Gonadal Toxicity of an Ethanol Extract of Psoralea Corylifolia in a Rat 90-Day Repeated Dose Study

The Journal of Toxicological Sciences, 97 Vol.27, No.2, 97-105, 2002

GONADAL TOXICITY OF AN ETHANOL EXTRACT OF PSORALEA CORYLIFOLIA IN A RAT 90-DAY REPEATED DOSE STUDY

Tamotsu TAKIZAWA1, Toshio IMAI1, Kunitoshi MITSUMORI1,2, Hisayoshi TAKAGI1, Hiroshi ONODERA1, Kazuo YASUHARA1, Makoto UEDA1, Toru TAMURA1 and Masao HIROSE1

1Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan 2Laboratory of Veterinary Pathology, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan

(Received November 7, 2001; Accepted January 30, 2002)

ABSTRACT — Ethanol extracts of seeds of Psoralea corylifolia are proposed as food additives for processed food preservation. An extract was administered by admixing into diet at concentrations of 0, 0.375, 0.75, 1.5 or 3.0% to 10 male and 10 female F344 rats each for 90 days to evaluate its toxicity. Body weight gain, food consumption and food conversion efficiency (body weight gain per food consumption) were lower in the extract-treated animals, except for the 0.375% males, as compared to the control animals. Absolute and/or relative testes weights in the 1.5 and 3.0% groups and those of ovaries in the 3.0% group were significantly (p<0.01) lower than in the control group. On histopathological examination, seminiferous tubular atrophy and Leydig cell atrophy in the testes, and epithelial cell atrophy in the seminal vesicles and prostate were observed in the 1.5 and 3.0% males. Decrease in the number of corpora lutea associated with frequent necrotic follicles in the ovaries in the 1.5 and 3.0% females and less frequent endometrial glands in the uterus in the 3.0% females were also detected. These results might suggest disruption of the hypothalamus-pituitary-gonadal axis in Psoralea corylifolia-treated rats as possible mechanisms underlying this gonadal toxicity.

KEY WORDS: Psoralea corylifolia, Gonadal toxicity

INTRODUCTION for preservation of some processed foods such as grilled or fried chicken or pickles in Japan. However, The seeds of Psoralea corylifolia (P. corylifolia) the biological activities listed above include adverse as are used as a folklore medicine in India and China, and well as desirable effects. Since the available toxicolog- a variety of biological effects of extract or constituents ical data for such an extract are limited, as part of a have been reported. These included antihyperglycemic comprehensive safety evaluation, a 90-day repeated (Krenisky et al., 1999) and anti-inflammatory effects dose toxicity study was performed in rats of both sexes. (Ferrandiz et al., 1996) as well as inhibition of mitochondrial lipid peroxidation (Haraguchi et al., 2000), MATERIALS AND METHODS antibacterial potential (Kaul, 1976) and cytotoxicity (Kubo et al., 1989, Iwamura et al., 1989). Modulation Test substance of the immune system and tumor growth (Latha and An ethanol extract from seeds of P. corylifolia Panikkar, 1999; Latha et al., 2000), inhibition of DNA was obtained from Higashimaru-shoyu Co. (Osaka, polymerase and topoisomerase II activities (Sun et al., Japan). The sample was a brown liquid with a herbal 1998), and antimutagenic effects (Wall et al., 1988a, medicine odor and comprised of approximately 57% 1988b) have been also shown. Ethanol extract of seeds liquid components (water and ethanol) and 43% resi- of P.corylifolia has been proposed for food additive use dues after drying. The extract was weekly admixed into

