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The aim of present study was to investigate hair growth promoting effects of extracts of Trigonella foenum-graecum leaves. The extracts of powdered leaves were obtained in various solvents (petroleum ether, chloroform, methanol, ethanol and distilled water) using hot and cold extraction methods. Phytochemical analysis of leaves was performed on powder and extracts of leaves using already reported methods. Mineral analysis was done using atomic absorption spectrophotometer. Hair growth promoting effects were examined using alopecia mouse model. Phytochemical analysis demonstrated the presence of higherquantities of carbohydrates, proteins and secondary metabolites in methanol compared to other extracting solvents. Moreover, only ethanol (5 and 10%) and petroleum ether (5%) demonstrated significant hair growth promoting effects (p < 0.05) compared to standard, i.e., 5% minoxidil and extracts in other solvents. Likewise, ethanol (5% and 10 %) and petroleum ether (5%) extract had significant impact (p < 0.05) on hair length in comparison to minoxidil, however, no significant differences were observed between ethanol (5% and 10%) extracts and minoxidil on mice hair diameter. Taken together, our data suggested that ethanol extracts of leaves of T. foenum-graecum had significantly higher growth promoting effects compared to standard, i.e., minoxidil. Thus, results from our study set the stage of further studies to identify the constituents in ethanol extracts, responsible for hair growth promoting effects.
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Impact of Trigonella foenum-graecum Leaves
Extract on Mice Hair Growth
Fariha Imtiaz1, Muhammad Islam1, Hamid Saeed2,*, Bushra Saleem1, Maryam Asghar1
and Zikria Saleem2
1Section of Pharmaceutical Chemistry, University College of Pharmacy, University
of the Punjab, Pakistan
2Section of Clinical Pharmacy, University College of Pharmacy, University of the
Punjab, Pakistan
Article Information
Received 13 December 2016
Revised 05 February 2017
Accepted 15 March 2017
Available online 21 July 2017
Authors’ Contribution
MI and HS design the study. FI and
MA conducted the experiments and
FI, HS and ZS analyzed data. HS and
BS wrote the article while MI and ZS
edited it.
Key words
Fenugreek, Hair growth, Hair
treatment, Trigonella foenum-
graecum, Alopecia, Zinc.
The aim of present study was to investigate hair growth promoting effects of extracts of Trigonella
foenum-graecum leaves. The extracts of powdered leaves were obtained in various solvents (petroleum
ether, chloroform, methanol, ethanol and distilled water) using hot and cold extraction methods.
Phytochemical analysis of leaves was performed on powder and extracts of leaves using already reported
methods. Mineral analysis was done using atomic absorption spectrophotometer. Hair growth promoting
effects were examined using alopecia mouse model. Phytochemical analysis demonstrated the presence
of higherquantities of carbohydrates, proteins and secondary metabolites in methanol compared to
other extracting solvents. Moreover, only ethanol (5 and 10%) and petroleum ether (5%) demonstrated
signicant hair growth promoting effects (p < 0.05) compared to standard, i.e., 5% minoxidil and extracts
in other solvents. Likewise, ethanol (5% and 10 %) and petroleum ether (5%) extract had signicant
impact (p < 0.05) on hair length in comparison to minoxidil, however, no signicant differences were
observed between ethanol (5% and 10%) extracts and minoxidil on mice hair diameter. Taken together,
our data suggested that ethanol extracts of leaves of T. foenum-graecum had signicantly higher growth
promoting effects compared to standard, i.e., minoxidil. Thus, results from our study set the stage of
further studies to identify the constituents in ethanol extracts, responsible for hair growth promoting
effects.
INTRODUCTION
Thehair, a vital part of human body, not only protects
scalp against detrimental effects of the environment
but also plays an important role in garnishing one’s
personality. They are derived from upper layer of the skin
and are evolved from epidermis of the embryo (Kaushik
et al., 2011). Hair growth is a complex process involving
extremely controlled cycles, i.e., hair generation,
prolongation and shedding, however, the exact process
is still unclear. It is estimated that approximately 85-
90% hairs on the scalp are in synthesis phase while the
remaining exist in shedding phase (Schulz et al., 2006).
