]; and P5, one of valid portion in pollen extract EA-10, P5, may have anti-oxidative effect owing to providing phenolic hydroxyl too. Nevertheless this view still needs to be confirmed by more investigation. It was reported that zinc content in prostatic secretion and semen was higher than in other organ and body fluid, which showed that zinc played an important role in keeping function of prostate and other accessory sex glands. Our studies showed that zinc content was increased accompanying with improvement of an illness state. EA-10, P5 can enhance zinc content in seminal plasma, which may be related to improve local circumstance.
In summary, all these results could provide us with a possible therapeutics approach to treat infertility
with CP. In order to improve therapeutic efficacy, anti-infection and anti-oxidation should be adopted
in the treatment of CP or infertility with CP.

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