Correspondence: Toshio IMAI

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T. TAKIZAWA et al. basaldiet(CRF-1®, Oriental Yeast Co., Tokyo, Japan) ing parameters were performed by SRL Inc. (Tokyo, for dietary exposure. Japan): total protein, albumin, albumin/globulin ratio, aspartate aminotransferase, alanine aminotransferase, Experimental animals alkaline phosphatase, γ-glutamyl transpeptidase A total of 50 male and 50 female 5-week-old (γ GTP), total bilirubin, total cholesterol, urea nitrogen F344/DuCrj rats were purchased from Charles River (BUN), creatinine, sodium, potassium, chloride, cal- Japan Inc. (Kanagawa, Japan) and used after a one- cium, and inorganic phosphorus. Complete necropsy week acclimatization. Animals were housed at a maxi- was performed and the brain, heart, lungs, liver, spleen, mum of five in a polycarbonate cage with soft chip adrenal glands, kidneys, testes, ovaries and uterus were bedding in a room controlled for light-dark cycle (12- weighed. These organs and the following organs and 12 hr, lights on 7:00-19:00), ventilation (air-exchange tissues were fixed in 10% neutral buffered formalin, rate of 18 times per hr), temperature (23-25°C) and rel- and then paraffin-embedded sections of all organs and ative humidity (50-60%) during the study. The cages tissues were stained with hematoxylin and eosin (H.E.) and the chip bedding were exchanged twice a week. for histopathological examination; skin, mammary Each animal had free access to a powdered diet (CRF- gland, sternum with marrow, femur with marrow, thy- 1®, Oriental Yeast Co., Tokyo, Japan) with or without mus, mandibular and mesenteric lymph nodes, sub- the test substance and tap water. The animals were ran- mandibular glands, sublingual glands, aorta, trachea, domly allocated to 5 groups of 10 male and 10 female tongue, esophagus, stomach, small and large intestine, rats each based on their body weights. pancreas, urinary bladder, epididymides, seminal vesicles, prostate gland, bulbourethral gland, vagina, pitu- Study design itary gland, thyroid glands, parathyroid glands, spinal In a 2-week preliminary study of the extract of P. cord, trigeminal nerve, optic nerve, sciatic nerve, Har- corylifolia at a dose of 5% in the diet, body weight gain derian glands, and femoral skeletal muscle. One of the and food consumption were significantly decreased testes for five animals in each group was fixed in compared to the control group. From these results, Bouin’s fixative for quantitative analysis of seminifer- male and female rats were fed a diet containing the ous tubules: counting numbers of round and elongated extract at concentrations of 0, 0.375, 0.75, 1.5 or 3.0% spermatids, and Sertoli cells for five seminiferous for 90 days in the present study. tubules of stage VII. Histopathological examination General condition was observed daily and body was in principle performed on the animals of the con- weights were measured once a week during the study trol and highest-dose groups, but the gonads and acces- period. The amounts of supplied and residual diet were sorygonadswerealsoexaminedinthelower-dose weighed weekly in order to calculate the average daily groups. consumption of each week, and then the overall mean throughout the treatment period was calculated from Statistical analysis the determined weekly food consumption. Rats were Body weight, food consumption, and organ fasted overnight at the completion of the treatment weights as well as the results of hematology, serum period, and then blood samples were collected from the chemistry and testicular morphometry were analyzed abdominal aorta under ether anesthesia for hematology by one-way analysis of variance for homogeneity fol- and serum chemistry. The following hematological lowed by the two-tailed Dunnett’s test (Dunnett, 1955) parameters were analyzed using an automatic analyzer against the basal diet control group. Significance was (Sysmex M-2000, Toa Medical Electronics Co., inferred at either the 5% or 1% levels. Hyogo, Japan): red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscu- RESULTS lar hemoglobin, mean corpuscular hemoglobin concentration, white blood cell count, and platelet count. Neither deaths nor deterioration in general condi- Blood smears were processed for Giemsa staining and tion were observed in any animals of the treated and counting of erythroblasts and differential leukocytes control groups. Body weight gain was significantly (band and segmented neutrophils, eosinophils, baso- lower in males of the 3.0, 1.5 and 0.75% groups and phils, lymphocytes and monocytes) using a semiauto- females of all treated groups as compared to the control matic analyzer (Microx, Tateishi Electric co., Tokyo, animals (Fig. 1). Data for food consumption, food con- Japan). Serum chemistry measurements of the follow- version efficiency (body weight gain per food con-

