CMU J. Nat. Sci. (2018) Vol. 17(1) 25
Enhanced VEGF Expression in Hair Follicle Dermal
Papilla Cells by Centella asiatica Linn.
Pahol Saansoomchai1, Apinun Limmongkon1, Damratsamon Surangkul1,
Teera Chewonarin2 and Metawee Srikummool1*
1Department of Biochemistry, Faculty of Medical Science, Naresuan University, Phitsanulok
2Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai
*Corresponding author. E-mail: firstname.lastname@example.org
Centella asiatica Linn. (C. asiatica) extract has been shown to possess high antioxidant
activity due to its phenols and avonoids. This study tested the ecacy of 70%-ethanol
(EtOH) crude extracts of C. asiatica and its fractions (H2O, EtOAc, CH2Cl2, and hexane)
to modulate human follicle dermal papilla cells. In addition, we analyzed the extracts for
major phytochemicals as well as free radical scavenging activity. Our results from ABTS
and DPPH assays showed that the amounts of phenolic and avonoid compounds in the
extracts were both related to its free radical scavenging activity. While the EtOAc fraction of
C. asiatica demonstrated the highest free radical scavenging activity, it was toxic to human
follicle dermal papilla cells. The cell viability test was positive when cells were treated with
EtOH crude extract and H2O fraction. VEGF gene expression, quantied by real-time PCR
analysis of the EtOH crude extract, showed a signicant level of induction, indicating that
the growth promotion eect in human follicle dermal papilla cells was related to VEGF
gene expression, which has a positive hair growth stimulating eect. The EtOH crude
extract of C. asiatica may oer potential in hair growth promoting products.
Keywords: Antioxidant activities, Centella asiatica, Phytochemical screening, Real-time
PCR, Gene expression
Hair protects the scalp from the environment, including heat, cold, and UV radiation,
and serves as a measure of beauty. As its loss can result in distress and psychological problems,
prevention or treatment strategies need to be investigated. So far, only two drugs, minoxidil
and nasteride, have been approved for the treatment of hair loss in men by the US Food and
Drug Administration (Park et al., 2012).
Hair follicles of any hair type have a unique life cycle comprised of three main stages –
anagen, catagen, and telogen, each of which leads to the destruction and regeneration of hair
follicles over a lifetime. The regulation of the hair cycle is complicated and involves several
CMU J. Nat. Sci. (2018) Vol. 17(1)26
factors (Hibino and Nishiyama, 2004) that are not well understood. Genetic factors, cytokine
imbalance, and oxidative stress can cause abnormal hair follicle cycling and subsequent hair
loss (Rho et al., 2005; Aron et al., 2013). Many cytokines and receptors are involved in the
cell cycle of human follicle dermal papilla cells, including the vascular endothelial cell growth
factor (VEGF) (Shin et al., 2014), vascular endothelial cell growth factor receptor (VEGFR)
(Li et al., 2012), broblast growth factor (FGF) (Rho et al., 2005), insulin-like growth factor
(IGF) (Panchaprateep and Asawanonda, 2014), epidermal growth factor (EGF) (Bressan et
al., 2014), keratinocyte growth factor (KGF) (Gopu et al., 2015), and transforming growth
factor (TGF) (Kang et al., 2013; Shin et al., 2014). Some hair regeneration has been achieved
by molecular eect and growth factors (Danilenko et al., 1996) and follicle dermal stem cells
(Rahmani et al., 2014). Some evidence has suggested that VEGF and VEGFR could induce
the proliferation of human follicle dermal papilla cells through ERK activation (Li et al.,
2012). TGF has been related to human follicle dermal papilla cell death involving free radicals
(Soma et al., 2003; Rho et al., 2005).
Medicinal plants, including Centella asiatica Linn. (C. asiatica), are natural sources
of bioactive compounds that possess health-promoting eects. C. asiatica has been used to
treat a range of ailments, including the common cold (Roy et al., 2013). In Thailand, its fresh
leaves have been used to treat wounds and burns and its extract has been used to reduce
swelling and infection. C. asiatica extract is widely available in Thailand and cost eective
(Taemchuay et al., 2009). Many scientic studies have researched traditional applications of
C. asiatica extract (Bylka et al., 2014; Hashim, 2014). C. asiatica extract contains several
bioactive compounds, including saponins, essential oils, avone derivatives, sesquiterpenes,
triterpenic acid, and triterpenic steroids (Roy et al., 2013). It also has been reported to contain
bioactive compounds, such as terpenes, avonoids, and polyphenols, that are related to its
potent antioxidative activities (Hashim et al., 2011; Nurlaily et al., 2012; Orhan et al., 2013).
