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Dysenzi^®

ABSTRACT

The in vitro anti-amoebicidal activity of FAME's "Dysenzi^®" ( water soluble 50% alcoholic extract of Brucea javanica ) was tested with two isolates of Entamoeba histolytica grown in two types of media. "Dysenzi^®" was observed to be amoebicidal at the concentrations of 50, 100, 200, and 400 µg/ml. Metronidazole was used as a control drug. The minimum amoebicidal concentration (MAC) with ED₉₅ was 50 µg/ml. There was only a slight variation between the two media used for the assay of drug effects. The variation between two isolates of E. histolytica was negligible and not significant.

INTRODUCTION

The search for new drug for any particular purpose is still very largely empirical and practically every new drug in clinical use has been discovered by the process of "screening" and subsequent follow-up works. In "screening", new compound of any type are examined for the particular property required. A compound (single or multiple) having such activity is regarded as a "lead", and chemical (constituents) modification of the molecule (proportion) is under taken in order to increase the activity, decrease the toxicity, and to eliminate undesirable side effects. Efficient screening methods must be capable of dealing with large numbers of compounds (extracts; constituents) in order to find as many leads as possible. It matters little if preliminary screening tests release relatively large numbers of compounds are discarded in later stages of testing, but it would be desirable to use screening procedure which might fail to detect activity in potentially useful drugs. The screening methods envisaged will, whenever possible, comprise first in vitro test, then a series of in vitro and in vivo tests in parallel, and finally a series of in vivo tests (including clinical trials).

The main requirement of the in vitro tests for any purpose is that they should be simple that they can be carried out mechanically by qualified technical staff with only general supervision. They should be reasonably specific, and drugs (to be used as controls) known to be active clinically must be active in tests. Of the in vitro methods described for testing drugs against E. histolytica, many workers have used diphasic media ^{(1,2,3,4,5,6,7)}, and others used monophasic medium ^{(8,9, 10 11)}. In our present experiment for testing of antimaoebicidal activity of "Dysenzi", we have used both monophasic and diphasic media to get a stronger conclusion⁽¹²⁾.

MATERIALS AND METHODS

Isolates of E. histolytica

Two isolates of E. histolytica of human origin obtained from subjects with amoebic dysentery were used. The isolates were designated as FEh 001/03 and FEh 002/03.

Culture methods

Two culture media were used for maintaining the test isolates of E. histolytica. These were inspissated horse serum-Ringer overlay method of Dobell and Laidlaw ⁽¹³⁾ and synthetic medium described by Hallman, Michaelson and DeLamater ⁽¹⁴⁾.

Routine subcultures were made at 48=2 hours intervals after adding approximately 10mg of rice starch to fresh culture medium at each passage. For initial two to three subcultures in Dobell and Laidlaw's modified medium, crystalline benzyl penicillin (1,000 IU/ml) was added to suppress gram-positive bacteria and to help establish the amoeba strains in the media.

Usually five to six subcultures were required in both media in order to obtain a suitable test population of E. histolytica (minimum 30,000 trophozoites/ml). The harvested culture was mixed with fresh medium and washed twice with sterile physiological saline. The washed amoebae were adjusted to contain approximately 10,000 trophozoites/ml of the medium overlay.

Counting procedure

The counting procedure of amoebae was done by using standardized cover-slip preparation method of Rawson and Hitchcock ⁽²⁾ and Botero⁽¹⁵⁾ with some minor modifications. The perfect use of this method had been tested statistically and have been found to show low variability. In counting, a 200 µl sample from the well mixed culture medium was placed at the center of a 1 x 3 inch microscopic slide and covered with an 30 x 22 mm cover slip. This was then sealed immediately with parafilm wax and ready for counting. Five replicates from each culture tube was counted. Multiplication of the average count by 50 gives the number of amoebae/ml. 

Test drug and control drug

Test drug was "Dysenzi", a total alkaloid water-soluble extract from the seed of Brucea javanica (Myanmar: Yar-dan-tze), produced by FAME Pharmaceutical Enterprise, Myanmar. Metronidazole produced by Myanmar Pharmaceutical Factory was used as control drug. Both the drugs were dissolved in medium overlay at desirable concentrations. 

