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

Authors:
  • Chiang Mai Univ. /Univ. of Phayao. Thailand

Abstract and Figures

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.
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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, 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 5m 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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Chem., 102: 764-770.
... The concentrated residue (1.5 ml) was later diluted in 3 ml methanol. Finally, RMs were syringe filtered (0.2 µm) before HPLC run to avoid contamination (Sher et al., 2015;Kumar et al., 2011). ...
... Isocratic elution was performed, making a mobile phase with methanol and water (80: 20) and filtering it to remove any contamination. The percentage inhibition of SRD5AII was observed from the peak height and the peak area ratio (r) applying the following formula (Kumar et al., 2011):(%) r sample − r reaction r standard − r reaction × 100 r Area of sample Area with least value ; Peak area (mm 2 ) height × width 1/2 . ...
... The prostate gland holds sufficient amount of isozyme SRD5AII, and its homogenate explains the phenomena of testosterone's conversion into DHT in RM (Steers, 2001). To study the inhibitory activity of SRD5AII, 17 hydroalcoholic Thai plant extracts were examined using HPLC analysis (Kumar et al., 2011). The in vitro study describes limiting the conversion of testosterone to DHT by lowering the catalytic activity of SRD5AII. ...
Article
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The screening of hair follicles, dermal papilla cells, and keratinocytes through in vitro, in vivo, and histology has previously been reported to combat alopecia. Ficus benghalensis has been used conventionally to cure skin and hair disorders, although its effect on 5α-reductase II is still unknown. Currently, we aim to analyze the phytotherapeutic impact of F. benghalensis leaf extracts (FBLEs) for promoting hair growth in rabbits along with in vitro inhibition of the steroid isozyme 5α-reductase II. The inhibition of 5α-reductase II by FBLEs was assessed by RP-HPLC, using the NADPH cofactor as the reaction initiator and Minoxin (5%) as a positive control. In silico studies were performed using AutoDock Vina to visualize the interaction between 5α-reductase II and the reported phytoconstituents present in FBLEs. Hair growth in female albino rabbits was investigated by applying an oral dose of the FBLE formulation and control drug to the skin once a day. The skin tissues were examined by histology to see hair follicles. Further, FAAS, FTIR, and antioxidants were performed to check the trace elements and secondary metabolites in the FBLEs. The results of RP-HPLC and the binding energies showed that FBLEs reduced the catalytic activity of 5α-reductase II and improved cell proliferation in rabbits. The statistical analysis (p < 0.05 or 0.01) and percentage inhibition (>70%) suggested that hydroalcoholic FBLE has more potential in increasing hair growth by elongating hair follicle’s anagen phase. FAAS, FTIR, and antioxidant experiments revealed sufficient concentrations of Zn, Cu, K, and Fe, together with the presence of polyphenols and scavenging activity in FBLE. Overall, we found that FBLEs are potent in stimulating hair follicle maturation by reducing the 5α-reductase II action, so they may serve as a principal choice in de novo drug designing to treat hair loss.
... The enzyme 5α-reductase metabolizes testosterone to dihydrotestosterone (DHT). However, imbalance between testosterone and DHT causes acne and benign prostatic hyperplasia [26,27]. In AV, sebum secretion, dehydration of the corneum, and transepidermal water loss (TEWL) are increased. ...
... Furthermore, the activated sebaceous gland increases the production of sebum leading to blockage of the sebaceous gland duct, causing acne. The enzyme 5αreductase metabolizes testosterone to DHT, but abnormally high 5α-reductase activity results in excessive DHT production [26]. .It is reported that caffeoyl derivatives from Adina rubella showed good 5α-reductase inhibitory activity [57]. ...
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Background: Carpinus tschonoskii (CT) has been previously studied for various activities in the improvement of skin diseases. In the present study, we examined the in vitro anti-acne vulgaris (AV) effect of CT leaves (CTL) and tellimagrandin I (TI), one of the main ellagitannins from CT, including skin barrier improvement and 5α-reductase inhibitory activity. Methods: To test the anti-AV activities of CTL and TI, firstly, anti-oxidative and anti-inflammatory activities including DPPH radical scavenging activity, nitric oxide (NO) inhibitory activity, and cytokines [interleukin (IL)-6 and IL-8] were tested. Skin barrier improvement experiments were tested using developing cornified envelope (CE) formation, and filaggrin mRNA expression level was determined by RT-PCR. The 5α-reductase inhibitory activity was determined by measuring the testosterone levels in rat liver microsomes. Results: CTL and TI showed potent anti-oxidative activity and anti-inflammatory activities. Especially, the cytokine production inhibitory activities of TI were found to be similar to the positive control, epigallocatechin gallate (EGCG). CTL and TI enhanced the CE formation and filaggrin mRNA expression levels and showed potent activities compared to that in the positive control, 1.5 mM Ca2+. In additionally, CTL and TI showed 5α-reductase inhibitory activities in a dose-dependent manner. Conclusion: The results showed that CTL and TI inhibit AV endogenous factors such as 5α-reductase and inflammatory cytokines and affect exogenous factors such as developing skin barrier function (CE and filaggrin levels). Therefore, CTL and TI may be plant-derived agent, promising in the treatment of acne vulgaris.
