Screening of steroid 5α-reductase inhibitory activity and total phenolic content of Thai plants

Abstract

Steroid 5-reductase is the enzyme responsible for changing androgen testosterone into the more potent androgen dihydrotestosterone (DHT). Overexpression of DHT can cause many disorders including androgenic alopecia and benign prostatic hypertrophy (BPH). The aim of this study is to determine which plants possess 5-reductase inhibitory activity, and to evaluate the correlation between 5-reductase inhibitory activity and total phenolic content of these plants. Ten kinds of Thai plants were collected from local areas and extracted with 95% ethanol. The yields of ethanolic extracts of these plants ranged from 2.22 to 16.05%, dry weight. In the present study the ability of the extracts to inhibit 5-reductase enzyme has, for the first time, been calculated as finasteride equivalent 5-reductase activity (FEA) value (mg finasteride per 1 g extract). FEA values are easier to understand and to compare their activity. FEA values of the extracts ranged from 5.56 to 17.59 mg finasteride per 1 g extract. The highest FEA value was found in Ocimum basilicum L. The red strain of Oryza sativa L. was the second most potent 5-reductase inhibitor, with FEA value of 16.72. Total phenolic content of the extracts ranged from 32.00 to 370.85 mg gallic acid equivalent per 1 g extract. There was no correlation between 5-reductase inhibitory activity and total phenolic content. Phytochemicals other than phenolic compounds may play an important role in enzyme inhibition. As the usual dosage regimen of finasteride for treating androgen-related disorders is 1 to 5 mg/d, regular intake of these fresh plants or their extracts may be beneficial in health promotion, prevention or treatment effect.

Full text

Journal of Medicinal Plants Research Vol. 5(7), pp. 1265-1271, 4 April, 2011
Available online at http://www.academicjournals.org/JMPR
ISSN 1996-0875 ©2011 Academic Journals
Full Length Research Paper
Screening of steroid 5-reductase inhibitory activity
and total phenolic content of Thai plants
Thapana Kumar1, Chaiyavat Chaiyasut1*, Wandee Rungseevijitprapa2 and Maitree Suttajit3
1Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Thailand.
2Department of Pharmaceutical Science and Technology, Faculty of Pharmacy, Ubon Ratchathani University, Thailand.
3School of Medical Science, Naresuan University at Phayao, Thailand.
Accepted 22 September, 2010
Steroid 5-reductase is the enzyme responsible for changing androgen testosterone into the more
potent androgen dihydrotestosterone (DHT). Overexpression of DHT can cause many disorders
including androgenic alopecia and benign prostatic hypertrophy (BPH). The aim of this study is to
determine which plants possess 5-reductase inhibitory activity, and to evaluate the correlation
between 5-reductase inhibitory activity and total phenolic content of these plants. Ten kinds of Thai
plants were collected from local areas and extracted with 95% ethanol. The yields of ethanolic extracts
of these plants ranged from 2.22 to 16.05%, dry weight. In the present study the ability of the extracts to
inhibit 5-reductase enzyme has, for the first time, been calculated as finasteride equivalent 5-
reductase activity (FEA) value (mg finasteride per 1 g extract). FEA values are easier to understand and
to compare their activity. FEA values of the extracts ranged from 5.56 to 17.59 mg finasteride per 1 g
extract. The highest FEA value was found in Ocimum basilicum L. The red strain of Oryza sativa L. was
the second most potent 5-reductase inhibitor, with FEA value of 16.72. Total phenolic content of the
extracts ranged from 32.00 to 370.85 mg gallic acid equivalent per 1 g extract. There was no correlation
between 5-reductase inhibitory activity and total phenolic content. Phytochemicals other than
phenolic compounds may play an important role in enzyme inhibition. As the usual dosage regimen of
finasteride for treating androgen-related disorders is 1 to 5 mg/d, regular intake of these fresh plants or
their extracts may be beneficial in health promotion, prevention or treatment effect.
