Reproductive Toxicology 29 (2010) 214–224 Contents lists available at ScienceDirect Reproductive Toxicology journal homepage: www.elsevier.com/locate/reprotox Inhibition of hyaluronidase activity of human and rat spermatozoa in vitro and antispermatogenic activity in rats in vivo by Terminalia chebula, a flavonoid rich plant  Archana Srivastav a,∗ , Abhishek Chandra a , Madhulika Singh a , Farrukh Jamal d , Preeti Rastogi b , Siron Mani Rajendran c , Falgun Wanganuji Bansode a , Vijai Lakshmi b a Division of Endocrinology, Central Drug Research Institute, Lucknow, India b Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow, India c Botany Division, Central Drug Research Institute, Lucknow, India d Department of Biochemistry, Dr. R.M.L. Avadh University, Faizabad, India article info Article history: Received 7 May 2009 Received in revised form 16 October 2009 Accepted 2 November 2009 Available online 10 November 2009 Keywords: T. chebula Flavonoids Human sperm Rat sperm Antihyaluronidase Antispermatogenic abstract Our interest in development of hyaluronidase inhibitors as male antifertility agents led to identification of Terminalia chebula (T. chebula) plant with hyaluronidase (HAase) inhibitory activity of human sperma- tozoa (∼93% inhibition) and rat caudal epididymal spermatozoa (∼86% inhibition) in vitro at 30 mg/ml. We further demonstrated inhibition of hyaluronidase activity of testis and epididymal spermatozoa in vivo coincident with antispermatogenic activity and contraceptive efficacy of TC extract administered at 50 and 100 mg/kg/day orally for 60 days in male albino rats. The significant decrease in motility, count and increase in morphological abnormalities of epididymal spermatozoa and severe reduction in fertility (-100%) of male rats treated with T. chebula fruit extract at 100 mg/kg dose could be attributed to either direct effect on testis or direct or indirect interference with sperm maturation in epididymis, and/or inhi- bition of testicular and epididymal sperm hyaluronidase enzyme in vivo probably caused by flavonoids like tannins present in T. chebula. © 2009 Elsevier Inc. All rights reserved. 1. Introduction In recent years, there has been growing interest in the field of herbal medicines research and search for promising male antifer- tility agents from natural products remains a potential area of investigation [1,2]. Terminalia chebula Retzius (T. chebula) belong- ing to Combretaceae family, is the native plant of India and South East Asia. The dried ripe fruit of T. chebula, commonly known as black myrobalan in English and Harad in Hindi, is being used either alone or as an adjuvant to other medicines like Triphala and Menosan, and is a popular folk medicine in India and Asia. T. chebula has been reported to exhibit a vari- ety of biological activities including antiviral [3], antibacterial [4], antidiabetic and renoprotective [5], radioprotective [6], anticancer  Supported by Ministry of Health (MOH), Council of Scientific and Industrial Research (CSIR) and Indian Council of Medical Research (ICMR), Government of India. ∗ Corresponding author at: Division of Endocrinology, Central Drug Research Institute, Post Box No. 173, Chattarmanzil Palace, MG Road, Lucknow 226 001, UP, India. Tel.: +91 0522 2612411–18x4404; fax: +91 0522 223405. E-mail addresses: archana1849@gmail.com, archana srivastav1@yahoo.com (A. Srivastav). [7], antioxidant free radical scavenging activity and adaptogenic activity [8,9]. Chemical constituents of Terminalia species have been identified as tannins, flavonoids, sterols, amino acids, fruc- tose, resin, and fixed oils. In varying compositions, compounds like anthraquinones, gallic acid, chebulinic and chebulagic acid, ellagic and ethaedioic acid, 4,2,4 chebulyl--d-glucopyranose, terpinenes and terpinenols have been described for this species [4,7,10]. Hyaluronidase (HAase), a family of -1,4-endoglucosaminidase are a class of enzymes (EC 3.2.1.35) reported to be involved in the spread of infections/toxins, ovum fertilization and cancer progres- sion [11]. To date, six hyaluronidase-like genes in the human and mouse genomes have been identified. Six hyaluronidases have been cloned, and three of them have been expressed in different cell lines [12]. The human enzymes, the PH-20 protein, Hyal-1, and Hyal-2, probably exist in several isoforms [13] and seem to play crucial roles in physiological and pathophysiological processes like fertilization, embryonic development, tumor growth and metastasis. Mammalian fertilization requires sperm to penetrate the cumu- lus mass surrounding the eggs to reach the zona pellucida (ZP) of the oocyte. The sperm acrosome contains several hydrolytic enzymes including hyaluronidase, one of the key enzymes neces- sary for penetration of the spermatozoon through viscous, gel-like matrix of the follicle cell layer, a major extracellular component of 0890-6238/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.reprotox.2009.11.001