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Gonadal toxicity of P. corylifolia extract in rats. sumption) and intake of the extract are shown in Table flattened epithelia. Furthermore a decrease in the num- 1. Food consumption and food conversion efficiency ber of corpora lutea associated with increased necrotic were lower in all treated groups except for the 0.375% follicles was observed in the ovaries of females in the males. There were no abnormalities attributable to 3.0 and 1.5% groups. In the uterus, endometrial glands treatment in the hematological findings. In serum were less frequent in the 3.0% group. There were no chemistry, there were increases in BUN and γ GTP in treatment-related changes evident in other tissues or males of the 3.0% group and females of the 3.0 and organs including the pituitary. 1.5% groups (Table 2). Elevated γ GTP values were also noted for the 0.75% females. Absolute and relative DISCUSSION weights of the testes and ovaries were significantly lowered in the 3.0% males and females, respectively, In the present study, an extract of P. corylifolia and absolute testes weights were decreased in the 1.5% exhibited distinct toxic effects on the gonads at con- males (Table 3). Increase in relative weights of the centrations of 1.5 and 3.0% in the diet. These included gonads reflecting the reduction of terminal body decrease in testicular weight, Leydig cell atrophy and weights was observed in the lower doses. Histopatho- seminiferous tubular atrophy of the testes, epithelial logical examination revealed Leydig cell atrophy and cell atrophy of the accessory gonads in males, and atrophy of seminiferous tubules (Table 4). In the most decrease in the weights and suppression of luteiniza- marked cases, round and elongated spermatids disap- tion of the ovaries and less frequent endometrial glands peared and spermatocytes were degenerated, with fea- in the uterus in females. A number of constituents of P. tures of pyknotic nucleus and uniformly condensed corylifolia extracts have been reported (Wall et cytoplasm, and exfoliated in seminiferous tubules of al.,1988a, 1988b; Kong et al., 2001). One of them, pso- the 3.0% males (Photo 1 and 2). In the 1.5% males, the ralen, is a possible testicular and ovarian toxicant. numbers of round and elongated spermatids were sig- Massive doses of 8-methoxypsoralen, a member of the nificantly decreased (Table 5), and degenerated fea- psoralen family, was found to induce testicular and tures of spermatocytes and round spermatids were prostate atrophy (NTP Technical Report, 1988), and evident. These testicular changes were accompanied psoralen is known to decrease numbers of implantation by atrophy of epithelial cells in the ventral and dorso- sites, pups and corpora lutea in pregnant females lateral prostate and seminal vesicles, characterized by (Diawara et al., 1999). Although the content of psor-

Fig. 1. Body weight changes for rats fed a diet containing a P. corylifolia extract for 90 days.

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T. TAKIZAWA et al. alen in the extract used here is uncertain, participation a few days after treatment, followed by disorder of of the constituent in the observed gonadal toxicity is other stages and ultimately reversible infertility (Kerr clearly conceivable. In addition, constituents such as et al., 1993; Morris, 1996). The findings for seminifer- bakuchiol, corylifolin and bakuchicin, which inhibit ous tubules in our present experiment resemble those DNA polymerase or topoisomerase II activities (Sun et induced by EDS. Deleterious effects of EDS on sper- al., 1998), should be taken into account as potential matogenesis are attenuated by testosterone supplemen- testicular toxicants. Among them, bakuchiol is a major tation (Kerr et al., 1993), and the prostate and seminal constituent of the extract used, and comprised as much vesicles are also androgen-dependent organs as 20% of the constituents. Since cell population- (Vanderschueren et al., 2000). Therefore, Leydig cells linked expression of DNA polymerase subtypes during are likely to be a prior target for testicular toxicity of spermatogenesis (Kamel et al., 1997; Garcia-Diaz et the extract in the present study, and the toxic effects on al., 2000) and preferential attack of primary spermato- the seminiferous tubules and accessory gonads may be cytes by etoposide, a topoisomerase II inhibitor, have a result of androgen deprivation. Androgen deprivation been reported (Russell et al., 1998), bakuchiol is a can- is also inducible by the disruption of the hypothala- didate for causing derangement of spermatogenesis. mus-pituitary-gonadal axis. Treatment with an antago- On the other hand, the atrophy of Leydig cells nist to gonadotropin-releasing hormone [GnRH] observed in the present study points to a decline in (Sinha-Hikim and Swerdloff, 1993), immunoneutral- function for maintaining spermatogenesis (O’Donnell ization of luteinizing hormone [LH] (Marathe et al. et al., 1994). Ethan-1,2-dimethansulfonate (EDS), a 1995) and hypophysectomy (Russell et al., 1993; toxicant which specifically targets Leydig cells in the Tapanainen et al., 1993) all result in atrophy of Leydig rats (Morris et al., 1986), elicits degeneration of cells and degenerative changes of the seminiferous pachytene spermatocytes and step 7 and 19 spermatids tubules as with EDS-treatment. Regarding gonadal at stages VII and/or VIII in seminiferous tubules within toxicity in females, the uterine change is likely related