Several synthetic hair tonics are available that promote
hair growth, yet with considerable side effects when used
for a longer period of time (Wijaya et al., 2013).
In this context, Trigonella foenum-graecum,
belonging to family Fabaceae, has been used traditionally
for various pharmacological effects, such as anti-diabetic,
anti-cancer, anti-fungal, anti-pyretic, anti-bacterial and
* Corresponding author: hamid.pharmacy@pu.edu.pk
0030-9923/2017/0004-1405 $ 9.00/0
Copyright 2017 Zoological Society of Pakistan
anti-oxidant (Yadav and Kaushik, 2011). Besides, the
seeds of T. foenum-graecum have been used as anti-lice,
anti-dandruff and for hair growth and soothing effects
(Didarshetaban et al., 2013; Ziyyat et al., 1997).
Currently, minoxidil, a synthetic hair tonic and
a vasodilator, is available in the market in various
concentrations and is used for hair growth promoting
effects. It increases the blood circulation to the scalp,
subsequently increases the hair growth.In rst year thehair
growth is at its peak, which decreases in subsequent years
along with some side effects, like burning, itching and
soreness of eyes (Purwal et al., 2008). Studies have shown
that natural sources are more reliable than Minoxidil, for
example, in male albino rats the use of Eclipta alba resulted
in enhanced hair growth activity than minoxidil (Roy et al.,
2008). Likewise, Citrullus colocynthis also demonstrated
improved hair growth promoting effects than the minoxidil
(Roy et al., 2007). It has been documented that treatment
effects with synthetic drugs are limited and transient, and
may cause toxicity on prolong usagethus advocating the
use of natural products for medical purpose (Paus, 2006).
However, despite numerous reports regarding hair growth
promoting effects of natural compounds, minoxidil is still
considered the gold standard synthetic hair tonic utilized
ABSTRACT
Pakistan J. Zool., vol. 49(4), pp 1405-1412, 2017. DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.4.1405.1412
1406
for hair growth effects.
According to literature evidence, the hair growth
promoting effects of seeds of T. foenum-graecum
were comparable with minoxidil (Wijaya et al., 2013).
However, no literature evidences exist that utilized T.
foenum-graecum leaves in hair growth promotion studies.
Thus, we aimed at comparing the hair growth promoting
effects of extracts of T. foenum-graecum leaves with that
of minoxidil using mice alopecia model.
MATERIAL AND METHODS
Plant material and chemicals
T. foenum-graecum plant, 20 kg, was purchased from
local market of Lahore, Pakistan. Leaves of the plant were
dried, crushedpulverized and stored in air tight containers.
Chemicals like solvents were purchased from BDH,
England, while quercetin and gallic acid were procured
from Sigma Life Sciences and Sinochem, respectively.
All other chemicals were procured from Merck,
Germany. Triton X and Folin and Ciocalteu’s reagents
were purchased from Uni-Chemicals, Ireland. Standard
solutions of different metals were purchased from Acros
Organics, USA, and minoxidil 5% were purchased form
Brookes Pharma Private Limited, Pakistan.
Extraction from leaves of T. foenum-graecum
Extraction was carried out using hot and cold method
(Handa et al., 2008). Briey, two fty grams powdered
material was sequentially extracted using solvents;
petroleum ether, chloroform and methanol, and twenty
ve grams powdered material was extracted in ethanol and
distilled water employinghot and cold extraction methods,
respectively.
Phytochemical analysis of leaves extract
Phytochemical analysis was done to identify chemical
compounds present in the leaves of T. foenum-graecum.
Primary metabolites
Total proteins were determined using a method
described by Lowry et al. (1951). Briey, after mixing
with distilled water and Triton-X, reagent C, reagent A
(2% Na2CO3 in 0.1 N NaOH), reagent B (0.5 % CuSO4 in
1% potassium sodium tartrate) and folin-ciocalteau’s were
mixed. Later, absorbance was measured at 600nm against
the blank, using bovine serum albumin (BSA) as standard.
Total lipids were estimated by extraction in n-hexane
and by subtracting weight of dried material from the
weight of a round bottom ask (Besbes et al., 2004).