Extracts from C. asiatica leaves consist of gallic acid and ferulic acid, which have antioxidant
and anti-inammatory eects (Ramesh et al., 2014). Another study has shown that C. asiatica
extracts exhibited antioxidant activity and UV protection eects (Hashim et al., 2011). Many
beauty products are currently available that incorporate C. asiatica extracts, such as cosmetic
creams, hand and body lotions, eye gel, and face mask products (Bylka et al., 2014). A previous
study found that C. asiatica extract enlarged hair follicles (Jain and Dass, 2015) and inhibited
the activity of 5α-reductase that causes hair loss (Jain et al., 2016). However, few hair care
or restoration products contain the extract, as its eect on the hair root remains unclear; the
molecular mechanisms involved in plant extracts modulating gene expression are not well
The in vitro treatment of human follicle dermal papilla cells could, possibly, provide
a gateway to hair regeneration and sustainably protect against hair loss. The objective of this
study was to search for any potential eect, especially involving antioxidant activity, of C.
asiatica extract on growth and molecular regulation in human follicle dermal papilla cells.
Positive ndings would indicate the potential for developing accessible and aordable value
added hair growth promoting products using ingredients extracted from natural sources rather
than synthetic drugs.
CMU J. Nat. Sci. (2018) Vol. 17(1) 27
MATERIALS AND METHODS
A C. asiatica plant was collected from Chiang Rai, Thailand and positively identied
by the taxonomist of Nareasuan University. The leaves were cleaned and dried in an oven at
40°C, then stored at -20°C until use.
Preparing the ethanol crude extract
One kilogram of dried samples were ground into powder and macerated in 4 L of 70%
(v/v) ethanol (EtOH) for 24 h at room temperature. The extraction was performed twice under
the same conditions. Chlorophyll was removed using the charcoal absorption method with
some modication (Limtrakul et al., 2004). Briey, each extract was bleached with 160 g
of activated charcoal. The chlorophyll-free extract was then ltered through Whatman’s
No.1 lter paper and the solvent was removed using a vacuum rotary evaporator (Buchi,
Switzerland) at room temperature. The concentrated aqueous portion was lyophilized (Christ
Alpha1-4 LD, UK) into a powder and further partitioned using four dierent solvents:
hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and water (H2O). The EtOH crude
extract and four fractions with the highest antioxidant activity were then used in subsequent
Evaluating the free radical scavenging activity of the crude extract
Two methods measured the free radical scavenging activity of C. asiatica crude extract:
1) a DPPH inhibition assay following the method of Padmanabhan and Jangle (2012) and
2) an ABTS inhibition assay as described by Gorjanović et al. (2012). With treatments of
various concentrations of the extract, the decrease in absorbance was measured at 517 nm for
the DPPH assay and 735 nm for the ABTS assay; the % inhibition and IC50 value were also
Measuring total phenolic and avonoid contents of the crude extract
Total phenolic content was determined using the Folin-Ciocalteu method. Quantication
was expressed as milligrams of gallic acid equivalent per gram of extract (mg GE/g of ext)
(Saikia et al., 2012). The total avonoid content (TF) was measured by aluminium chloride
colorimetric assay and expressed in milligrams of catechin equivalent per gram of extract (mg
CE/g of ext) (Saikia et al., 2012).
Human follicle dermal papilla cell cultures and cell viability testing
Human follicle dermal papilla cells cultures were obtained from PromoCell, Germany.
