Evaluation of hair growth properties of Topical Kombucha tea extracts

Abstract

Consuming functional foods and drinks such as kombucha tea is believed to give many beneficial effects for promoting immunity and preventing cancer. The fermented tea with sugar and symbiotic culture of yeast and bacteria is also useful in the topical application for hair re-growth. This study aims to investigate hair re-growth properties of kombucha black tea and chamomile with and without methylsulfonylmethane. Treatments were applied topically to denuded Balb/c mice and observed for hair growth initiation time, density and length in 15 and 30 days in addition to microbiological identification of the main yeast and bacteria. Results showed significantly better hair density and length in groups treated with kombucha black tea with and without MSM, followed by kombucha chamomile tea with and without MSM respectively. In addition, it has been found that MSM promotes hair growth significantly when combined with kombucha ferments. Microbiological analysis indicated the presence of Zygosaccharomyces bailii and Acetobacter genus in the tea broth. Black tea kombucha with MSM can be used as a natural alternative therapy for hair fall and a cost-effective treatment with fewer side effects compared to the synthetic drugs.

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Advances in Traditional Medicine
https://doi.org/10.1007/s13596-020-00534-7
RESEARCH ARTICLE
Evaluation ofhair growth properties ofTopical Kombucha tea extracts
MohammedAyadAlboreadi1· ManalMamdouhAl‑Najdawi1· QaisBashirJarrar1· SaidMoshawih2
Received: 6 October 2020 / Accepted: 29 November 2020
© Institute of Korean Medicine, Kyung Hee University 2021
Abstract
Consuming functional foods and drinks such as kombucha tea is believed to give many beneficial effects for promoting
immunity and preventing cancer. The fermented tea with sugar and symbiotic culture of yeast and bacteria is also useful in
the topical application for hair re-growth. This study aims to investigate hair re-growth properties of kombucha black tea
and chamomile with and without methylsulfonylmethane. Treatments were applied topically to denuded Balb/c mice and
observed for hair growth initiation time, density and length in 15 and 30days in addition to microbiological identification
of the main yeast and bacteria. Results showed significantly better hair density and length in groups treated with kombucha
black tea with and without MSM, followed by kombucha chamomile tea with and without MSM respectively. In addition,
it has been found that MSM promotes hair growth significantly when combined with kombucha ferments. Microbiological
analysis indicated the presence of Zygosaccharomyces bailii and Acetobacter genus in the tea broth. Black tea kombucha
with MSM can be used as a natural alternative therapy for hair fall and a cost-effective treatment with fewer side effects
compared to the synthetic drugs.
Keywords Kumbocha· Black tea· Chamomile· Hair growth· Zygosaccharomyces· Acetobacter
Introduction
Tea fungus or “Kombucha” is dated back to thousands of
years in Manchuria, China and still widely-consumed in
Europe and Russia. The traditional drink is considered as a
probiotic with functional properties that has been reported
to have many biological benefits upon drinking or topical
application in different diseases such as arthritis, hyperten-
sion, cancer, AIDS, atherosclerosis, anorexia, hemorrhoids,
indigestion. In addition, it possesses energizing, detoxifi-
cation and hair re-growth properties observed personally
and through testimonials, however, most of these claims
are yet to be confirmed clinically (Ernst 2003; Sreeramulu
etal. 2000). Kombucha is a symbiotic growth of acetic acid
bacteria and yeast strains such as Schizosaccharomyces and
Zygosacchromyces species cultured in a sugared tea in order
to produce a sparkling apple cider taste after final fermenta-
tion by tea fungus.
The traditional way to prepare kombusha is by adding
tea leaves to boiling water and allowed to infuse after which
the leaves are removed. Sucrose is dissolved in the hot tea to
provide the substrate for fermentation process along with the
tea leaves. Vinegar or already prepared Kombucha is added
to the mixture to increase its acidity with tightly closed con-
tainer. The preparation is allowed to incubate at room tem-
perature for one to two weeks, meanwhile, a new tea fungus
is formed at the surface of the mixture and the fermented
tea can be sieved and kept at 4 C to stop the fermentation
process (Dufresne and Farnworth 2000). The combination
of yeast-bacteria symbiosis produces different acids, xan-
thine, amino acids, sugars, elements such as Cu, Fe, Mn, Ni,
and Zn;vitamin C, vitamin B complex and ethanol that are
essential to provide biological benefits endowed by kombu-
cha in addition to that prevent harmful organism’s growth
(Kapp and Sumner 2019). Also, the high level of glucuronic
Supplementary information The online version of this article
(https ://doi.org/10.1007/s1359 6-020-00534 -7) contains
supplementary information, which is available to authorized
users.
* Manal Mamdouh Al-Najdawi
manalnajdawi@yahoo.com
Said Moshawih
saeedmomo@hotmail.com
1 Department ofApplied Pharmaceutical Sciences, Faculty
ofPharmacy, Al-Isra University, Amman, Jordan
2 Jordan Center forPharmaceutical Research, Postal code
11817, Amman, Jordan

