A New Method for Evaluating Mucolytic Expectorant Activity and its Application

II. Application to two proteolytic enzymes, serrapeptase and seaprose*

By Y. Kase, H. Seo, Y. Oyama, M. Sakata, K. Tomoda, K. Takahama, T. Hitoshi, Y. Okano, and T. Miyata

Arzneim.-Forsch. / Drug Res. 32 (1), Nr. 4 (1982)

From the Department of Chemico-Pharmacology. Faculty of Pharmaceutical Sciences, Kumamoto University, Kumamoto (Japan)

Summary: Using our new method described in a preceding paper, in vivo effects of two proteolytic enzymes such as serrapeptase and seaprose (SAP) on sputa collected from bronchitis rabbits were examined. Serrapeptase (20 mg/kg) and SAP (30 mg/kg) significantly reduced the viscosity of sputum (P 0.05) at the 1-3-h periods and the 4-6-h periods, respectively, after intraduodenal administration. 50 mg/kg of serrapeptase also significantly decreased not only viscosity (P 0.001) but also amount of freeze-dried substance (P 0.05) of sputum at the 1-3-h periods, but SAP did not affect the amount of dried substance. Both enzymes significantly increased the volume of sputum, probably as the result of liquefaction. Thus, mucolytic expectorant activity of both enzymes can be demonstrated first by the reduction in viscosity and next by the increase in volume of sputa. However, the decrease in amount of freeze-dried substance is not always in accord with the reduction in viscosity.

Key words: Bromhexine o Bronchitis o Mucolytic expectorants o Proteolytic enzymes o Seaprose o serrapeptase

1. Introduction

In this previous paper [1], we reported a new method which seems to be applicable to examine the in vivo effect of mucolytic expectorants. By the use of this method, the expectorant effect of a drug can be evaluated from the changes in both quantity and quality of sputa, which were quantitatively collected from the rabbits suffering from subacute bronchitis caused by long-term exposure to SO2 gas. The purpose of the present study is to ascertain whether this method is well applicable to the evaluation of mucolytic expectorant effect of the reference drugs as was expected, whose clinical efficacy was already well established. Two proteolytic enzymes, serrapeptase and seaprose, were chosen for such a purpose. Though their chemical properties differ, both enzymes have so far been used as the effective mucolytics in the treatment of various disorders related to viscous sputum or pus, and their efficacies have been war-ranted to be more potent and reliable than those of a-chymotrypsin and others. Therefore, they have widely been used not only in Japan but also in. some other countries. Nevertheless, the pharmacological evidence which sub-stantiates their clinical efficacies, in particular, mucolytic expectorant effect, is insufficient, though they exhibit potent mucolytic activity in in vitro experiments [2, 3]. Bromhexine, a representative of the expectorants, was used as a control drug, because its mechanism of action is quite different from that of proteolytic enzyme, that is, it does not exhibit in vitro mucolytic activity and its main effect is known only by the increase in the volume of respiratory tract fluid (RTF) when it was examined by Perry and Boyd's method [4-7] using normal healthy rabbits. Further pharmacological study, for instance, the acting mechanism of mucolytic expectorant effect of intraduodenally administered enzymes will be described in the subsequent paper.

2. Materials and methods

2.1. Animals and drugs

Male rabbits of New Zealand White-strain, weighing 1.8 to 2.5 kg, were used. Serrapeptase (Danzen*, hereafter abbreviated as SER), a proteolytic enzyme (endopeptidase) prepared from the culture broth of. genus Serratia sp. E-15 (one of enteric bacilli in silkworm) which comes as grayish powder, was provided.