Key words: Androgenic alopecia, benign prostatic hyperplasia, dihydrotestosterone, steroid 5-reductase,
testosterone, Thai plants, total phenolic content.
INTRODUCTION
Steroid 5-reductase (5aR, EC 1.3.99.5; 4-3-oxo-steroid
5-oxidoreductase) is a microsomal enzyme that
catalyzes the NADPH-dependent reduction of 4,5 double
bond of a variety of 3-oxo-4steroids such as
testosterone, progesterone and corticosterone. The
*Corresponding author. E-mail: chaiyavat@gmail.com. Tel:
+6653944340. Fax: +6653894163.
important role of 5aR is to metabolize testosterone into a
more potent androgen, dihydrotestosterone (DHT), which
can bind firmly to androgen receptors with higher affinity
and slower dissociation rate than testosterone. In
humans, DHT is necessary for normal male growth; but
high expression of DHT can cause many diseases such
as acne, hirsutism, androgenic alopecia, benign prostatic
hyperplasia (BPH), and prostate cancer (Bruchovsky et
al., 1968; Liu et al., 2006; McGuire et al., 1960). In all
animals, including humans, two different 5aR isozymes

1266 J. Med. Plant. Res.
have been characterized. They are 5- reductase type 1
(5aR1) and 5-reductase type 2 (5aR2). In humans, the
two isozymes share less than 50% sequence identity.
Moreover, the two isozymes differ in their biochemical
properties and specific organ distribution. 5aR1 has a
broad basic optimum pH and a lower affinity for
testosterone (Km > 1µm), but higher capacity (high Vmax);
whereas 5aR2 prefers a slightly acidic pH and has higher
affinity for testosterone (Km < 10nm), but lower capacity
(low Vmax). 5aR1 can be found in the brain, liver, non-
genital skin, and in the dermal papilla of hair follicles,
while 5aR2 can be found only in androgen-dependent
tissues such as the prostate, epididymis, and seminal
vesicles (Eicheler et al., 1998; Iehlé et al., 1999; Liu et
al., 2008b).
In treating DHT-related disorders, many synthetic 5aR
inhibitors have been studied. For example, finasteride
(MK-906, ProscarTM) has been a drug of choice to treat
BPH (Robinson et al., 2003). However, finasteride has a
number of unfavorable side effects including impotence
(erectile dysfunction), abnormal ejaculation, decreased
ejaculatory volume, abnormal sexual function,
gynecomastia, testicular pain, and myalgia (Lacy et al.,
2008). To avoid these side effects, natural products may
be used instead. In recent years, many researchers have
found that some phytochemical classes possess an anti-
5-reductase activity. For example, aliphatic
polyunsaturated fatty acids such as γ-linoleic acid can
inhibit 5aR enzymes (Liang et al., 1992). Phenolic
compounds such as tannin, isoflavones and chalcones
have also been found to be effective in inhibiting 5aR
enzymes in vitro (Hiipakka et al.; 2001, Liu et al., 2008a;
Shimizu et al., 2000). Moreover, some triterpenoids
isolated from Ganoderma lucidum can also inhibit 5aR
(Liu et al., 2006).
Some plants with reported 5aR inhibitory activity are
Serenoa repens (saw palmetto) fruit (Niederprûm et al.,
1994), Myrica rubra (red bayberry) bark (Matsuda et al.,
2001a), Boehmeria nipononivea (Shimizu et al., 2000a),
Artocarpus incisus (Thai breadfruit) leaf (Shimizu et al.,
2000b), Alpinia officinarum (lesser galangal) rhizome
(Kim et al., 2003), Lygodium japonicum (Japanese
climbing fern) spore (Matsuda et al., 2002), Pleurotus
ostreatus (oyster mushroom) fruiting body, and Lentinula
edodes (shiitake) fruiting body (Fujita et al., 2005).
