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J Thai Trad Alt Med Vol. 20 No. 1 Jan-Apr 2022 109

erythrocyte suspension. was performed, and the Heinz body was ob-
2.4 inhibition of Heinz body induction served in at least 1,000 red blood cells under a
Two milliliters of extract (100, 500, and microscope (1,0005).

1,000 μg/mL) was mixed with 0.1 mL blood 2.5 hemolysis test
and incubated for 2 hours, and then 2 mL The properties of L. leucocephala extract

acetylphenylhydrazine (APH) was added. The to prevent oxidative hemolysis was evaluated
mixture was incubated at 37˚C for another 2 by spectrophotometer method. A volume of 0.5
hours, and then Heinz bodies were counted. mL of erythrocyte suspension was added to

Positive control was prepared by adding 2 mL 1 mL of the plant extract (100, 500, and 1,000
of APH into 0.1 mL of packed red blood cells. μg/mL). Then 0.5 mL of hydrogen peroxide

Negative control was prepared by adding 2 solution was added. The reaction mixtures
mL of buffer solution into 0.1 mL of packed were incubated (4 hours, 37˚C) and then
red blood cells. The positive and negative centrifuged. The free hemoglobin found in

control were incubated at 37˚C for 2 hours. the supernatant was used for measurement at
We performed the counterstain of Heinz body 540 nm. Hydrogen peroxide solution and PBS
by transferring solutions from each test and were used as positive and negative hemolytic

mixed it with crystal violet solution at equal controls, respectively. The percentage hemo-
volume. The mixture was left undisturbed at lysis and protection were calculated as
room temperature for 5 minutes. A thin smear follows .
[17]

(Absorbance sample –Absorbance neg control
% Hemolysis = 5 100
Absorbance pos control –Absorbance neg control
% Protection=100 % Hemolysis

2.6 microorganisms preparation ism in the stock was sub-cultured into freshly

Five pathogens in total, (Staphylococ- prepared nutrient broth aseptically using a
cus aureus, Escherichia coli, Pseudomonas flamed wire loop and then incubated at 37˚C

aeruginosa, Proteus mirabilis, and Klebsiella for 24 hours in the incubator. This culture was
pneumoniae), were kindly provided and identi- used for the antimicrobial assay.
fied by Maharaj Nakorn Chiang Mai Hospital, 2.7 antimicrobial assay

Thailand. These bacteria were isolated from Disk diffusion assay was performed for
clinical samples. An overnight culture of each antimicrobial screening. The inoculums were
bacterial organism was prepared. Each organ- adjusted to contain approximately 108 cells/