Secondary metabolites
Total polysaccharides were determined according
to the protocol described previously (Hussain et al.,
2008). Briey, extract was mixed with 80% hot ethanol,
centrifuged and mixed with anthrone reagent. Final residue
was dried and extracted in 1:1 (v/v) mixture of 25 % HCl
and water, centrifuged and supernatant was again mixed
with distilled water and 4 ml of anthrone reagent followed
by absorbance measurement at 630 nm against the blank.
Glucose solution was used as standard solution.
Total glycosaponins were estimated according to
protocol described previously (Hussain et al., 2008).
Briey, extract was reuxed with methanol for 30 min
andprecipitated in a tarred beaker containing 50 ml of
acetone. Total glycosapoins were calculated using formula:
Total glycosaponins (%) = weight of precipitate /
weight of sample x 100
Total polyphenols were determined by using formula
described by Slinkard and Singleton (1977). Briey,
dilutions of gallic acid and extract solution (200µL) were
prepared and were added to 200µL of Folin–Ciocalteau’s
reagent and 1 ml of 15 % Na2CO3to make the volume upto
3ml with methanol. After incubation for 90 min at room
temperature, absorbance was measured at 760 nm against
the blank.
For avonoids estimation, quercetin was used
as standard. Briey, 200 µg/ml of each quercetin and
extract solution were added to methanol to make the nal
volume of 1 mL followed by the addition of 100µl of 1M
potassium acetate, 100 µl of 10% aluminium nitrate and
4.6 ml distilled water. Solutions were incubated for 45 min
at room temperature followed by absorbance measurement
at 415 nm against blank (Chang et al., 2002).
Mineral estimation
Mineral estimation procedure was carried out using
method described by Ahmad et al. (2014). Estimation
of calcium, iron, zinc, magnesium and potassium was
performed using atomic absorption spectrophotometer.
Study animals
Thirty-two male albino mice weighing 20±10 g were
purchased from University of Veterinary and Animal
Sciences, Lahore, Pakistan. Mice were acclimatized for
seven days in University College of Pharmacy animal
housing facility under 12 h dark light cycle on standard
chow. The study protocol was approved by Animal
Research Ethical Committee, Punjab University College
of Pharmacy, University of the Punjab, Lahore, Pakistan,
reference No. AEC/UCP/1040/4313.
Hair tonic formulation and evaluation
The components and concentrations of hair tonic
formulations are described in Table I. All the extracts of T.
F. Imtiaz et al.
1407
foenum-graecum leaves were diluted with distilled water
and later homogenized with 98% ethanol using ultra sonic
mixer. Finally butylene glycol was added to hair tonic
(Wijaya et al., 2013). Evaluation of hair tonic was done
through organoleptic test, homogeneity test and pH test.
Studies on hair growth promoting effects of extracts
Mice were randomly segregated into eight groups,
four in each, i.e., positive control group; treated with
5% minoxidil solution, negative control; vehicle only,
untreated and test groups. Extracts were used in two
different concentrations; 5% and 10%. Mice were shaved
on the dorsal side using hair removing cream (VEET®
cream) and were kept for 24 h before applying treatments
for any untoward reactions. Thereafter, 100µl of test
samples were applied on designated area, left and right
dorsal side, and day one was considered as zero time point
(Wijaya et al., 2013).
Qualitative evaluation of hair growth
The qualitative evaluation was done by visual
comparison of time taken to cover the shaved area in a
stipulated time that is 21 days (Adhirajan et al., 2003).
Hair length measurements
Ten hair strands were randomly plucked from
each side of dorsal area on day 7, 14 and 21. Hair was
straightened out and length was measured using digital
vernier caliper as described previously (Adhirajan et al.,
2003).
Measurement of hair diameter
Hair diameter was measured using microscope with
ocular micrometer as described previously (Adhirajan
et al., 2003). The data was presented as an average hair
diameter, representative of three biological replicates.
RESULTS
Metabolites and minerals of leaves of T. foenum-graecum
The percentage contents of primary and secondary
metabolites in the leaves of T. foenum-graecum are
summarized in Table II. The leaves of T. foenum-graecum
were found to be rich in carbohydrates (52.31 % ± 2.46),
proteins (18.54% ± 0.80) and lipids (5.443% ± 0.07)
(Table II). Among all the extracts, methanol extract
demonstrated maximum concentration of all the secondary
metabolites, including total polysaccharides (30.54% ±
0.06), total polyphenols (107.35% ± 0.20), total avonoids
(14.25% ± 0.06) and total glycosaponins (88.73% ± 0.15)
(Table II). The ethanol extract contained 28.74% total
polysaccharides, 105.86% total polyphenols, 13.70%
total avonoids and 74.4% total glycosaponins (Table II).