Thailand is located in Southeast Asia and has
thousands of varieties of plants, one of which might prove
useful as a medicinal supplement to treat androgen-
related disorders. Ten kinds of plants were randomly
selected for screening tests. This study proposed to
screen the 5-reductase inhibitory activity of certain Thai
plants, in order to find new sources of potential agents
against several symptoms caused by excess 5-
reductase activity, and to determine the relationship
between phenolic content and 5-reductase inhibitory
activity.
MATERIALS AND METHODS
Plant materials
Ten kinds of plants were purchased from local markets in Chiang
Mai, Thailand. They were then identified by comparison with the
herbarium specimens at the Faculty of Pharmacy, Chiang Mai
University.
Animals
Six-week-old male Sprague-Dawley (SD) rats were obtained from
the National Laboratory Animal Center, Bangkok, Thailand, and
housed under a 12 h light/dark cycle with free access to food and
water. This study was approved by the Animal Research Ethical
Committee of the Faculty of Pharmacy, Ubon Ratchathani
University, Ubon Ratchathani, Thailand.
Reagents
Dithiothreitol, sucrose, testosterone, finasteride and NADPH were
purchased from Sigma (St. Louis, MO). Methanol, dichloromethane
and absolute ethanol were purchased from Fisher Chemical (Fair
Lawn, NJ). Other chemical compounds were purchased from Wako
Pure Chemical Industry (Osaka, Japan).
Extraction of plants
Each plant was ground and dried in a hot air oven at 40°C for 48 h,
and then extracted by maceration in 95% ethanol for 3 d. Each
marc extract was re-macerated in 95% ethanol for another 3 d. The
ethanol phase was evaporated to dryness under controlled
pressure by using a rotary evaporator (Eyela, Tokyo, Japan).
Preparation of rat microsomes
Rat microsomal suspensions were prepared by following the
method reported by Liu et al. (2006), with some modifications.
Three male SD rats were sacrificed. The livers were removed and
rinsed with cold normal saline solution. Specimens were then
minced with scissors and homogenized in a solution composed
of 0.32 M sucrose and 1 mM dithiothreitol in 0.02 M phosphate
buffer (pH6.5). The homogenate was then centrifuged twice at 4500
x g, 0°C for 30 min each time. All of the supernatants were
collected. The resulting supernatants containing microsomal
particles were tested for soluble protein by the Lowry method
(Lowry et al., 1951) and kept at -50°C until use.
Measurement of steroid 5-reductase inhibitory activity
5-reductase assay was performed according to the method of
Matsuda et al. (2001b) with some modifications. The 3.0 ml reaction
solutions each contained 0.2 ml of various plant extracts in 50%
ethanol solution, 1.0 ml of 0.02 mM phosphate buffer (pH 6.5), 0.3
ml of freshly prepared 500 ppm testosterone solution in 50%
ethanol solution, and 1.0 ml of microsomal suspension. Reactions
were then initiated by the addition of 0.5 ml of 0.77mg/ml NADPH in
phosphate buffer; samples were then incubated at 37 °C for 30 min.
The reactions were then stopped by adding 5.0 ml
dichloromethane, followed by adding 0.5 ml of 100ppm propyl p-
hydroxybenzoate in 50% ethanol (as an internal standard for

Kumar et al. 1267
Table 1. Plants used and their percentage yield of extraction.
Scientific name Family Part used % Yield of the ethanolic extract
Centella asiatica (L.) Urb. Apiaceae Leaf 10.26
Terminalia chebula Retz. Combretaceae Fruit 11.50
Terminalia bellirica (Geartn.) Roxb. Combretaceae Fruit 16.05
Oryza sativa L. Poaceae Grain 2.22
Garcinia mangostana L. Guttiferae Peel 14.78
Ocimum basilicum L. Lamiaceae Whole plant 2.74
Piper nigrum Wall. Piperaceae Fruit 13.60
Citrus reticulata Blanco Rutaceae Peel 14.06
Houttuynia cordata Thunb. Saururaceae Whole plant 2.59
Curcuma longa L. Zingiberaceae Rhizome 7.84
HPLC). Samples were shaken for 60 s, and then centrifuged at 400
x g for 10 min. The water phase was frozen at -50°C. Four ml of
organic phase was decanted and evaporated to dryness. The
residue was redissolved in 5.0 ml methanol. An aliquot of 10.0 µl
was analyzed for remaining testosterone content using high
pressure liquid chromatography (HPLC). Samples were injected
into an analytical Hypersil®-ODS column (Thermo Scientific, USA)
250 x 4.6 mm i.d. with 5m internal particle size, using testosterone
(>98% pure) as a standard. The mobile phase used was 65%
methanol with a flow rate of 1 ml/min and detected by UV
absorbance at 242 nm. The temperature of the column was
controlled at 40°C.