Table 1. Average food consumption, food conversion efficiency and test substance intake in rats fed a diet containing P. corylifolia extract for 90 days. Male Female Dose level (%) 0 (Control) 0.375 0.75 1.5 3.0 0 (Control) 0.375 0.75 1.5 3.0 Food consumption (g/rat/day) Week 112.5 11.9 9.5 7.7 5.7 9.1 7.8 6.5 5.7 3.6 Week 214.2 14.4 13.0 11.2 9.7 9.5 8.7 8.5 9.1 8.6 Week 315.3 15.0 13.1 11.2 11.0 10.3 8.5 7.8 9.5 8.4 Week 415.8 15.5 15.8 12.9 9.0 9.8 8.7 8.5 8.0 6.9 Week 59.1 15.4 9.9 7.6 7.2 8.1 6.7 7.2 6.1 4.3 Week 66.0 18.7 10.1 4.9 6.3 8.4 7.2 7.6 7.6 7.5 Week 718.2 14.9 13.6 8.4 9.1 9.0 8.4 9.3 7.7 6.9 Week 816.4 11.9 7.3 6.3 7.9 9.4 6.4 7.7 7.0 7.6 Week 915.0 13.0 14.8 12.2 10.2 9.5 8.6 8.2 8.2 8.8 Week 1011.2 12.3 11.8 9.2 9.9 8.6 7.2 8.2 7.4 8.2 Week 1117.4 14.7 14.5 12.0 9.5 9.5 8.0 8.3 7.7 9.2 Week 128.5 13.3 10.4 5.6 8.5 9.0 5.6 7.3 6.3 6.5 Overall average13.3 14.3 12.0 9.1 8.7 9.2 7.6 7.9 7.5 7.2 Food conversion efficiency 0.167 0.159 0.144 0.107 0.074 0.110 0.095 0.094 0.079 0.057 Test substance intake (mg/kg/day) 0 253 486 890 1938 0 237 486 997 2115

Food conversion efficiency; body weight gain/food consumption.

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Gonadal toxicity of P. corylifolia extract in rats. to the ovarian changes. Atrophic changes of the ovaries al., 1984, Dukes et al., 1994). In addition, ovarian and uterus are produced by various chemicals known functions are regulated by pituitary-derived LH and as anticancer and antiestrogenic agents (Shiromizu et follicle stimulating hormone, and immunological or biological antagonism to GnRH suppresses ovulation Table 2. Data for γ GTP and BUN in the serum of rats of the follicles (Arai et al., 1998; Kovacs et al., 1989). treated with P. corylifolia extract for 90 days. Therefore, a hypothesis of the disruption of the hypo- Doses N GTP BUN thalamus-pituitary-gonadal axis draws an attention as a (%) (IU/L) (mg/dL) possible common mechanism that couples the gonadal toxicity of the extract in both sexes. Further studies Male should be done to clarify the mechanisms of the 0 (control) 10 2.0 0.0 17.2 8.0 gonadal toxicity of the extract. 0.375 10 2.0 0.0 16.6 1.1 Administration of the extract to rats induced a 0.75 10 2.0 0.0 16.7 0.7 reduction of body weight gain in males given 0.75% 1.5 10 3.0 1.3 20.1 5.2 and above and females given 0.375% and greater 3.0 10 12.0 2.1** 24.3 4.0** doses. This became more pronounced with increases in Female the concentration and was principally associated with a 0 (control) 9 2.0 0.0 19.5 1.7 decrease in food consumption. Reduced palatability of 0.375 10 2.2 0.4 19.7 3.0 the diet, perhaps due to the peculiar odor of the extract, 0.75 10 5.0 0.7** 21.9 1.8 might thus be an important factor. However, as food 1.5 10 6.2 0.8** 23.1 3.3** efficiency was also decreased in the higher dose 3.0 10 14.2 0.9** 25.8 1.9** groups, some effects on metabolism such as anti- Values are mean S.D. hyperglycemic action (Krenisky et al., 1999) could N: number of animals. account at least in part for the reduction in body weight **: Significantly different from the control at p<0.01. gain. The decreased body weight gain is unlikely to be related to gonadal toxicity, since a similar reduction in