Table I.- Components and concentrations of different hair tonic formulation.
Ingredient Hair tonic formulation
Positive
control
Negative
control (*% )
Untreated Petroleum
ether (%)
Chloroform
(%)
Methanol
(%)
Ethanol
(%)
Aqueous
(%)
Extract - - - 5 10 5 10 5 10 5 10 5 10
Minoxidil 5 - - - - - - - - - - - -
Butylene glycol 10 10 - 10 10 10 10 10 10 10 10 10 10
Ethanol 98% 25 25 - 25 25 25 25 25 25 25 25 25 25
Distilled water 60 65 - 60 55 60 55 60 55 60 55 60 55
*, w/w.
Table II.- Estimation of primary and secondary metabolites of powdered leaves of Trigonella foenum-graecum.
Sample powder Primary metabolites
Total proteins (% contents ± SD) Total lipids (% contents ± SD) Total carbohydrates (% contents ± SD)
18.54 ± 0.80 5.443 ± 0.07 52.31 ± 2.46
Sample extracts Secondary Metabolites
Total polysaccharides
(% contents ± SD)
Total polyphenols
(% contents ± SD)
Total avonoids
(% contents ± SD)
Total glyco-saponins
(% contents ± SD)
Petroleum ether 19.99 ± 0.10 90.74 ± 2.96 4.66 ± 0.09 34.76 ± 0.05
Chloroform 21.74 ± 0.06 91.43 ± 0.13 11.20 ± 0.09 52.2 ± 0.20
Methanol 30.54 ± 0.06 107.35 ± 0.20 14.25 ± 0.06 88.73 ± 0.15
Ethanol 28.74 ± 0.06 105.86 ± 0.46 13.70 ± 0.18 74.4 ± 0.30
Water 17.77 ± 0.08 102.76 ± 2.29 4.26 ± 0.12 59.83 ± 0.25
Impact of Trigonella foenum-graecum Leaves Extract on Mice Hair Growth 1407
1408
While, the secondary metabolites in petroleum ether
extract, include19.99% total polysaccharides, 90.74%
total polyphenols, 4.66% total avonoids and 74.4% total
glycosaponins (Table II). In aqueous and chloroform
extract the concentration of secondary metabolites were
as follows; 17.77% and 21.74% of total polysaccharides,
102.76% and 91.43% of total polyphenols, 4.26% and
11.2% of total avonoids and 59.83% and 52.2% of total
glycosaponins, respectively (Table II). We also found
considerable concentration of calcium (0.80 mg/g),
magnesium (5.79 mg/g), potassium (1.42 mg/g), iron
(7.66 mg/g), and zinc (2.32mg/g) using atomic absorption
spectrophotometer in sample of leaves of T. foenum-
graecum (Table III).
Characteristics of hair tonic
Organoleptic evaluation of hair tonics resulted in
pungent smell while aqueous extract hair tonic had rotten
egg smell. There was difference in color of different
extracts hair tonic (Table IV). Homogeneity test showed
that all hair tonics were homogenous and were smoothly
spreading on skin. The pH test results are summarized in
Table IV. The pH of minoxidil hair tonic was 6.11. The
acidity of all hair tonics were within the pH range of a
normal human skin, i.e., 4.5-6.5 (Tranggono, 2007).
Table III. Mineral analysis of powder of leaves of
Trigonella foenum-graecum.
Element Concentration
(mg/g)
Element Concentration
(mg/g)
Iron 7.6 Calcium 0.8
Magnesium 5.7 Zinc 2.3
Potassium 1.4
Table IV.- Organoleptic evaluation of fenugreek hair
tonic.