The cells were cultured and maintained in Follicle Dermal Papilla Cell Growth Medium
(PromoCell, Germany) at 37°C in 5% (v/v) CO2. Cytotoxicity of the extract of the human
follicle dermal papilla cells was performed using the Presto-blue (Invitrogen, USA) assay
according to the PrestoBlueTM cell viability reagent protocol. Briey, 2 × 103 of the human
follicle dermal papilla cells were seeded into a 96-well, at-bottomed, microliter plate and
cultured for 24 h. A 100-μl sample of C. asiatica extract at dierent concentrations was
CMU J. Nat. Sci. (2018) Vol. 17(1)28
added to each well and the cells were further cultured for 24 h for one group of cells, and
48 h for another. Then, 20 μl of Presto-blue solution was added to each well and the cells
were incubated for 20 min. The C. asiatica extracts were compared to 1% standard minoxidil
(Sigma-Aldrich, USA) as the control. The absorbance was measured at 570 nm. The eective
time of incubation to human follicle dermal papilla cells was used for studying the mRNA
Detecting VEGF mRNA expression in C. asiatica–treated human follicle dermal papilla
The human follicle dermal papilla cells were treated with the indicated concentrations of
C. asiatica extract for 24 h. Total RNA was isolated using RNAZol® RT (Molecular Research
Center Inc., USA) according to the manufacturer’s protocol. RNA quality was assessed by
RNA/Protein sample PCR amplication of cDNA performed in a RevertraAce®qPCR RT
Master Mix (Toyobo, Japan). cDNA was obtained from 2 μg/ml of RNA by one cycle of reverse
transcription. Gene expression was quantied using real-time PCR (RT-PCR). Targeted genes
and the details are presented in Table 1. The PCR cycle steps consisted of denaturation at 94°C
for 1 min, annealing at 58°C for 1 min, and a nal extension step at 72°C for 1 min within
40 cycles. Each gene expression was calculated according to the threshold cycle (CT) value,
normalized using the value of sample with the lowest level for each product, and the data were
corrected according to the level of β-actin.
Table 1. Sequences of gene specic primers used in RT-PCR.
Genes Sequences Size Reference
VEGF forward 5’- ATGACGAGGGCCTGGAGTGTG -3’ 91 Soulitzis et al.,
reverse 5’- CCTATGTGCTGGCCTTGGTGAG -3’
β-actin forward, 5’- CTTCCAGCCTTCCTTCCTGG -3’ 162 Soulitzis et al.,
reverse, 5’- TTCTGCATCCTGTCGGCAAT -3’
To verify the RT-PCR, PCR products were analyzed by electrophoresis in 2% agarose
gels. They were then stained with ethidium bromide and photographed on a UV light
transilluminator. PCR product length for VEGF growth factor was analyzed, as well as β-actin.
Each experiment was performed in triplicate. All values were presented as a mean
value (Mean ± SD). The statistically signicant dierences between the means of the samples
were calculated by one-way ANOVA and the dierences were considered signicant at a level
of p<0.05 (*).
CMU J. Nat. Sci. (2018) Vol. 17(1) 29
Antioxidant activity of C. asiatica extracts
The antioxidant activity of the C. asiatica extract was measured by its ability to
scavenge DPPH and ABTS radicals.
Figure 1 shows the DPPH (A) and ABTS (B) free radical scavenging assays. The free
radical scavenging activity of C. asiatica extract showed the highest activity in the EtOH
crude extract for DPPH assay and the highest activity in the EtOAc fraction for ABTS assay.
Figure 1. The free radical scavenging activities of C. asiatica extracts.
Note: *The dierences were considered signicant at p<0.05.
Table 2. IC50 of the C. asiatica extracts against DPPH and ABTS radicals.
IC50 of C. asiatica extract (μg/ml)
DPPH assay ABTS assay
Hexane >200 >200
CH2Cl2>200 173.20 ± 3.47
EtOAc 134.76 ± 12.09 122.22 ± 7.49
EtOH 34.63 ± 0.76 >200
H2O >200 >200
Ascorbic acid 13.95 ± 0.01 -
Trolox - 6.41 ± 0.03
Note: The presence of IC50 of C. asiatica and standards against DPPH and ABTS radicals are presented as
mean ± SD.
CMU J. Nat. Sci. (2018) Vol. 17(1)30
As shown in Figure 1 and Table 2, the free radical scavenging activity of C. asiatica
extract at 0-200 μg/ml was found to scavenge the DPPH radicals and ABTS radicals in a dose
dependent manner when compared with the positive control, ascorbic acid and Trolox.
The EtOH crude extract of C. asiatica at a concentration of 200 μg/ml displayed the
highest inhibitory eect; it inhibited DPPH radicals at 97.01 ± 0.42% of the ascorbic acid and
also inhibited IC50 at 34.63 ± 0.76 μg/ml. CH2Cl2, hexane, and H2O fractions also displayed
inhibitory eects at the same concentration levels of IC50 (>200 ug/ml).
C. asiatica extract in the EtOAc fraction displayed the highest inhibitory eect
on ABTS radicals at a concentration of 200 μg/ml, inhibiting 65.33 ± 2.09% of Trolox with
IC50 at 122.22 ± 7.49 μg/ml. The CH2Cl2 fraction inhibited at IC50 of 173.20± 3.47 μg/ml.