... Study of the reductase inhibition activity of dutasteride with nanocarriers should therefore be undertaken, and this will give an idea of how the cell proliferation activity of the nanoparticles formulations has been achieved. This work should be undertaken using rat microsomal suspension from the liver as described previously (Kumar et al., 2011). DST-NLCs or empty NLCs, uncoated and coated with CSO-SA or CSO-LA will be reacted with the supernatants from the homogenated-liver with the addition of testosterone. ...
... DST-NLCs or empty NLCs, uncoated and coated with CSO-SA or CSO-LA will be reacted with the supernatants from the homogenated-liver with the addition of testosterone. 5α-reductase inhibition activity can be calculated, with finasteride used as a standard enzyme inhibitor (Kumar et al., 2011). The results will be expressed as finasteride equivalent 5α-reductase inhibitory activity value (units of mg finasteride equivalent per 1 g sample). ...
Thesis
INTRODUCTION: Androgenic alopecia (AA) patients usually have high levels of dihydrotestosterone on their balding scalp area. Currently, dutasteride (DST) is given orally and has systemic adverse effects; diminished sexual desire, increased depression and ejaculation disorder. Topical administration of DST is an appropriate drug-delivery strategy with the potential to reduce systemic side effect, skin irritation and cytotoxicity effects. MATERIALS AND METHOD: Chitosan oligomer (CSO) conjugated with stearic acid (SA) or lauric acid (LA) was synthesised and characterised. Dutasteride-loaded nanostructured lipid carriers (DST-NLCs) were prepared using a melt-dispersion ultrasonication method. DST-NLCs were optimised using a design of experiments approach. DST-NLCs, uncoated and coated with CSO-SA or CSO-LA were characterised for particle size distribution, surface charge and morphology. In vitro release and permeation studies were performed. Cytotoxicity was investigated using human hair follicle dermal papilla cells, and skin irritation was performed using an EpiDerm™ RHE model. Cou-6 loaded NLCs were prepared and characterised before proceeding with the cell and skin uptake study. RESULTS: CSO-SA and CSO-LA were successfully synthesised; confirmed using 1H NMR and FTIR. The mean size of DST-NLCs was significantly increased (p<0.05) when coated with 5% CSO-SA but not with 5% CSO-LA (p>0.05). The zeta potential changed from negative to positive charge when coating DST-NLCs with CSO-SA or CSO-LA. All formulations were physically stable over six months when stored at 4-8°C. However, DST-NLCs coated with CSO showed aggregation. All formulations exhibited rapid drug release. No dutasteride permeated through pig ear skin after 48 h for all formulations. The cytotoxicity (IC50) for DST nanoparticles, coated and uncoated, was greater than for DST alone (p<0.05). The in vitro skin irritation study indicated no irritation for all nanoparticle preparations. For the cell and skin uptake studies, all samples showed time-dependent skin and cell uptake. CONCLUSIONS: These stable, low cytotoxic and irritant, positively-charged DST-NLCs with CSO-SA or CSO-LA, represents a promising strategy for topical/ transfollicular delivery of DST.
... Thus, it is contraindicated in women who are currently pregnant or hope to become pregnant in the future (Sallout & Al Wadi, 2009). In addition, many patients prefer to avoid oral medications due to possible adverse effects, such as decreased libido and erectile dysfunction in men (Kumar et al., 2011). Topical minoxidil is commonly used as an off-label treatment for male-and female-pattern baldness. ...