Formulations Color pH
Petroleum ether 5% Light brown 4.78
10% Brown 5.09
Chloroform 5% Light brown 4.77
10% Brown 5.42
Methanol 5% Light reddish brown 5.83
10% Dark reddish brown 6.09
Ethanol 5% Yellowish brown 6.19
10% Brown 6.27
Aqueous 5% Green 6.21
10% Blackish green 6.45
Fig. 1. Qualitative assessment of hair growth effects of various extracts of leaves of Trigonella foenum-graecum.
F. Imtiaz et al.
1409 Impact of Trigonella foenum-graecum Leaves Extract on Mice Hair Growth 1409
Hair growth promoting effects of leaves extracts
Mice representing each group and at different time
points (Day 7, 14 and 21) are shown in Figure 1. Data
suggested that ethanol extract, group 7, demonstrated
maximum hair growth promoting effects not only when
compared with standard (group 1) but also compared to all
other groups having different solvents (Fig. 1).
Qualitative evaluation of hair growth on different
groups is given in Figure I. Percentage of shaved area
covered with hairs in all the groups with in a stipulated
timeof 21 days was as follows; group 1, 75%; group 2,
60%; group 3, 70%; Group 4, 90%; group 5, 80%; group 6,
90%; group 8, 90% and only group 7 demonstrated 100%
hair coverage with in a stipulated time of 21 days (Fig. 1).
Table V.- Effect of fenugreek leaves extract on mice
hair length.
Test groups Treatment Average length (mm) ± SD
7th day 14th day 21st day
Group 1 P control 3.81 ± 0.38 4.65 ± 0.86 6.96 ± 0.72
Group 2 N control 3.81 ± 1.90 4.17 ± 0.68 6.10 ± 1.11
Group 3 Untreated 2.54 ± 0.27 3.90 ± 0.26 5.17 ± 0.61
Group 4
Petroleum
ether
5% 3.80 ± 0.64 6.64 ± 1.54 8.04 ± 0.38
p – values 0.98 0.08 0.03*
10% 4.80 ± 1.20 5.94 ± 0.51 7.44 ± 1.31
p – values 0.24 0.07 0.54
Group 5
Chloroform
5% 4.38 ± 0.61 5.46 ± 0.91 6.96 ± 0.53
p – values 0.24 0.28 0.98
10% 4.50 ± 0.43 5.65 ± 0.86 6.75 ± 0.56
p – values 0.62 0.19 0.69
Group 6
Methanol
5% 4.09 ± 0.64 4.73 ± 0.10 8.40 ± 0.86
p – values 0.55 0.88 0.06
10% 3.85 ± 0.06 4.83 ± 0.34 7.85 ± 0.33
p – values 0.88 0.75 0.11
Group 7
Ethanol
5% 5.81 ± 1.62 7.44 ± 1.55 8.52 ± 1.02
p – values 0.09 0.01* 0.04*
10% 6.19 ± 0.50 7.79 ± 1.51 8.86 ± 1.40
p – values 0.001** 0.02* 0.04*
Group 8
Aqueous
5% 5.15 ± 0.47 5.32 ± 0.37 6.87 ± 0.83
p – values 0.01* 0.27 0.89
10% 4.85 ± 0.76 5.61 ± 0.88 7.39 ± 0.57
p – values 0.10 0.21 0.51
p values were obtained by comparing with test groups to positive control;
*, p value < 0.05 – 0.01; **, p value < 0.0009 – 0.001; P control, positive
control; N control, negative control.
Data on hair length measurements are summarized
in Table V. At day 7, standard and petroleum ether
(5%) groups demonstrated similar hair growth patterns,
while only ethanol 10% (6.19±0.50 mm, p=0.001) and
aqueous 5% extracts (5.15±0.47 mm, p=0.01) exhibited
signicantly higher hair growth effects on hair length in
comparison to standard (3.81±0.38 mm) and petroleum
ether (Table V). Likewise, in the second week (day 14),
ethanol group (5 and 10%) showed signicantly higher
growth promoting effects (5%; 7.44±1.55 mm, p=0.01,
10%; 7.79 ± 1.51 mm, p=.0.02) compared to positive and
all other test groups, including group 4; petroleum ether
5% (6.64 ± 1.54 mm) and petroleum ether 10% (5.94 ±
0.51 mm), group 5; chloroform 5% (5.46 ± 0.91 mm),
chloroform 10% (5.65 ± 0.86 mm), group 6; methanol 5%
(4.73 ± 0.10 mm) and methanol 10% (4.83 ± 0.34 mm) and
group 8; aqueous 5% (5.32 ± 0.37 mm) and aqueous 10%
(5.61 ± 0.88 mm) (Table V). On the 21st day petroleum
ether 5% (8.04 ± 0.38 mm, p= 0.03) along with ethanol,
5% (8.52 ± 1.02 mm, p=0.04) and 10 % (8.86 ± 1.40 mm,
p= 0.04), exhibited highly signicant results in comparison
to positive group (6.96 ± 0.72 mm) (Table V).