EtOH crude extract, hexane fraction, and H2O fraction had the same concentration levels
The two active fractions, the EtOH crude extract and EtOAc fraction, have been linked
to solvent polarity that can extract dierent fractions of polar/nonpolar constituents from the
plant. This nding agreed well with the total phenolic and avonoid contents of each fraction,
as shown in Table 3.
Total phenolic and avonoid content in C. asiatica extracts
The amount of phenols and avonoids contained in C. asiatica extracts are shown in
Table 3. The EtOAc fraction contained the most phenolic compounds, at 19.72 ± 0.02 mg
GE/g of extract, closely followed by the CH2Cl2 fraction. The EtOAc fraction contained the
most avonoids, at 6.79 ± 0.12 mg CE/g of extract, with all other fractions containing only
small or trace amounts.
Table 3. Total phenolic (TP) and avonoid (TF) content of C. asiatica extracts.
C. asiatica Hexane CH2Cl2EtOAc EtOH H2O
TP (mg GE/g of ext) 7.71 ± 0.01 17.04 ± 0.01 19.72 ± 0.02 2.68 ± 0.00 0.13 ± 0.00
TF (mg CE/g of ext) 1.10 ± 0.64 1.51 ± 0.36 6.79 ± 0.12 0.54 ± 0.14 0.00 ± 0.00
Note: The present chemical components, including TP and TF, are presented as mean ± SD.
Cytotoxicity of the C. asiatica extracts
The cytotoxicity of the C. asiatica extract from EtOH crude extract and each fraction
were further examined by treating human follicle dermal papilla cells with the extract at
dierent concentrations. The EtOH crude extract and H2O fraction were evaluated for
cytotoxic eects on human follicle dermal papilla cells at dierent doses up to 1,000 μg/ml
extract for 24 h and 48 h (Figure 2). The cell viability was 100% or more compared with the
non-treated cells (0 μg/ml extract). The C. asiatica extract at concentrations of 500 μg/ml and
1,000 μg/ml slightly induced cell proliferation. From this, it can be concluded that the EtOH
crude extract and H2O fraction of C. asiatica did not show any toxicity to the human follicle
dermal papilla cells, and in this regard were as eective as 1 μg of minoxidil (control). No
statistical dierences were found between the viability of the cells treated with minoxidil,
CMU J. Nat. Sci. (2018) Vol. 17(1) 31
and the viability of the cells in the control. Unfortunately, the screening data revealed that the
EtOAc fraction, which possessed the greatest antioxidant activity, was toxic to human follicle
dermal papilla cells (data not show). Therefore, we did not use this fraction for further study.
We then focused on the extract from the safer solvents – EtOH and H2O.
Figure 2. Cell viability of EtOH crude extract and H2O fraction of C. asiatica extract at 24 h
and 48 h. The EtOH crude extract and H2O fraction did not display cytotoxicity to
human follicle dermal papilla cells.
Note: *The dierences were considered signicant at p<0.05.
VEGF gene expression
The EtOH crude extract and H2O fraction (the safer solvents) of C. asiatica at
concentrations of 500 μg/ml and 1,000 μg/ml were used for a gene expressivity assay by RT-
PCR. As shown in Figure 3, both concentrations of EtOH crude extracts of C. asiatica induced
VEGF gene expression (p<0.05), with 500 μg/ml the most at 37.30 ± 9.47. This far exceeded
the slightly induced VEGF gene expression of minoxidil (1.99 ± 0.07); the H2O fraction did
not induce gene expression (data not shown).
The size of the PCR products corresponded to the data shown in Table 1. The band of
VEGF growth factor was presented after incubating the C. asiatica extract with human follicle
dermal papilla cells. C. asiatica extract at the concentration of 500 μg/ml showed a more
intense band than the C. asiatica extract at the concentration 1,000 μg/ml. Similarly to the
β-actin, these concentrations showed the same result as VEGF growth factor (data not show).
The PCR products coresponded to RT-PCR.
CMU J. Nat. Sci. (2018) Vol. 17(1)32
Figure 3. VEGF expression of human follicle dermal papilla cells by the induction of EtOH
crude extract of C. asiatica.
Note: *The dierences were considered signicant at p<0.05.
