Determination of Bioactive Compounds in Roots of Different Ages

Pueraria mirifica, Airy Shaw Suvatabhandhu and Butea superba, Roxb.
from Various Locations in Thailand

A. Manosroi and J. Manosroi
Pharmaceutical-Cosmetic Raw Materials and Natural Products
Research and Development Center (PCRNC)
Institute for Science and Technology Research and Development

Keywords: puerarin, daidzein, genistein, miroestrol, sites, HPLC, finger prints

Abstract


The bioactive compounds from the roots of White Kwao Krua (Pueraria
mirifica) and Red Kwao Krua (Butea superba) extracted by organic solvent were
determined from the HPLC finger prints compared with the standard isoflavonoids
(puerarin, daidzein and genistein). Roots of White Kwao Krua from Chiang Mai
province harvested at the age of 6 years gave the highest amount of isoflavonoid
compounds of 290, 89 and 16 mg/kg of the dried root, respectively. Roots that were
younger or older than 6 years old appeared to contain less amount of the active
compounds. Red Kwao Krua from Chiang Mai had the highest amount of the active
compounds. Puerarin contents in Red Kwao Krua were less whereas daidzein and
genistein were more than those found in White Kwao Krua. The contents of
puerarin, daidzein and genistein in Red Kwao Krua from Chiang Mai were 1.9, 37.2
and 4.5 mg/kg of the dried root, respectively. For miroestrol contents, the highest
amount of 45.0 mg/kg of the dried root was found in White Kwao Krua collected
from Chiang Mai province at the age of 5.5 years old. No miroestrol can be
determined from roots of Red Kwao Krua. This study suggested that both
isoflavonoids and miroestrol contents in the two plants depended on ages and
locations of cultivation in Thailand. Informations from this study can be applied for
the selection of sources and ages of the plants that contain high amounts of the
bioactive compounds for further herbal formulation.


INTRODUCTION


White Kwao Krua and Red Kwao Krua are Thai medicinal plants which have been
widely used for generations by the native northern Thais. They have been used as tonics
for longer life, rejuvenation, improving the muscle tonic and firming of the body. The
juice or powder from sun dried flesh of White Kwao Krua root are mixed with cow milk
or honey and prepared as pills in northern Thai folk medicines (Anusarnsunthorn, 1932).
White Kwao Krua and Red Kwao Krua are two of the four Kwao Krua types which have
been included in the recipe of northern Thai traditional medicines for centuries. Previous
research has reported that at least three important groups of chemicals were found in
White Kwao Krua (Ingham et al., 1986): coumarins, flavonoids (daidzein, daidzin,
genestin, genistein, puerarin) and chromene (miroestrol). The claim of antiaging and
rejuvenation effects of this plant appears to be from these compounds, especially
miroestrol which is a known potent estrogenic principle (Lakshnakara et al., 1952; Cain,
1960). Red Kwao Krua has been used traditionally for men as a phytoandrogen in many
Thai folk medicinal recipes. It has been found to contain butenin and butin as well as
other compounds that are also found in White Kwao Krua (Manosroi et al., 2002). The
bioactivity of these two plants is correlated to the amounts of these bioactive compounds
which depend on the ages of the roots and locations of cultivation. The present study
compared the contents of some bioactive compounds found in roots of White Kwao Krua
and Red Kwao Krua which were harvested at different ages from various locations in
Proc. WOCMAP III. Vol. 4: Targeted Screening of MAPs, Economics & Law
Eds. C. Franz, Á. Máthé, L.E. Craker and Z.E. Gardner
Acta Hort. 678, ISHS 2005 136 Thailand.

MATERIALS AND METHODS


Eleven root samples of White Kwao Krua were collected from Chiang Mai,
Chiang Rai and Kanjanaburi provinces in Thailand. The average ages of the plant were
determined from the annual rings of at least three tuberous roots of the same plant. Two
root samples of Red Kwao Krua were collected from Chiang Mai and Kanjanaburi
provinces. The root samples were chopped into small pieces, dried at 50±2°C and ground
into powder. The powder was extracted by methanol (AR grade) using a soxhlet
apparatus. The extract was filtered through a filter paper (Whatman No. 42). The filtrate
was dried by a rotary evaporator (Buchi, Rotavapor R-124, Switzerland). Percentage
yields and physical characteristics of the dried crude extract were examined. The finger
print chromatogram of each crude extract was performed by HPLC (Thermo Separation
Products, TSP, UV 1000/P2000). The HPLC conditions for puerarin were mobile phase:
methanol/water (2:3), flow rate: 1.0 ml/min, column: Luna 10 µ C18 100° A (250 x 4.6
mm) and UV detector at 254 nm. For daidzein, genistein and miroestrol, the conditions
were mobile phase: acetonitril/water (1:1), flow rate: 1.0 ml/min, column: Luna 10 µ C18
100° A (250 x 4.6 nm) and UV detection at 254 nm. Amounts of puerarin (P5555),
daidzein (D7802) and genistein (G6649) in each extract were analysed by comparing the
HPLC finger print chromatogram of the extracts with the standard isoflavonoids from
Sigma Co. Contents of these compounds were calculated in percentages of the dried root.
Purified miroestrol (about 85% purity as determined by HPLC and identified by NMR)
was prepared by the method previously described with some modifications (Ingham et al.,
1986; Jones and Pope, 1961). The amounts of miroestrol in each extract were determined
by comparing the HPLC finger print chromatogram with the purified miroestrol. Since no
standard miroestrol was available in the market, we isolated this compound in purified
form according to the method previously descrided (Jones and Pope, 1961). This purified
compound was identified by NMR, IR, spectrophotometry indicating the maximum
absorption at 254 nm and one single peak by HPLC at 254 nm (data not shown). This
purified miroestrol was used as the reference standard to determine the amount of this compound in the crude extract samples.
Contents of miroestrol in mg/kg of the dried root was calculated.