Table 3. Organ weights for rats fed a diet containing P. corylifolia extract for 90 days. Dose level (%) 0 (control) 0.375 0.75 1.5 3.0

Male N 10 10 10 10 10

Body weight (g) 280.2 16.5 287.7 17.8 240.5 9.7** 178.8 14.0** 155.9 10.8** Testes Abs (g) 3.00 0.13 3.04 0.10 2.99 0.10 2.29 0.43** 0.53 0.31** Rel (%) 1.08 0.07 1.06 0.04 1.25 0.06* 1.27 0.18** 0.33 0.18**

Female N 10 10 10 10 10

Body weight (g) 163.0 4.8 140.7 8.4** 143.2 3.3** 131.9 4.4** 115.0 5.1** Ovaries Abs (g) 0.051 0.006 0.060 0.010 0.057 0.014 0.043 0.005 0.021 0.002** Rel (%) 0.031 0.003 0.042 0.007** 0.040 0.009** 0.033 0.004 0.018 0.002** Uterus Abs (g) 0.41 0.09 0.26 0.06** 0.31 0.06** 0.33 0.04* 0.34 0.02* Rel (%) 0.26 0.06 0.18 0.04** 0.22 0.04 0.25 0.03 0.30 0.02 Values are mean S.D. N: number of animals, Abs: absolute weight, Rel: relative weight. *, **: Significantly different from the control at p<0.05 and p<0.01, respectively.

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Table 4. Incidences of histopathological findings in rats fed a diet containing P. corylifolia extract for 90 days.

Dose level (%) 0 (Control) 0.375 0.75 1.5 3.0

Male Testis Atrophy of Leydig cells 0/10 0/10 0/10 6/10 10/10 Atrophy of seminiferous tubules 0/10 0/10 0/10 4/10 10/10

Seminal vesicles Atrophy of epithelial cells 0/10 0/10 0/10 6/10 10/10

Prostate gland Atrophy of epithelial cells 0/10 0/10 0/10 6/10 10/10

Female Ovaries Decreased corpora lutea with 0/10 0/10 0/10 9/10 10/10 frequent necrotic follicles

Uterus Less frequent endometrial glands 0/10 0/10 0/10 0/10 10/10 Data are numbers of animals with lesions/total examined.

Photo 1. Seminiferous tubules and interstitium (Leydig cells) of a control male, H.E., ×80.

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Photo 2. Seminiferous tubules and interstitium of a 3.0% group male. Interstitial Leydig cells are atrophied. The seminiferous tubules are small in size and their numbers of epithelial cells are severely decreased. Some remaining degenerated germ cells have a pyknotic nucleus and are exfoliated into the lumen of the seminiferous tubules, H.E., ×80.

Table 5. Morphometry of seminiferous tubules in male rats fed a diet containing P. corylifolia extract for 90 days.

Doses No. of animals Number of spermatids per Sertoli cell

(%) examined Round Elongated

0 (Control) 5 7.44 0.50 6.43 0.48 0.375 5 7.53 0.34 6.85 0.34 0.75 5 7.02 0.53 6.44 0.33 1.5 5 6.32 0.65** 4.97 1.75 a(5.96 0.59)** a(4.12 1.84)**

3.0 5NENE Values are mean S.D. a; Mean S.D calculated with data for 3 animals with atrophy of seminiferous tubules. NE; Not examined. **: Significantly different from the control at p<0.01.