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To prepare a topical formulation of bimatoprost (BIM) with high skin permeability, we designed a solvent mixture system composed of ethanol, diethylene glycol monoethyl ether, cyclomethicone, and butylated hydroxyanisole, serving as a volatile solvent, nonvolatile co-solvent, spreading agent, and antioxidant, respectively. The ideal topical BIM formulation (BIM–TF#5) exhibited 4.60-fold higher human skin flux and a 529% increase in dermal drug deposition compared to BIM in ethanol. In addition, compared to the other formulations, BIM–TF#5 maximally activated human dermal papilla cell proliferation at a concentration of 5 μM BIM, equivalent to 10 μM minoxidil. Moreover, BIM–TF#5 (0.3% [w/w] BIM) significantly promoted hair regrowth in the androgenic alopecia mouse model and increased the area covered by hair at 10 days by 585% compared to the vehicle-treated mice, indicating that entire telogen area transitioned into the anagen phase. Furthermore, at day 14, the hair weight of mice treated with BIM–TF#5 (5% [w/w] BIM) was 8.45- and 1.30-fold greater than in the 5% (w/w) BIM in ethanol and 5% (w/v) minoxidil treated groups, respectively. In the histological examination, the number and diameter of hair follicles in the deep subcutis were significantly increased in the BIM–TF#5 (0.3 or 5% [w/w] BIM)-treated mice compared to the mice treated with vehicle or 5% (w/w) BIM in ethanol. Thus, our findings suggest that BIM–TF#5 is an effective formulation to treat scalp alopecia, as part of a novel therapeutic approach involving direct prostamide F2α receptor-mediated stimulation of dermal papilla cells within hair follicles.
... Several reports suggest that male pattern hair loss or AA are linked to dihydrotestosterone (DHT) [4,5]. DHT on the hair follicles was metabolized from testosterone by 5αreductase [6]. Two isoforms of 5α-reductase are Type I and Type II, the former present in the skin and outer root sheath of the hair follicles, and the latter most evident in the prostate, seminal vesicles, and inner epithelial root sheath of hair follicles. ...
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The study aimed to develop the finasteride-loaded proniosome (FLP) to enhance the transfollicular delivery of finasteride (FN). The response surface methodology (RSM) combined with central composite design (CCD) with three independent variables (FN concentrations, total lipid content, and cholesterol content) was used to optimize the FLP preparation. The particles size, zeta potential, entrapment efficiency, and drug loading capacity of the FLP were analyzed. The transfollicular delivery of the optimum formulation was investigated in vitro. In vivo hair growth stimulation study was performed on C57BL/6Mlac mice dorsal areas. The Draize primary skin irritation test for erythema and edema was performed in the New Zealand white rabbit skin. The optimum FLP consists of 5.0 mM of FN, 10.1 mM of total lipid content, and 50.0% of the cholesterol in the total lipid. The prepared proniosome delivered the FN significantly (p < 0.05), compared to the naked finasteride solution in a dose- and time-dependent manner. The FLP treatment significantly increases the number and size of hair follicles in a dose-dependent manner. The efficiency of 1% FLP was comparable to the 2% minoxidil solution. The FLP exhibited no skin irritation after 72 h. Therefore, the results demonstrated that the FLP could stimulate hair growth via a transfollicular delivery system.
... Oxypeucedanin isolated from A. koreana exhibited a weak inhibitory activity against 5α-reductase type I, an enzyme responsible for changing androgen testosterone into its activated form, dihydrotestosterone (DHT) [112]. This compound, with an IC 50 higher than 20 µg/mL, demonstrated inactivity against LNCaP (androgen-sensitive human prostate adenocarcinoma) cells, although an IC 50 of 19.8 µg/mL was recorded for finasteride as the control drug [37]. ...
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The present review comprehensively gathered phytochemical, bioactivity, and pharmacokinetic reports on a linear furanocoumarin, namely oxypeucedanin. Oxypeucedanin (OP), which structurally contains an epoxide ring, has been majorly isolated from ethyl acetate-soluble partitions of several genera, particularly Angelica, Ferulago, and Prangos of the Apiaceae family; and Citrus, belonging to the Rutaceae family. The methanolic extract of Angelica dahurica roots has been analytically characterized as the richest natural OP source. This naturally occurring secondary metabolite has been described to possess potent antiproliferative, cytotoxic, anti-influenza, and antiallergic activities, as assessed in preclinical studies. In order to explore potential drug candidates, oxypeucedanin, its derivatives, and semi-synthetically optimized analogues can be considered for the complementary assessments of biological assays.