To determine inhibitory activity, two special reactions must be
completed: firstly, a complete reaction (rxn) containing 0.2% of
50%ethanol instead of the extract; secondly, an enzyme blank (ctrl)
that receives 5.0 ml dichloromethane before the addition of
NADPH, so that the conversion of testosterone into DHT does not
occur. The % inhibition was calculated using peak area ratio (r) of
testosterone/internal standard following the equation:
% inhibition = [(rsample - rrxn)/(rctrl - rrxn)]x 100
Finasteride, a well-known 5-reductase inhibitor, was used as a
standard enzyme inhibitor. The IC50 of finasteride was calculated.
From the remaining testosterone content in each sample,
finasteride equivalent anti-5-reductase activity of each extract was
calculated and recorded in terms of finasteride equivalent 5-
reductase inhibition activity (FEA) as a unit of mg finasteride
equivalent per 1 g extract.
Determination of phenolic content
Total phenolic content (TPC) was determined using Folin-Ciocalteu
reagents with gallic acid as a standard, following the method of
Stoilova (2007) with some modification. Briefly, 0.2 ml of diluted
plant extracts was added to 1.0 ml of 0.2 N Folin-Ciocalteu phenol
reagent in a test tube and kept for 5 min. After that, 3.0 ml of 7.5%
sodium carbonate solution was then added. Reactions were kept in
a dark place for 2 h, and then read for UV absorbance at 750 nm.
Gallic acid was used as a standard. TPC of each sample was
expressed as mg gallic acid equivalent (GAE) per 1 g extract.
Statistics
All samples were analyzed in triplicate. All values were expressed
as mean ± SD. To compare several groups, analysis of variance
was used. Significant differences between means were determined
by Duncan’s multiple range tests. Pearson’s correlation coefficient
was used to predict the relationship between 5-reductase
inhibitory activity and TPC. A probability value of p < 0.05 was
adopted as the criteria for the significant differences.
RESULTS
The plants and the parts used in this research are shown
in Table 1. The plants were randomly selected from a
variety of families. All of these plants are easily acquired
and widely used in Thailand. The % yields of their
ethanolic extracts ranged from 2.22 to 16.05%.
Terminalia bellirica (Geartn.) Roxb. had the highest %
yield of extraction at 16.05%, followed by Garcinia
mangostana L. (14.78%) and Citrus reticulata Blanco
(14.06%), while the lowest yields were found in the red
strain of Oryza sativa L. (2.22%), Houttuynia cordata
Thunb. (2.59%) and Ocimum basilicum L. (2.74%).
Rat microsomal suspensions appeared opaque pinkish
in color, and contained 4.71 mg/ml soluble protein as
assessed by the Lowry method. With given HPLC
conditions, propyl p-hydroxybenzoate (an internal
standard) and testosterone gave retention times of
around 5 and 8 min, respectively. HPLC chromatograms
of the complete reaction, enzyme blank, 0.5µM
finasteride, and O. basilicum are shown in Figures 1A, B,
2A, and B, respectively.
5-reductase inhibitory activity of finasteride was
calculated as IC50 of 0.39 µM. The correlation between
inhibitory activity (as % inhibition) and concentration of
finasteride was expressed as:
y = 166.78x - 15.285 (R2 = 0.999)
with y representing % inhibition and x the concentration
of finasteride. Based on the given equation, FEA values
of the extracts were calculated and expressed in Table 2.