Table VI.- Effect of fenugreek leaves extract on mice
hair diameter.
Test groups Treatment Average diameter (µm) ± SD
7th day 14th day 21st day
Group 1 P control 5.00 ± 0.41 5.20 ± 1.29 7.29 ± 0.86
Group 2 N control 5.41 ± 0.34 5.83 ± 0.41 6.66 ± 0.22
Group 3 Untreated 5.00 ± 0.33 5.27 ± 0.63 6.80 ± 0.24
Group 4
Petroleum
ether
5% 4.69 ± 0.59 5.00 ± 1.22 6.38 ± 0.48
p – values 0.50 0.82 0.46
10% 5.52 ± 0.62 6.25 ± 0.58 6.25 ± 0.58
p – values 5.83 0.35 0.21
Group 5
Chloroform
5% 4.30 ± 0.24 4.86 ± 1.04 4.86 ± 0.86
p – values 0.06 0.72 0.01*
10% 4.86 ± 0.63 5.20 ± 0.88 6.11 ± 0.48
p – values 0.76 1.0 0.09
Group 6
Methanol
5% 4.58 ± 0.41 4.72 ± 0.24 5.97 ± 1.57
p – values 0.28 0.55 0.21
10% 5.00 ± 1.44 5.13 ± 0.63 5.62 ± 1.47
p – values 1.0 0.93 0.14
Group 7
Ethanol
5% 5.10 ± 0.52 5.83 ± 1.17 6.25 ± 1.44
p – values 0.78 0.50 0.26
10% 5.41 ± 1.44 5.52 ± 0.62 7.29 ± 0.24
p – values 0.65 0.67 1.0
Group 8
Aqueous
5% 5.13 ± 0.63 5.69 ± 2.29 6.66 ± 2.35
p – values 0.76 0.73 0.63
10% 5.00 ± 0.83 5.41 ± 2.53 6.25 ± 1.17
p – values 1.0 0.89 0.27
p values were obtained by comparing with test groups to positive control;
*, p value < 0.05 – 0.01; P control, positive control; N control, negative
control.
1410 F. Imtiaz et al.
Comparison of hair diameter after ethanol and minoxidil
treatment
Next we evaluated the impact of different extracts on
hair strand diameter in comparison to standard and controls.
As shown in Table VI, at day 7 no signicant difference
between group 1 (5.00 ± 0.41 µm), group 2(5.41 ± 0.34
µm) and group 3 (5.00 ± 0.33 µm) were observed, while
comparison of these groups with other groups showed
signicant results except for extracts formulated in 10 %
methanol (5.00 ± 1.44 µm), ethanol (5.41 ± 1.44 µm) and
aqueous base (5.00 ± 0.83 µm) (Table VI). On Day 14,
no signicant differences were observed between group 1
(5.20 ± 1.29 µm), group 2 (5.83 ± 0.41 µm) and group 3
(5.27 ± 0.63 µm), and also with rest of the groups (Table
VI). On 21st day, group 1 (7.29 ± 0.86 µm) demonstrated
signicant differences in mouse hair diameter compared
to group 2 (6.66 ± 0.22 µm), group 3 (6.80 ± 0.24 µm)
and group 5 in 5% chloroform (4.86 ± 0.86 µm, p=0.01)
only, but not with 10% chloroform extract. However, no
signicant differences in hair diameter were observed
when all other groups were compared with group 1, group
2 and group 3 (Table VI).
