An in vitro study of theaflavins extracted from black tea to neutralize bovine rotavirus and bovine coronavirus infections K.J. Clarka, P.G. Grantb, A.B. Sarrb, J.R. Belakereb, C.L. Swaggertya, T.D. Phillipsb, G.N. Woodea,* aDepartment of Veterinary Pathobiology, College of Veterinary Medicine, Texas A accepted 6 July 1998 Abstract Crude theaflavin was extracted from black tea and then fractionated by HPLC into five components (initial peaks (IP), TF1, TF2A, TF2B, and TF3). The crude extract and the various fractions of theaflavin were collected and tested, individually and in combination, for antirotaviral activity. The mean effective concentration (EC50) was calculated and compared. Activity varied from the most active being the uncharacterized theaflavin-like initial peaks (IP) with an EC50of 0.125 mg/ml to the least active being theaflavin-3 monogallate (TF2A) with an EC50of 251.39 mg/ ml. The combination of TF1?TF2A?TF2B?TF3was more active than the sum of the activities of these four fractions individually, indicating synergism among the peaks. Only the crude extract was assayed for activity against coronavirus; the EC50was 34.7 mg/ml. # 1998 Elsevier Science B.V. All rights reserved. Keywords: Tea; Theafl avin; Theafl avin gallate; Neutralization; Rotavirus; Coronavirus 1. Introduction Gastroenteritis due to rotavirus and coronavirus infections has a significant economical impact on agriculture. Yet, despite numerous trials and decades of research on these two Veterinary Microbiology 63 (1998) 147157 * Corresponding author. Tel.: +1-409-845-5941; fax: +1-409-845-9231; e-mail: mezemanekcvm.tamu.edu 0378-1135/98/$ see front matter # 1998 Elsevier Science B.V. All rights reserved. PII: S0378-1135(98)00242-9 viruses, effective vaccines are still not available. Although research efforts have appeared promising, vaccine field trials continue to produce inadequate results or fail entirely (de Leeuw et al., 1980; Myers and Snodgrass, 1982; Snodgrass et al., 1982; Waltner-Toews et al., 1985). Following infection the only treatment available is supportive therapy such as fluid replacement. Thus, viral gastroenteritis is still largely uncontrolled and alternative methods to control infection appear necessary. One of the goals of our laboratory has been to investigate a broad spectrum antiviral agents that is not discriminatory among various serotypes and strains of viruses. Provided that these agents could be used as food additives, they would have a distinct advantage over vaccines in prevention of infection and/or disease due to their broad spectrum of activity and because most agents that cause gastroenteritis are contracted orally, usually via contaminated food and water. The broad spectrum antiviral agents previously investigated in this laboratory included phyllosilicate clays and charcoal as viral adsorbents. Extensive research on these adsorbent materials indicates that they have high affinity adsorbing properties, although the adsorbed virus retains some infectivity in vitro. Therefore, although the use of phyllosilicate clays and charcoal are an ideal method of concentrating virus particles, they do not appear to be an effective method of eliminating the infectivity of rotavirus or coronavirus as judged from in vitro studies (Clark et al., 1998). To pursue further the idea of using broad spectrum methods to inhibit infection, we looked for a natural substance that could neutralize the infectivity of both bovine rotavirus and bovine coronavirus. The intent was to find a substance that is active either alone or in conjunction with a phyllosilicate clay to reduce or eliminate viral infectivity. Among the chemicals considered for testing were phenol, formalin, theaflavin and theaflavin gallate derivatives. Theaflavins are extracted from black tea and were selected because they are derived from a natural source. The oral administration of tea is reported to be used as a therapy for enteric diseases; a Japanese folk legend discusses the medicinal value of tea for curing gastroenteritis in children (Mukoyama et al., 1991). Theaflavin is thought to be responsible for the medicinal value of black tea and can be fractionated into four parts: theaflavin (TF1), theaflavin-3-monogallate (TF2A), theaflavin-30-monogallate (TF2B), and theaflavin-3,30 digallate (TF3) (Hara et al., 1987). TF3has been found to have antiviral activity against influenza A and B (Green, 1949; Nakayama et al., 1990, 1993), poliovirus 1 (strain Sabin 1) coxsackie virus B3 (strain Nancy), and human rotavirus group A strains of serotypes 14, and 8 (Mukoyama et al., 1991). In our studies, the crude extract of theaflavin and each of the purified derivatives were studied (individually and in combination) for their antiviral effect against rotavirus and coronavirus. 2. Materials and methods 2.1. Extraction procedure Theaflavin and theaflavin gallate derivatives were extracted from three sources of black tea, one sample each from the United States