The 5aR inhibitory activity of each extract can be

1268 J. Med. Plant. Res.
Figure 1. HPLC chromatogram of: (A) complete reaction control and (B) enzyme blank. a, b
and c represent dithiotreitol, propyl p-hydroxybenzoate, and testosterone, respectively.
arranged from higher to lower, as follows: O. basilicum,
O. sativa, H. cordata, Curcuma longa L., Centella asiatica
(L.) Urb., Terminalia chebula Retz., G. mangostana,
T.bellirica, Piper nigrum Wall., and C. reticulata,
respectively. FEA values of the extracts ranged from
17.59 to 5.56 mg finasteride equivalent per 1 g extract.
The best inhibitory activity was achieved by O. basilicum
extract, and the lowest inhibitory activity was found in
Citrus reticulata extract. There were no significant
differences in 5aR inhibitory activity in C. longa and C.
asiatica, or in G. mangostana, T. bellirica, and P. nigrum.
TPC of each extract (Table 3) ranged from 32.00 to
370.85 mg GAE per 1 g extract. They can be arranged
from higher to lower as follows: T. bellirica, T. chebula, C.
longa, G. mangostana, O. sativa, C. reticulata, H.
cordata, P. nigrum, O. basilicum, and C. asiatica,
respectively. T. bellirica had the highest TPC, followed by
T. chebula (286.04 mg GAE per 1 g extract). C. asiatica
had the lowest TPC. There were no significant
differences in TPC among C. longa and G. mangostana,
or among O. sativa, C. reticulata, H. cordata, P. nigrum,
and O. basilicum.

Kumar et al. 1269
Figure 2. HPLC chromatogram of: (A) 0.5 µM finasteride and (B) Ocimum basilicum. a, b,
and c represent dithiotreitol, propyl p-hydroxybenzoate, and testosterone, respectively.
DISCUSSION
Ten kinds of plants from different families were randomly
selected. The parts of the plants used in this experiment
are common used by Thai people for cooking, and also
by traditional practitioners for medicinal treatment. After
the processes of extraction, it was found that the crude
extracts of all plants seemed to have a sticky, semi-solid
appearance.
Rat microsomal suspensions consisted of a 5α-
reductase enzyme and other enzymes that may be able
to metabolize the substrate testosterone. Therefore, a
control reaction was necessary to minimize errors. Two
control reactions were evaluated. Firstly, the completed
reaction control was one in which 5aR had full activity
and could metabolize testosterone into DHT. Secondly,
the enzyme blank was a reaction in which 5aR had none
of the activity which is acquired by denaturing the
enzyme; this helps in determining the total amounts of
testosterone in the reactions studied. When the reactions
were complete, the reacted tubes were further treated as
described, and injected into a HPLC system. Generally,
the determination 5α-reductase inhibitory activity is
performed by radioimmunoassay (RIA); but the RIA

1270 J. Med. Plant. Res.
Table 2. Activity of plant extract on inhibition of 5-reductase enzyme.
Plants Finasteride equivalent 5-reductase inhibition activity
(mg finasteride/ 1 g crude extract)1
O. basilicum L. 17.59 ± 1.00a
O. sativa L. 16.72 ± 0.95b
H. cordata Thunb. 15.37 ± 1.50c
C. longa L. 13.83 ± 1.03d
C. asiatica (L.) Urb. 13.73 ± 1.05d
T. chebula Retz. 12.74 ± 0.84e
G. mangostana L. 11.62 ± 1.18f
T. bellirica (Geartn.) Roxb. 11.58 ± 0.84f
P. nigrum Wall. 11.18 ± 0.81f
C. reticulata Blanco 5.56 ± 1.12g
1-value in table expressed as mean ± SD of triplicate experiments. Means in column with different letters are significantly
different (p<0.05).
Table 3. Total phenolic content of extracts.