RESULTS AND DISCUSSION


The maximum percentage yield of the crude extract was 25.72% of White Kwao
Krua found in the sample from Chiang Mai province. Among the three bioactive
isoflavonoid compounds in all samples, puerarin was the highest (Table 1). Genistein was
the lowest and not found in some root samples. This may be due to the instability of this
compound. White Kwao Krua at 6 years old collected from Chiang Mai province Site No.
4 and Site No. 2.4 gave the highest amount of puerarin 29 mg/kg and daidzein 8.9 mg/kg,
respectively, while at the age of 5.5 years old at Site No. 3 gave the highest amount of
genistein of 1.6 mg/kg. Isoflavonoid contents in tuberous roots of White Kwao Krua
collected from Chiang Mai (Site No. 2) at different ages determined by HPLC finger print
chromatogram in comparing to the reference standards obtained from Sigma Co. No
genistein was found in all ages (Table 1). It appeared that the amounts of isoflavonoids
(puerarin and daidzein) depended on ages of the roots. The highest amount of 15.2 mg/kg
of puerarin was obtained at 5 years old in Site No. 2.2 whereas the highest amount of
daidzein of 8.9 and 7.3 mg/kg were found at 6 years old Site No. 2.4 and 14 years old of
Site No. 2.6, respectively. However, only minute amounts of these three isoflavonoid
compounds were found in Red Kwao Krua collected from Kanjanaburi province. But,
Red Kwao Krua roots from Chiang Mai province had isoflavonoid contents, especially
daidzein more than that from Kanjanaburi province. Red Kwao Krua from Chiang Mai
province appeared to contain less puerarin, but more daidzein and genistein than that
found in White Kwao Krua collected from the same province.
The amounts of miroestrol ranged from 2.40 to 45.00 mg/kg of the dried root of 137
White Kwao Krua. No miroestrol was found in the two Red Kwao Krua samples collected
from Chiang Mai and Kanjanaburi.

Fig. 2 compared the amounts of 29.80 to 45.00 mg of miroestrol per kg of the dried root

in White Kwao Krua collected from Chiang Mai province (at Site No. 2, i.e. Site Nos. 2.1 to 2.5)
at the ages of 4.5 to 6.5 years old. This amount was close to that was previously reported
(Jones and Pope, 1961).
Interestingly, at the age of 5.5 years old of White Kwao Krua root collected from Chiang Mai
Site No.2.3, the amount of miroestrol found in the crude extract was the highest of 45 mg/kg.

The miroestrol contents appeared to depend on sites of cultivation more than the ages, since
roots at the age of 6 years old but from different sites of Chiang Mai (Site No. 4) and
Chiang Rai had lower amount of miroestrol of only 5.80 and 13.40 mg/kg of the dried
root, respectively in comparing to 29.80 mg/kg of the dried root from Chiang Mai (Site
No. 2.4).

This study indicated that isoflavonoid and miroestrol contents in White KwaoKrua
depended on locations of cultivation and ages of its tuberous roots. Puerarin and
miroestrol seemed to be the main bioactive compounds in White Kwao Krua while
daidzein and genistein were isoflavonoids that were found more in Red Kwao Krua. This
study suggested the proper source and age of White Kwao Krua and Red Kwao Krua
cultivated in Thailand. This information will be beneficial for the selection of these two
plants in future formulation development.

ACKNOWLEDGEMENTS


Thanks to Mathana Phanit Chiangmai Co., Ltd. for supporting some root samples
of the plants and Mr. Somsak Tharatha for assisting the experiments in this study.

Literature Cited
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Cain, J.C. 1960. Miroestrol: an oestrogen from the plant Pueraria mirifica. Nature
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Ingham, J.L., Tahara S. and Dziedzic, S.Z. 1986. A chemical investigation of Pueraria
mirifica roots. Z. Naturforsch. 41C:403-408.

Jones, H.E. and Pope, G.S. 1961. A method for the isolation of miroestrol from Pueraria
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Lakshnakara, K., Suvatabandhu, K. and Airy Shaw, H.K. 1952. A new species of
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