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T. TAKIZAWA et al. body weight gain caused by other factors including eukaryotic DNA polymerase with a potential feed restriction (Seki et al., 1997; Levin et al., 1993) is role in meiosis. J. Mol. Biol., 301, 851-867. not associated with a decrease in relative gonadal Haraguchi, H., Inoue, J., Tamura, Y. and Mizutani, K. weights or histopathological changes observed in the (2000): Inhibition of mitochondrial lipid peroxi- present study. In the present study, serum biochemistry dation by bakuchiol, a meroterpene from Psor- revealed increases in γ GTP and BUN in the treated alea corylifolia. Planta Med., 66, 569-571. groups. However, histopathological examination Iwamura, J., Dohi, T., Tanaka, H., Odani, T. and Kubo, revealed no changes attributable to the treatment in the M. (1989): Cytotoxicity of corylifoliae fructus. liver or kidney. Therefore, the changes of the parame- II Cytotoxicity of bakuchiol and the analogues. ters are of uncertain toxicological significance. Yakugaku Zasshi, 109, 962-965 (in Japanese). In conclusion, the present study demonstrated an Kamel, D., Mackey, Z.B., Sjöblom, T., Walter, C.A., ethanol extract of P. corylifolia to exhibit toxic effects McCarrey, J.R., Uitto, L., Palosaari, H., on gonads and accessory gonads at 1.5% or higher Lähdetie, J., Tomkinson, A.E. and Syväoja, J.E. doses. The non observed adverse effect level (NOAEL) (1997): Role of deoxyribonucleic acid poly- was estimated to be 0.375% (253 mg/kg body weight/ merase ε in spermatogenesis in mice. Biol. day) for males and lower than 0.375% (237 mg/kg Reprod., 57, 1367-1374. body weight/day) for females on the basis of reduction Kaul, V.R. (1976): Kinetics of the anti-staphylococcal in body weight gain and food consumption. activity of bakuchiol in vitro. Arzneim.-Forsch./ Drug Res., 26, 486-489 REFERENCES Kerr, J.B., Millar, M., Maddocks, S. and Sharpe, R.M. (1993): Stage-dependent changes in spermato- Arai, K., Ohshima, K., Watanabe, G., Arai, K., genesis and Sertoli cells in relation to the onset Uehara, K. and Taya, K. (1998); Inhibition of of spermatogenic failure following withdrawal ovarian follicular development associated with a of testosterone. Anat. Rec., 235, 547-559. decrease in luteinizing hormone levels during Kong, L.D., Tan, R.X., Woo, A.Y. and Cheng, C.H. the estrous cycle of the rat. Endocr. J., 45, 539- (2001): Inhibition of rat brain monoamine oxi- 546. dase activities by psoralen and isopsoralen: Diawara, M.M., Chavez, K.J., Hoyer, P.B., Williams, Implications for the treatment of affective disor- D.E., Dorsch, J., Kulkosky, P. and Franklin, ders. Pharmacol. Toxicol., 88, 75-80. Kovacs, M., Mezö, I., Seprödi, J., Csernus, V., Teplan, M.R. (1999): A novel group of ovarian toxi- I. and Flerko, B. (1989): Effects of long-term cants, the psoralens. J. Biochem. Mol. Toxicol., administration of a superactive agonistic and an 13, 195-203. antagonistic GnRH analog on the pituitary- Dukes, M., Chester, R., Yarwood, K. and Wakeling, gonad system. Peptides, 10, 925-931. A.E. (1994): Effects of a non-steroidal pure Krenisky, J.M., Luo, J., Reed, M.J. and Carney, J.R. antiestrogen, ZM 189,154, on oestrogen target (1999): Isolation and antihyperglycemic activity organs of the rat including bones. J. Endocrinol., of bakuchiol from Otholobium pubescens 141, 335-341. (Fabaceae), a Peruvian medical plant used for Dunnett, C.W. (1955): A multiple comparison proce- the treatment of diabetes. Biol. Pharm. Bull., dure for comparing several treatments with con- 22, 1137-1140. trol. J. Am. Statist. Assoc., 50, 1096-1121. Kubo, M., Dohi, T., Odani, T., Tanaka, H. and Ferrandiz, M.L., Gil, B., Sanz, M.J., Ubeda, A., Erazo, Iwamura, J. (1989): Cytotoxicity of corylifoliae S., Gonzalez, E., Negrete, R., Pacheco, S., Paya, fructus. I Isolation of effective compound and M. and Alcaraz, M.J. (1996): Effect of baku- the cytotoxicity. Yakugaku Zasshi, 109, 926- chiol on leukocyte functions and some inflam- 931 (in Japanese). matory responses in mice. J. Pharm. Pharmacol., Latha, P.G. and Panikkar, K.R. (1999): Inhibition of 48, 975-980. chemical carcinogenesis by Psoralea corylifolia Garcia-Diaz, M., Dominguez, O., Lopez-Fernandez, seeds. J. Ethnopharmacol., 68, 295-298. L.A., de Lera, L.T., Saniger, M.L., Ruiz, J.F., Latha, P.G., Evans, D.A., Panikkar, K.R. and Parraga, M., Garcia-Ortiz, M.J., Kirchhoff, T., Jayavardhanan, K.K. (2000): Immunomodula- Del Mazo, J., Bernad, A. and Blanco, L. (2000): tory and antitumour properties of Psoralea DNA polymerase lambda (Pol λ), a novel corylifolia seeds. Fitoterapia, 71, 223-231.