Plants Total Phenolic content (mg GAE/ 1 g extract)1
T. bellirica (Geartn.) Roxb. 370.85 ± 26.80a
T. chebula Retz. 286.04 ± 3.37b
C. longa L. 218.26 ± 14.90c
G. mangostana L. 205.90 ± 6.05c
O. sativa L. 75.48 ± 6.04d
C. reticulata Blanco 75.53 ± 5.46d
H. cordata Thunb. 67.67 ± 3.43d
P. nigrum Wall. 60.75 ± 3.26d
O. basilicum L. 63.12 ± 0.57d
C. asiatica (L.) Urb. 32.00 ± 0.91e
1-value in table expressed as mean ± SD of triplicate experiments. Means in column with different letters are
significantly different (p<0.05).
method has many limitations, such as the dangerous
from radioactive compounds and requiring complex
equipment. The HPLC method was developed by
Matsuda et al. (2001) to replace the RIA method. This
method for determination of 5α-reductase inhibition
activity is comparable to RIA and GC-MS. In our
experiment, the IC50 of finasteride was 0.39 µM, which is
comparable to the previous report of 0.34 µM (Park et al.,
2003) which was assessed by the RIA method. According
to HPLC conditions in this experiment, it was found that
propyl p-hydroxybenzoate and testosterone have a good
resolution and selectivity. Finasteride was used as a
standard enzyme inhibitor in the 5-reductase inhibition
experiment because it is a well-known drug of choice to
treat DHT-related disorders. In the process of comparing
the activities of samples, we formulated a new term –
finasteride equivalent 5-reductase inhibitory activity, or
FEA value – based on the inhibitory activity of selected
plants at selected concentrations, converted into a
finasteride equivalent in units of mg finasteride equivalent
per g extract. FEA value is proportionally related to 5-
reductase inhibition activity. The higher the FEA value,
the higher the 5-reductase inhibition activity.
All of the plant extracts used in this report had a
different ability to inhibit 5α-reductase enzyme. Among
the extracts, O. basilicum or basil, the most potent 5aR
inhibitor, contained volatile compounds in a class of
terpenoids and aliphatic alcohols (Politeo et al., 2007).
These compounds may be responsible for the highest
FEA value. The red strain of O. sativa, or red rice, was
used in this experiment. It contains a high level of
anthocyanin, which was classified as one of the phenolic
compounds, reported to be 5aR inhibitors (Hiipakka,
2001). The lowest activity of 5α-reductase inhibition was

found in C. reticulata, or tangerine, with the FEA value of
5.56. This was surprising because tangerine peel
contains several flavonoids which have been reported to
be 5aR inhibitors (Hiipakka et al.; 2001).
To determine whether phenolic compounds in the
plants studied were the main active phytochemicals
involved in enzyme inhibition, TPC of these plant extracts
were determined. The results showed that each plant
extract had different TPC. Unfortunately, there was no
correlation between 5α-reductase inhibitory activity and
TPC; Pearson’s correlation coefficient was -0.169, p =
0.373. This suggested that phytochemicals other than
phenolic compounds may play an important role in
enzyme inhibition. As seen in O. basilicum, the plant with
the highest FEA value has lower TPC.
The usual doses of finasteride to treat alopecia and
benign prostatic hypertrophy (BPH) are 1 and 5 mg/d,
respectively. From FEA values of each plant, it may be
assumed that regular intake of these fresh plants or their
extracts may be beneficial in preventing and treating
symptoms related to excess 5aR activity. Further
investigations of other bioactive phytochemical classes,
which may play a role in enzyme inhibition and the in vivo
activity of these extracts, will be conducted.
ACKNOWLEDGMENTS
The author would like to thank the Office of Higher
Education Commission, Thailand for supporting by grant
fund under the program Strategic Scholarships for
Frontier Research Network for the Join Ph.D. Program
Thai Doctoral degree for this research. This research was
also supported by Office of the National Research
Council of Thailand and Faculty of Pharmacy, Ubon
Ratchathanee University.
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