DISCUSSION
It has already been demonstrated in a case study
involving human volunteers that the oral intake of seeds of
T. foenum-graecum as a food supplement can increase hair
growth in these volunteers (Schulz et al., 2006). Similarly,
the seeds of T. foenum-graecum have been shown to
improve the hair growth in mice alopecia model (Wijaya et
al., 2013). Interestingly, use of ointment, containing seeds
of T. foenum-graecum, demonstrated positive hair growth
promoting effects in chemotherapy induced alopecia
(Gupta et al., 2013). In the present study we found that in a
stipulated time of 21 days, extracts made from the leaves of
T. foenum-graecum; ethanol (5% and 10%) and petroleum
ether 5%, exhibited signicant hair growth promoting
effects in alopecia mouse model. Data further revealed
that the leaves of T. foenum-graecum are rich source of
carbohydrates, proteins, glycosaponins, known to possess
anti-diabetic, anti-hypercholeterolemic, hepatoprotective
and anti-cancer effects (Laila et al., 2014; Manivannan
et al., 2013), avinoids and polyphenols, known to have
anti-oxidants properties and protect against cancer, aging
and cardiovascular diseases (Huang et al., 2009; Pandey
and Rizvi, 2009). Interestingly, all these metabolites were
favorably present in methanol extract compared to other
extracting solvents.
Seemingly, the benecial effects of T. foenum-
graecum against cancer and aging related infections may
be attributable to the presence of avonoids known to
act against various diseases caused by viruses, bacteria,
fungus, they protects against oxidation, inammation
and cancer (Nazni and Dharmaligam, 2014; Priya et al.,
2011; Tanwar and Modgil, 2012). Additionally, we found
quantiable concentrations of several minerals in the
extracts of T. foenum-graecum as leaves, such as iron,
magnesium, potassium, calcium and zinc. Several lines of
evidences suggest a strong relationship between vitamins,
minerals and other phytonutrients with cancer and their
protection against cancer (Ghiringhelli et al., 2012; Nazni
and Dharmaligam, 2014; Priya et al., 2011; Tanwar and
Modgil, 2012). Likewise, potassium plays a pivotal role in
regulating arrhythmias, hypertension, while its deciency
leads to many problems in human body (Ekinci et al.,
2004). Similarly, calcium can be helpful in combating
heart diseases, cancer and prevents osteoporosis (Khan et
al., 2011). Interestingly, deciency of zinc has been shown
to cause hair loss, growth retardation, delays in wound
healing and psychiatric problems (Khan et al., 2011).
Despite documented evidences of positive effects of
T. foenum-graecum on hair growth, the exact mechanism
is still obscure (Schulz et al., 2006). We found that among
all the extracts and compared to minoxidil, only ethanol
and petroleum ether extracts of leaves of T. foenum-
graecum demonstrated signicant effects on mice hair
growth and length, which may be due to rich phenolic
contents having reducing power, anti-oxidant activity
and free radical scavenging properties, protecting against
genotoxic insults, thus rejuvenating the cells to sustain cell
proliferation (Gupta et al., 2013). Anti-oxidant effects of
leaves of T. foenum-graecum have been demonstrated in
various models, such as drug induced hepatotoxicity goat
model and streptozotocin induced diabetic mouse (Annida
and Prince, 2005; Devatkal et al., 2012; Meera et al.,
2009). Another potential constituent that might contribute
towards hair growth promoting effects of extracts of
leaves of T. foenum-graecum, could be the presence of
considerable amount of Zinc, known to promote healthy
hair and nail growth (Khan et al., 2011). Another betting
hypothesis could be the presence of certain proteins and
amino acids in the leaves of T. foenum-graecum that
may promote hair growth (Purwal et al., 2008). It is
also plausible that Trigonelline, an alkaloid present in
the leaves of T. foenum-graecum, which physiologically
interacts and increases blood circulation to the scalp,
maypromote hair growth in mice with alopecia (Gupta
et al., 2013). Additionally, estrogen has been shown to
modulate several transcription factors known to control
hair growth (Ohnemus et al., 2006), thus, it is highly likely
that steroidal saponins like diosgenin having estrogen
like effects in T. foenum-graecum leaves might contribute
towards hair growth promoting effects in our mice (Sharma
1411 Impact of Trigonella foenum-graecum Leaves Extract on Mice Hair Growth 1411
et al., 2009). Possibly, these phytoestrogen interacts with
dihydrotestosteron (DHT) metabolism and increases hair
growth (Schulz et al., 2006). Besides, it has also been
proposed that rather than synthetic hormone, which causes
side effects, phytoestrogens can be used as an alternative
to promote hair growth with minimal side effects (Dixon,
2004; Ross et al., 2000).