Vo l . 2 7 N o . 2 105

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Levin, S., Semler, D. and Ruben, Z. (1993): Effects of tive and toxicological parameters in rats- in two weeks of feed restriction on some common search of suitable feeding regimen in long-term toxicologic parameters in Sprague-Dawley rats. tests-. J. Toxicol. Sci., 22, 427-437. Toxicol. Pathol., 21, 1-14. Shiromizu, K., Thorgeirsson, S.S. and Mattison, D.R. Marathe, G.K., Shetty, J. and Dighe, R.R. (1995): (1984): Effect of cyclophosphamide on oocyte Selective immunoneutralization of luteinizing and follicle number in Sprague-Dawley rats, hormone results in the apoptotic cell death of C57BL/6N and DBA/2N mice. Pediatr. Pharma- pachytene spermatocytes and spermatids in the col., 4, 213-221. rat testis. Endocrine, 3, 705-709. Sinha-Hikim, A.P. and Swerdloff, R.S. (1993): Tem- Morris, I.D., Phillips, D.M. and Bardin, C.W. (1986): poral and stage-specific changes in spermatoge- Ethylene dimethanesulfonate destroys Leydig nesis of rat after gonadotropin deprivation by a cells in the rat testis. Endocrinology, 118, 709- potent gonadotropin-releasing hormone antago- 719. nist treatment. Endocrinology, 133, 2161-2170. Morris, I.D. (1996): Leydig cell toxicology. In The Sun, N.J., Woo, S.H., Cassady, J.M. and Snapka, R.M. Leydig Cells (Payne, A.H., Hardy, M.P. and (1998): DNA polymerase and topoisomerase II Russell, L.D., eds.), 1st ed., pp.573-596, Cache inhibitors from Psoralea corylifolia. J. Nat. River Press, Vienna, IL. Prod., 61, 362-366. NTP Technical Report (1988): Toxicology and car- Tapanainen, J.S., Tilly, J.L., Vihko, K.K. and Hsueh, cinogenesis studies of 8-methoxypsoralen in A.J.W. (1993): Hormonal control of apoptotic F344/N rats (gavage studies). Report No. 359. cell death in the testis: Gonadotropins and O’Donnell, L., McLachlan, R.I., Wreford, N.G. and androgens as testicular cell survival factors. Robertson, D.M. (1994): Testosterone promotes Mol. Endocrinol., 7, 643-650. the conversion of round spermatids between Vanderschueren, D., Vandenput, L., Boonen, S., Van stages VII and VIII of the rat spermatogenic Herck, E., Swinnen, J.V. and Bouillon, R. cycle. Endocrinology, 135, 2608-2614. (2000): An aged rat model of partial androgen Russell, L.B., Hunsicker, P.R., Johnson, D.K. and deficiency: Prevention of both loss of bone and Shelby, M.D. (1998): Unlike other chemicals, lean body mass by low-dose androgen replace- etoposide (a topoisomerase-II inhibitor) pro- ment. Endocrinology, 141, 1642-1647. duces peak mutagenicity in primary spermato- Wall, M.E., Wani, M.C., Manikumar, G., Abraham, P., cytes of the mouse. Mutat. Res., 400, 279-286. Taylor, H., Hughes, T.J., Warner, J. and Russell, L.D., Corbin, T.J., Borg, K.E., de Franca, McGivney, R. (1988a): Plant antimutagenic L.R., Grasso, P. and Bartke, A. (1993): Recom- agents, 2 Flavonoids. J. Nat. Prod., 51, 1084- binant human follicle-stimulating hormone is 1091. capable of exerting biological effect in the adult Wall, M.E., Wani, M.C., Manikumar, G., Hughes, T.J., hypophysectomized rat by reducing the num- Taylor, H., McGivney, R. and Warner, J. bers of degenerating germ cells. Endocrinology, (1988b): Plant antimutagenic agents, 3 Cou- 133, 2062-2070. marins. J. Nat. Prod., 51, 1148-1152. Seki, M., Yamaguchi, K., Marumo, H. and Imai, K. (1997): Effects of food restriction on reproduc-

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