... Ferulic acid, γ-oryzanol, and phytic acid appeared to have unfavourable interactions. Similarly to a previous study, there was no correlation between SRD5A inhibitory activity and the TPC [88]. Strikingly, tocopherols displayed a higher affinity towards SRD5A2 compared with dutasteride and other active constituents. ...
Article
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Steroid 5-alpha reductases (SRD5As) are responsible for the conversion of testosterone to dihydrotestosterone, a potent androgen, which is the aetiologic factor of androgenetic alopecia. This study aimed to compare the SRD5A gene expression suppression activity exerted by Thai rice bran extracts and their components and investigate the interactional mechanism between bioactive compounds and SRD5A2 using molecular dynamics (MD) simulation. Bran of Oryza sativa cv. Tubtim Chumphae (TRB), Yamuechaebia Morchor (YRB), Riceberry (RRB), and Malinil Surin (MRB), all rice milling by-products, was solvent-extracted. The ethanolic extract of TRB had the highest sum of overall bioactive compounds (γ-oryzanol; α-, β-, and γ-tocopherol; phenolics; and flavonoids). Among all extracts, TRB greatly downregulated the expression of SRD5A1, SRD5A2, and SRD5A3; there were no significant differences between TRB and finasteride regarding SRD5A suppression. The linear relationship and principal component analysis supported that the α-tocopherol content was correlated with the SRD5A suppression exerted by TRB. Furthermore, MD simulation demonstrated that α-tocopherol had the highest binding affinity towards SRD5A2 by interacting with residues Phe118 and Trp201. Our findings indicate that α-tocopherol effectively downregulates the expression of SRD5A genes and inhibits SRD5A2 activity, actions that are comparable to standard finasteride. TRB, a source of α-tocopherol, could be developed as an anti-hair loss product.
... About 250 g portions of the dried material were separately macerated with exhaustion with hexane, ethyl acetate, methanol and distilled water. Combined hexane, ethyl acetate and methanol extracts were concentrated in vacuo (Heodolph, Schwabach, Germany) and then air-dried while combine aqueous extracts were lyophilized (KD Freeze Dryer, Zhengzhou Keda Machinery, Mainland, China) (Kumar et al., 2011;Hossain et al., 2013). ...
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Context: The prevalence of alopecia around the world is high and awareness continuously increased due to social stigma. There are limited commercially-available medications for the management of different forms of baldness, most of which are prohibitively expensive and presents with various adverse effects. Aims: To evaluate the flavonoid-rich ethyl acetate extract of Equisetum hyemale stem for its hair-regenerative properties in chemically-induced alopecia in Sprague Dawley rats. Methods: Various concentrations of the flavonoid-rich extract of E. hyemale were applied in depilated areas in dorsal rat skin after chemical induction of alopecia. Evidence of hair growth was observed for 28 days in a weekly basis. Histopathological analysis of rat skin was performed to demonstrate evidence of follicular hair growth. Results: The abundance of flavonoids in the ethyl acetate extract was established by total flavonoid contents and LC-MS analyses. The 2.5% E. hyemale extract exhibited hair-regenerative properties with high tensile strengths, combined masses and follicular growth, which was comparable to the positive control, 5% minoxidil (p>0.05). The highest rate of hair follicular development was observed in the telogen phase in rat skin after 28 days of treatment. Conclusions: The flavonoid-rich ethyl acetate extract of E. hyemale exhibits in situ hair-regenerative properties in chemically-induced alopecia in rats at 2.5% w/v concentration.
... About 250 g portions of the dried material were separately macerated with exhaustion with hexane, ethyl acetate, methanol and distilled water. Combined hexane, ethyl acetate and methanol extracts were concentrated in vacuo (Heodolph, Schwabach, Germany) and then air-dried while combine aqueous extracts were lyophilized (KD Freeze Dryer, Zhengzhou Keda Machinery, Mainland, China) (Kumar et al., 2011;Hossain et al., 2013). ...
Article
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Context: The prevalence of alopecia around the world is high and awareness continuously increased due to social stigma. There are limited commercially-available medications for the management of different forms of baldness, most of which are prohibitively expensive and presents with various adverse effects. Aims: To evaluate the flavonoid-rich ethyl acetate extract of Equisetum hyemale stem for its hair-regenerative properties in chemically-induced alopecia in Sprague Dawley rats. Methods: Various concentrations of the flavonoid-rich extract of E. hyemale were applied in depilated areas in dorsal rat skin after chemical induction of alopecia. Evidence of hair growth was observed for 28 days in a weekly basis. Histopathological analysis of rat skin was performed to demonstrate evidence of follicular hair growth. Results: The abundance of flavonoids in the ethyl acetate extract was established by total flavonoid contents and LC-MS analyses. The 2.5% E. hyemale extract exhibited hair-regenerative properties with high tensile strengths, combined masses and follicular growth, which was comparable to the positive control, 5% minoxidil (p>0.05). The highest rate of hair follicular development was observed in the telogen phase in rat skin after 28 days of treatment. Conclusions: The flavonoid-rich ethyl acetate extract of E. hyemale exhibits in situ hair-regenerative properties in chemically-induced alopecia in rats at 2.5% w/v concentration.