Design of Experiment

Amoebicidal activity of "Dysenzi" was assayed by the method of Dennis, Berberian and Hanson ⁽¹⁶⁾ with some modifications. Approximately 10,000 trophozoites/ml of the medium overlay was added to the 4ml of the culture medium previously incubated at 37^oC so that the initial count of amoebae at 0-hr was approximately 2,000 trophozoites/ml of the total medium. The counting was made at every 12-hr interval up to 72-hr of incubation at 37^oC. The test and control drugs dissolved in DMSO and diluted to desired concentration in medium overlay were added after the 24-hr counting was made. Only 0.3ml of suspension containing drug (approximately 2.0mg) was added to the culture medium (5ml total volume) so as to contain 400 µg/ml. In counting, as described above, a 200 µl sample from the well mixed culture medium was placed at the center of a 1 x 3 inch microscopic slide and covered with a 30 x 22 mm cover slip and this was sealed immediately with parafilm wax. The counting was made under low power (10X) and occasionally under high dry (40X) lens when necessary. If a tested drug was found to be active at 400 µg/ml, the dilution was made to decrease the concentration of the drug to 200 µg/ml. If amoebae multiplicated well in the untreated control tubes but not in the tested tubes, further dilutions of the test drug were made and continue until the minimum effective (amoebicidal) concentration was determined. The minimum amoebicidal concentration of known control drug metronidazole was adjusted at 10 µg/ml.

Growth rates of the strains tested against "Dysenzi" and metronidazole were calculated from the trophozoites count per ml.  Mortality rates of E. histolytica with respect to "Dysenzi" at various concentrations was obtained as follows:

Mortality Rate (%) = 100 – 100 x count/ml treated with"Dysenzi"                                                                     count/ml (untreated control)

RESULTS

The in vitro amoebicidal activity of "Dysenzi" on two isolates of E. histolytica in two types of media is shown in Table 1. "Dysenzi" is observed to be amoebicidal at the minimum concentration (MAC) of 50 µg/ml on both isolates grown in two types of media. The in vitro average mortality rates of two isolates of E. histolytica exposed to "Dysenzi" at various concentrations in two media are shown in Figure 1. The highest mortality rates are observed at the concentrations of 100 µg/ml, 200 µg/ml, and 400 µg/ml with an average mortality rates of 99.4-99.9% whereas 50 µg/ml concentration also shows an average mortality rate of 96.5%. 

Therefore, the effective dose ED₉₅ is taken as 50 µg/ml from our experiment with two isolates of E.histolytica grown in two types of media.

DISCUSSION

Of in vitro methods described for testing of unknown drugs against E. histolytica, many use diphasic media, and it is known that absorption of drugs at the interface of the components makes this system unreliable⁽¹⁵⁾. Few of the tests distinguish between direct action on the amoebae and action of bacterial associates in the cultures with consequent secondary effects on the amoebae. The method of choice appears to be that described originally by Dobell and Laidlaw (1926), and used subsequently by others ^(16,17). In the present experiments, Dobell and Laidlaw's (1926) diphasic medium is used simultaneously with Hallman, Michaelson and DeLamater's (1950) synthetic medium for reliability and comparison. There is no significant different between the two methods of cultivation ⁽¹⁸⁾. 

Regarding the drug "Dysenzi", the seeds of several species of Brucea, a simaroubaceous plant native to Southeast Asia, have long been used as a remedy for diarrhoea and dysentery. The seed of this plant, known as ko-sam, ya-tan-tzu or koo-sheng-tse in Chinese, has been studied in Asia, with preliminary results that can only be regarded as inconclusive. The drug ya-tan-tzu (Chinese) appears to have an antiamoebic activity comparable to that of "chaparro amargoso"  and "glaucerubin", but little detailed pharmacology is available during early 1960s ⁽¹⁹⁾. It is not clear which of the substances that have been isolated from the plant (Brucea spp.) is the amoebicidal. So far, there is one published report on the in vitro activity of extract of Brucea spp. ⁽²⁰⁾ and one paper presentation on clinical trial ⁽²¹⁾. Both the scientific groups ^(20,21) have suggested the efficacy of the total alkaloid extract of Brucea spp. The present study on the in vitro amoebicidal activity of "Dysenzi" is in agreement with the previous findings of DMR Scientific Groups ^(20,21). Thaung Hla, May Mya Win and Aye Than (2000)⁽²²⁾ has also reported the sub-acute toxicity study of yar-dan-tze (Brucea spp.. They observed that the dried powder of the defatted kernel produce no significant changes in blood urea levels, liver function values, blood, bone-marrow, and tissue pathology.

REFERENCES

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12. Myint Oo, Margaret Tu & Sein Gwan (1972). In vitro effects of some indigenous plant extracts on Entamoeba histolytica. Seventh Myanmar Research Congress. Abstract of paper, p.117

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22. Thaung Hla, May Mya Win & Aye Than (2000). Sub-acute toxicity study of yar-dan-tze. Paper presented at Myanmar Health Research Congress, December 2000. Abstract, p. 89

Table 1. In vitro amoebicidal activity of  "DYSENZI" on two strains of Entamoeba histolytica in two types of culture media

* Dysenzi dissolved in medium was administered after 24-hr amoeba counting
** All survived on sub-cultivation
# The remaining amoebae were vacuolated and not survived on sub-cultivation