Page 19 - วารสารกรมการแพทย์แผนไทยฯ ปีที่ 10 ฉบับที่ 2

Journal of Thai Traditional & Alternative Medicine Vol. 10 No. 2 May-August 2012 95

respectively; KK samples and other plant The extracts of each sample were pre-

materials from Tai-hua-jan drugstore; and a pared using two kinds of solvents, 1 M NH OH
4
batch of HNK sample was prepared by the in methanol and distilled water. The samples
Herbal Medicine and Products Manufactur- were extracted by vortex mixing for 30 min-
ing Unit Ayurved Siriraj, Center of Applied utes and then centrifuged at 15,000 rpm for
Thai Traditional Medicine (CATTM), Faculty 10 minutes at 4˚C. The supernatant was fil-
of Medicine Siriraj Hospital, Mahidol Univer- tered through a 0.2 μm polyvinylidene fluo-
sity, Bangkok, Thailand. ride (PVDF) membrane filters for the analy-
sis.
2. Instruments
3.3 TLC analysis
The HPTLC system (CamagMuttenz, By modifying the method of Ohno T et
Switzerland) consists of a semi-automatic al, 2006, the mobile phases of chloroform/
sample spotter (Linomat 5), an automatic TLC methanol (7:3, v/v), chloroform/methanol/ace-
plate developing equipment (ADC2) with a tic acid (65:20:1, v/v), and chloroform/metha-
twin trough glass chamber and a densitom- nol/ammonium hydroxide (10:10:1, v/v) were
eter (TLC Scanner 3). DigiStore 2 Documen- used for HPTLC analysis and acetonitrile/
tation system was also used for the docu- methanol/water (3:0.5:1, v/v) was used for RP-

mentation. WINCATS software was used to TLC analysis. The extracted solutions of KK,
control the system and analyze the results. HNK, AA-I and AA-I sodium salt were sub-
jected to HPTLC silica gel 60 F and TLC
254
3. Methods
silica gel 60 RP-18F plates which were
254s
3.1 Preparation of standard solutions pre-saturated in mobile phase for 5 minutes.
The stock solutions of AA-I and AA-I The saturation time was 20 minutes and the
sodium salt were prepared by dissolving 1 developing distance for HPTLC and RP-TLC
mg of AA-I in 1 ml of 1 M ammonium hy- plate was 6.0 cm. After development, the
droxide (NH4OH) and 1 mg of AA-I sodium plates were dried by cold air and detected

salt in 1 ml of distilled water. To determine under the ultraviolet light of 254, 323 and 366
the lower limit of detection (LOD), each stock nm. The existence of AA-I and AA-I salt in
solution of marker was diluted until the con- each sample was identified by comparing the
centration of the analyte exhibited the signal Rf value and absorption spectrum of the
height approximately three times the signal sample with that of the AA-I and AA-II
heights of blank samples. markers. To determine LOD, each known con-
3.2 Preparation of sample solutions centration of marker was diluted and run on

Amounts KK and HNK powder were ac- the TLC plate until the lowest concentration
curately weighed for 5.49 mg and 300 mg, exhibited approximately 3 times of noise
respectively. response level.