... Rosemary extract (R. officinalis) is found to have antiaging potential attributed to its active ingredient carnosic acid (Calabrese et al. 2000;Birtić et al. 2015). Anthocyanins extracted from Ocimum basilicum possess antiandrogenic action and is used in anti-hair loss preparations (Kumar et al. 2011). A formulation of cinnamon with centella and tamarindus was proved to increase the moisture content and decrease the melanin content of the skin (Saraf et al. 2012). ...
Chapter
The cosmetic industry is a high-valued and evergreen multibillion dollar industry with more specialized and advanced products adding up every year. The major product categories in the cosmetic industry are skin care, hair care, perfumes, deodorants, toiletries, and make-up. Of these, skin care products top the list, accounting around 36 % of the global cosmetic market. Natural products, a treasure of medicinally active compounds are used for treating various skin ailments, infections, inflammation and as a protectant of UV irradiation and pollution. The hybrid of cosmetic and pharmaceutical compounds, known as cosmeceuticals, possesses therapeutic as well as beautification potential based on its key ingredients. Natural products are well regarded as a rich source of cosmeceuticals. Different classes of natural compounds originating from animal, plant, and marine algal sources are placed under the category of high-valued cosmetic ingredients. The extraction of fatty acid components from botanicals and other natural sources opens up a big market in the cosmetic industry. The present chapter introduces the recent advancement and strategies followed in the cosmeceutical industry, and the role of plant tissue culture in enhancing the production of pharmaceutically valued natural products along with the current regulatory policies.
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We have reported the presence, in rat liver microsomes, of many A4-3-ketosteroid reductases (50() which catalyze the hy-drogenation of the 4-5 double bond of various unsaturated ster-oids to yield the corresponding & dihydrosteroids (1, 2). In this communication some characteristics of these enzymes, par-ticularly those relating to substrate specificity, will be described. EXPERIMENTAL PROCEDURE Materials and Methods Preparation of Microsomal Fraction of Rat Liver-Female Sprague-Dawley rats were decapitated and their livers removed and placed on ice immediately. The microsomal fraction, pre-pared by differential centrifugation (3), was resuspended so that 1 ml of suspension represented the microsomes in 1 g of liver. The most active preparations catalyzed the reduction of 450 mpmoles of cortisone1 per minute per gram of original liver. Fresh microsomes varied almost 2-fold in activity. Steroid sub-strates and metabolites were chromatographed and located on paper as previously described (3). Assay of A4-S-Ketosteroid Reductase (5a)-Reductase activity was determined by extracting, with dichloromethane or cyclo-hexane, aliquots of the reaction mixture which were removed be-fore and after incubation, and by measuring the decrease in opti-cal density that occurred with the saturation of the 4-5 double bond (3, 4). In cyclohexane, the X,,, of the substrates was at 232 and in dichloromethane at 240 rnp. In most experiments, excess TPNH was generated by coupling TPN reduction to the oxidation of isocitric acid in the presence of pig heart isocitric acid dehydrogenase (5). In experiments designed to determine the effect of pH, the affinity of the reductases for TPNH, or in-hibition by various reagents, TPNH as such was added to a mix-ture of steroid, microsomes, and buffer in a Thunberg tube at OO. iln aliquot was removed, the tube evacuated, and, after an appropriate incubation, a second aliquot was removed. These aliquots were extracted with dichloromethane as in the regular assay and their optical density at 240 rnp determined. Inhibition by Steroids-The inhibitory effect of one steroid on the reduction of another was measured for many different steroid pairs. When the inhibitory steroid whose effect was to be meas-ured had no absorption at 240 mp, the reduction of the 4-5-un-saturated 3-ketosteroid substrate could be measured as usual.