ABTS and DPPH assays determined the anti-oxidative activity of the 70%-EtOH extract
of C. asiatica and its partition fractions (hexane, CH2Cl2, EtOAc, and H2O fractions). The
dierent fractions inhibited the ABTS and DPPH radicals dierently. The EtOH crude extract
showed the strongest inhibitory eect on DPPH radicals, while the EtOAc fraction had the
strongest inhibitory eect on the ABTS radicals. These results indicated that the free radical
scavenging activities correlated to the phenolic and avonoid content in the extract. Moreover,
the inhibitory eect of both the EtOH crude extract and the EtOAc fraction depended on not
only their phytochemical ingredients, but also the solvents used to generate the radicals. Water
was used as the solvent in the ABTS assay, representing the polar solvent borne radicals
(Gorjanović et al., 2012), while methanol used as the solvent in the DPPH assay, representing
the organic solvent borne radicals (Padmanabhan and Jangle, 2012). Rahman et al. (2013)
and Shalaby and Shanab (2013) found that the free radical scavenging activity of an extract
related to the polarity of the solvent, which occurs because the antioxidant molecules engage
in strong interactions with free radicals.
The results showed that the free radical scavenging activity of the C. asiatica extracts
related to the variety of chemicals in the phenols and avonoids, which included many
lipophilic phytochemicals or hydrophilic phytochemicals. Many studies have reported levels
and activities of phenols and avonoids using a chlorophyll-free extraction method (Limtrakul
et al., 2004; Paula et al., 2012). In our study, the C. asiatica extracts and fractions showed total
phenols ranging from 0.13-19.72 mg GE/g of extract. This result agreed with Frederico et al.
(2009) for dierent parts of C. asiatica. The avonoids levels in the C. asiatica extracts were
CMU J. Nat. Sci. (2018) Vol. 17(1) 33
high, up to 6.79 mg CE/g extract. The EtOAc fraction showed the highest amount of phenols
and avonoids. This was consistent with previous reports that indicated that the moderate
polarity of a solvent, such as EtOAc, yields more phenols and avonoids than other solvents
(Wang et al., 2016) and that the polarity of a solvent aects the amount of each (Rahman et
The cytotoxicity tests showed that the EtOAc fraction harmed human follicle dermal
papilla cells. Natural glycosides were extracted from the plant by the low polarity of the
solvent, causing the harm. Podolak et al. (2010) also described the haemolytic activity and
cytotoxicity to cells of these natural glycosides.
RT-PCR analysis tested the stimulating eect of treating cells with EtOH crude extract
of C. asiatica, observed as the expression of VEGF mRNA. The EtOH crude extract of C.
asiatica induced VEGF expression in human follicle dermal papilla cells, possibly leading to
cell proliferation. Our results agreed with other studies of plant extracts (such as Asiasari radix
and Panax ginseng) in enhancing the expression of VEGF (Rho et al., 2005; Shin et al., 2014).
Other reports showed that the proliferation of human follicle dermal papilla cells was involved
with cytokines signaling (Soma et al., 2003; Rho et al., 2005). β-catenin causes the signaling
of human follicle dermal papilla cell proliferation (Driskell et al., 2011). The β-catenin activity
in the human follicle dermal papilla cells regulates a number of other signaling pathways,
including the phosphorelation of downstream signalling, such as the VEGF pathway, that
stimulates cell proliferation (Lachgar et al., 1999; Driskell et al., 2011). VEGF also plays an
important role in angiogenesis in follicle dermal papilla cells (Yano et al., 2001).
Previously, minoxidil was reported to have a concentration-dependent, biphasic eect
on proliferation and dierentiation, as well as on growth stimulation in low doses, and to be
an anti-proliferative through the expression of cytokines (Kwon et al., 2007). In solution at
less than 5%, Minoxidil can safely be applied to the human scalp; this equals a concentration
of 1 mM (Han et al., 2004). Our results showed that minoxidil in solution stimulated the
expression of VEGF, corresponding to Lachgar et al. (1998) and Li et al. (2001). The higher
VEGF expression after treatment with the EtOH crude extract of C. asiatica more eciently
promoted human follicle dermal papilla cells than minoxidil.
While many reports have studied C. asiatica extracts, few have looked at its eects
on human follicle dermal papilla cells and the molecular mechanisms that promote the
proliferation of the cells. This study focused on gene expression after incubating the cells with
the C. asiatica extract. The EtOH crude extract of C. asiatica induced the expression of VEGF
mRNA in human follicle dermal papilla cells. Moreover, the phenols and avonoids found
in the C. asiatica extracts demonstrated antioxidant activity that could maintain the growth
of human follicle dermal papilla cells. This study has clearly indicated that the EtOH crude
extract of C. asiatica will be of benet in the development of hair care products and hair loss
CMU J. Nat. Sci. (2018) Vol. 17(1)34
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