CONCLUSION
Thus, in conclusion, our data suggested that the
extracts of leaves of T. foenum-graecum signicantly
improved hair growth in mice in comparison to standard
and most frequently chosen synthetic hair tonic i.e.,
minoxidil, in ethanol (5% and 10%) and petroleum ether
(5%) extracts. Presumably, the effects of leaves extracts are
superior in comparison to extracts made from T. foenum-
graecum seeds. However, it would be nice to perform a
side by side comparison between leaves and seeds extracts
of T. foenum-graecum. Besides, further studies are
required to un-earth the underlying mechanisms of hair
growth promoting effect of T. foenum-graecum leaves,
particularly, by identifying constituents of the extracts
responsible for hair growth promoting effects.
ACKNOWLEDGEMENT
Authors are extremely thankful to Animal Technician,
Mr. Javed for the help.
Statement of conict of interest
Authors have declared no conict of interest.
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https://doi.org/10.1016/S0378-8741(97)00077-9
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A fully automated-continuous flow 40-sample/ hour procedure was adapted from the Singleton-Rossi method of analysis for total phenols in wine and other plant extracts. It was compared with small-volume manual and semiautomated versions of this analysis. The agreement in mg of gallic acid equivalent phenol (GAE) per liter among a series of dry wines was excellent by all three procedures. The coefficients of variation in replicate analyses averaged 5.8% for the manual, 6.2% for the semi-automated and 2.2% for the automated procedure. This greater reproducibility, plus savings of about 70% in labor and up to 40% in reagents, makes the automated procedure attractive for laboratories doing enough total phenol analyses to recoup the cost of the automating equipment. For continuous flow, color development with the Folin-Ciocalteu reagent in alkaline solution must be hastened by heating compared to slower room temperature development for the manual methods. Heating of sugar-containing samples in the alkaline solution gives interference presumably from endiol formation. Examples are given of corrections which were used successfully to estimate the true phenol content of sweet wines.
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BACKGROUND: Hormone replacement therapy (HRT) given as unopposed estrogen replacement therapy (ERT) gained widespread popularity in the United States in the 1960s and 1970s. Recent prescribing practices have favored combination HRT (CHRT), i.e., adding a progestin to estrogen for the entire monthly cycle (continuous combined replacement therapy [CCRT]) or a part of the cycle (sequential estrogen plus progestin therapy [SEPRT]). Few data exist on the association between CHRT and breast cancer risk. We determined the effects of CHRT on a woman's risk of developing breast cancer in a population-based, case-control study. METHODS: Case subjects included those with incident breast cancers diagnosed over 4½ years in Los Angeles County, CA, in the late 1980s and 1990s. Control subjects were neighborhood residents who were individually matched to case subjects on age and race. Case subjects and control subjects were interviewed in person to collect information on known breast cancer risk factors as well as on HRT use. Information on 1897 postmenopausal case subjects and on 1637 postmenopausal control subjects aged 55-72 years who had not undergone a simple hysterectomy was analyzed. Breast cancer risks associated with the various types of HRT were estimated as odds ratios (ORs) after adjusting simultaneously for the different forms of HRT and for known risk factors of breast cancer. All P values are two-sided. RESULTS: HRT was associated with a 10% higher breast cancer risk for each 5 years of use (OR5 = 1.10; 95% confidence interval [CI] = 1.02-1.18). Risk was substantially higher for CHRT use (OR5 = 1.24; 95% CI = 1.07-1.45) than for ERT use (OR5= 1.06; 95% CI = 0.97-1.15). Risk estimates were higher for SEPRT (OR5 = 1.38; 95% CI = 1.13-1.68) than for CCRT (OR5= 1.09; 95% CI = 0.88-1.35), but this difference was not statistically significant. Conclusions: This study provides strong evidence that the addition of a progestin to HRT enhances markedly the risk of breast cancer relative to estrogen use alone. These findings have important implications for the risk-benefit equation for HRT in women using CHRT.