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The acetone extract of Boehmeria nipononivea showed both potent 5 alpha-reductase inhibitory activity and hair regrowth promotion effects on mice. 5 alpha-Reductase inhibitory activity-guided fractionation led to six active fatty acids: alpha-linolenic, linoleic, palmitic, elaidic, oleic and stearic acids. The extract of B. nipononivea, and alpha-linolenic, elaidic and stearic acids exhibited a hair regrowth effect.
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The diethyl ether extract of Anemarrhenae Rhizoma (rhizomes of Anemarrhena asphodeloides BUNGE) showed testosterone 5α-reductase inhibitory activity. Two major constituents, cis-hinokiresinol (1) and 2,6,4'-trihydroxy-4-methoxybenzophenone (2) were identified as the active principles. The inhibitory activity of 1 was superior to that of ethinylestradiol, but that of 2 was weak.
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This paper describes the rational design and synthesis of novel inhibitors of human steroid 5α-reductase. Steroidal nitrones were synthesised via an eight-step sequence from epiandrosterone and were tested for activity against type I and II 5α-reductase isozymes. Judicious placement of the nitrone into the steroid A-ring provided an effective and stable transition-state mimic of the postulated enolate intermediate involved in the conversion of testosterone into dihydrotestosterone.
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In humans, steroid 5α-reductase is involved in the development of benign prostatic hyperplasia (BPH). We tested the steroid 5α-reductase inhibitory activity of the 70% acetone extract of woody plants. The tannin polymer prepared from the 70% acetone extract showed the highest steroid 5α-reductase inhibitory activity. Furthermore, the steroid 5α-reductase inhibitory activity of tannins was not affected by the addition of superoxide dismutase or catalase. It was concluded that the steroid 5α-reductase inhibitory activity of condensed tannin was caused by binding to the steroid 5α-reductase, rather than from the peroxide/superoxide produced by tannins. The tannins, prepared from tree barks, with potential for steroid 5α-reductase inhibitory activity might be advantageous in therapy for steroid 5α-reductase diseases such as BPH or prostate cancer.
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Hair growth depends on a close interaction of different cell populations of the hair follicle. In certain regions of the body, androgens interfere with this highly regulated cooperation in a yet poorly understood manner. The response of hair follicles to androgens can be categorized as androgen-dependent, e.g. in the beard, androgen-sensitive, e.g. in the frontal scalp of affected individuals, or androgen-independent, e.g. in the occipital scalp. At the target cell level, the balance between 5α-reductase, 17β-hydroxysteroid-dehydrogenase (17β-HSD) and 3α-hydroxysteroid dehydrogenase (3α-HSD) yields metabolites with different androgenic potential. We examined this target cell-specific androgen metabolism in microdissected intact subunits of dermal papillae, connective tissue sheaths (CTS) and root sheaths. In dermal papillae, 5α-reductase predominated with an accumulation of the strong androgen 5α-dihydrotestosterone. The specific activity of 5α-reductase in the papillae exceeded those in the other hair follicle compartments by a factor of at least 14 in the scalp (5.4, 0.4 and 0.1 pmol/h per mm 3 in the papilla, CTS and root respectively and at least 80 in the beard (16.0, 0.2 and 0.4 pmol/h per mm 3 in the papilla, CTS and root respectively). The root sheath keratinocytes expressed low 5α-reductase levels, but high 17βHSD levels, with androstenedione as the major metabolite. The CTS expressed both 5α-reductase and 17β-HSD, resulting in androstenedione, 5α-androsterone and 5α-androstanedione. In the CTS and the root sheath, only minor amounts of 5α-DHT were found. In beard papillae, the 5α-reductase activity was three times that in the occipital scalp papillae. These results indicate that the androgen response of hair follicles depends on a differentiated intrafollicular androgen metabolism and that the dermal papilla might be a primary target in this process.
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The present paper examines the chemical composition and antioxidant capacity of free volatile aglycones from basil compared to their essential oil. The comparison of chemical composition of volatile aglycones with the chemical composition of essential oil reveals four common compounds: eugenol, chavicol, linalool and α-terpineol. For the evaluation of the mentioned antioxidant capacities, two different methods were performed: the 2,2′-diphenyl-1-picrylhydrazyl radical scavenging method (DPPH) and ferric reducing/antioxidant power assay (FRAP). DPPH method shows that free volatile aglycones possess good antioxidant properties comparable with that of the essential oil and well-known antioxidant butylated hydroxytoluene (BHT), but less than pure eugenol. The results obtained by FRAP method show that these compounds are some less effective antioxidants than essential oil and BHT.