M.A.Alboreadi et al.
1 3
acid in the ferment avoid unexpected microbial contami-
nation (Nguyen etal. 2015). Acetic acid bacteria convert
glucose to gluconic acid and fructose into acetic acid. Caf-
feine and other xanthines of the tea stimulate the cellulose
synthesis by the bacteria which float onto the surface of the
mixture that form a suitable floated platform for the growth
of both bacteria and yeast (Balentine 1997). Furthermore,
yeasts metabolize fructose into ethanol and carbon dioxide,
while ethanol in turn is oxidized to acetic acid by Aceto-
bacter bacteria strains. Organic acids produced during the
fermentation protects the colony from contamination with
harmful foreign microorganisms (Sreeramulu etal. 2000).
The diversity of chemicals available in the kombucha
drink that were previously mentioned in addition to poly-
phenols derived from tea leaves implies different pharma-
cological benefits such as hair re-growth properties. For
instance, Hair follicles diameter and length were increased
when NMRI mice skin injury treated with kombucha (Pari-
var, Yaghmaei and Heidari 2012). Many ways through which
a drug might promote hair growth, such as; modifying the
hair cycle, enlargement of the hair fiber, prolongation of the
anagen phase or shortening the telogen phase, or a com-
bination of these mechanisms. Infusions from other plants
have been reported to be promising alternative to black tea
as studies have indicated (Villarreal-Soto, Beaufort, Boua-
jila, Souchard, and Taillandier 2018). Therefore, the present
research focuses on the assessment of the natural kombucha
extract from black and chamomile tea as an alternative natu-
ral agent for management of hair loss.
Materials
Chemicals and equipment used are: minoxidil (Dar Al
Dawa, Jordan), sugar (Nader, Jordan), chamomile flowers
(Kabatilo, Jordan), pure black tea (Ceylon tea, Sri Lanka),
methyl sulfonyl methane (MSM) (Nutricost, China), hair
removal cream Veet (Reckitt, France), glass jars (Mix glass,
Jordan) and cotton (Cotton land, Jordan). Digital pH meter
(SPMI, China), incubator (LW Scientific, USA), autoclave
(Alibaba, China), digital caliper (Rs PRO, China), electric
shaver (Kemei, China), electric balance (CGOLDENWALL,
China), camera EOS 80d (Canon, Japan) used with lense
18–135.
Methods
Preparation ofkombuch tea samples
andmicrobiological evaluation
Four different preparations of komucha tea were prepared in
accordance to previously described procedures (Jayabalan
etal. 2007), with some modifications. All preparations,
distinctive by different type of herbal tea and presence of
MSM, were prepared under room temperature (25 ± 1) and
protected from direct light exposure. Pure black tea (Ceylon
tea, Sri Lanka) 10% was added to boiling water and allowed
to infuse for 15min; the infusion was then filtered through
a sterile sieve. Sucrose 20% was added and dissolved in
the above mixture, and the preparation was allowed to cool
down to 25°C.
The cooled mixture was poured into a glass jar sterilized
at 121°C for 20min, 3% (w/v) freshly grown tea fungus
which was cultured with black tea for 14days was introduced
into the tea broth and 10% (v/v) of previously fermented liq-
uid tea broth was added too. The Fermentation incubated in
an incubator (LW Scientific, USA) at 25°C covered with a
clean cloth and tightly closed to prevent contamination and
left for 15days. The fermentation was watched until the pH
level drops to about 2.7 which were measured using digital
pH meter (SPMI Co.Ltd., China), filtration was performed
to the fermented mixture using a funnel with cotton to sepa-
rate the filtrate from the symbiotic bacteria and yeast. The
filtrate was then kept in another jar at subzero temperature
to deactivate the fermenting bacteria and yeast in the liquor.
Kombucha chamomile tea was prepared using the same pro-
cedure described above with chamomile flowers. Kombucha
black tea and chamomile with MSM were prepared using
the ratio of MSM: Kombucha black tea/chamomile were 5%
w/v. Later, a sample from the extract was sent to the National
Agricultural Research Center, Mafraq, Jordan to be analyzed
for microbiological contents.
Selection andhousing ofexperimental animals
A total of 48 adult male BALB/C mice were obtained from
the animal house of Al-Isra University and were of the same
age 7weeks, to make sure that all hair follicles were syn-
chronized in the telogen phase (Rho, Park, Hwang, Lee, and
CD Kim 2005). The average weight was around (26–33gm).
The mice were provided with standard pellet diet and tap
water adlibitum. All animal handling in the present study
were conducted according to the guidelines of Institutional
Animal Care and Use Committee (IACUC) and the research
ethics committee of the faculty of pharmacy, Isra University
has approved it under approval No. 55–5/2019–2020.
The mice were randomly distributed into eight groups
(n = 6/group) and acclimatized for a period of 7days. The
animal groups were made distinctive by the topical treat-
ments administrated to the mice as follows: Group 1 Nega-
tive control, Group 2 Positive control: Mice were treated
with 2% minoxidil solution, Group 3 Black tea control: Mice
were treated with black tea solution, Group 4 Chamomile tea
control: Mice were treated with chamomile solution, Group
5 Mice were treated with kombucha black tea, Group 6:

Evaluation ofhair growth properties ofTopical Kombucha tea extracts
1 3
Mice were treated with kombucha chamomile tea, Group
7: Mice were treated with a mixture of kombucah black tea
and MSM, Group 8: Mice were treated with a mixture of
kombucha chamomile tea and MSM.
Assessment andevaluation ofhair growth activity
inmice
Following acclimatization period, an area of the hair (4cm2)
from the dorsal part was shaved from each mouse using elec-
trical razor and hair removal cream and then it was wiped
with surgical spirit. Evaluation of hair growth activity was
performed according to procedures describe previously
(Park etal. 2015) as follows:
No change = 0 score.
< 30% darkening of the affected area = 1 score.
30–70% darkening of the affected area = 2 scores.
> 70% darkening of the affected area or hair growth
in < 30% of the affected area = 3 scores.
> 70% darkening of the affected area and hair growth in
30–70% of the affected area = 4 scores.
> 70% darkening of the affected area and hair growth
in > 70% of the affected area = 5 scores.
Hair growth in > 90% of the affected area = 6 scores.
Later on, the shaved area was then sprayed with 3 puffs
(equals to 250µL) of respective treatments, which was per-
formed immediately after shaving and on a daily basis for
30days. On days 1, 15, and 30 post-shaving, hair growth
activity was determined at three test parameters: Hair den-
sity, length, and initiation time.
Statistical analysis
The results were presented as mean ± S.D. Data obtained
was analyzed using the ANOVA followed by Tukey’s test
with p < 0.05 were considered statistically significant.
Results
Hair growth initiation time
The minimum time for the growth of hair from the denuded
dorsal area was observed and the time taken for visible sign
of hair was recorded in Table1. The results undoubtedly
indicated that treatment with kombucha/black tea with MSM
(G7) reduced the time for hair growth initiation by 7days,
groups (G5, G6, and G8) reduced the time by 6days, while
the positive control group (G2) by 4days compared to the
negative control group, that suggests the beneficial effect of
kombucha in promoting hair growth and pushing the hair
cycle to enter the anagen phase quickly. Although some
reduction in the time for initiation of hair re-growth was
documented in group (G3, and 4) but it was not as noticeable
as with kombucha treated groups.
Effect ofdifferent kombucha tea extracts onhair
density
On days 15 and 30 of checking hair densities, it is so obvi-
ous that mice treated with kombucha/black tea with MSM
and without MSM have gained the highest hair densities
among all mice groups treated with other treatments. The
results presented in Fig.1 and supplementary Table1 indi-
cate that there is a significant enhancement in hair density
(p < 0.0001) approximately 70%, 50%, 38%, 20% in groups
G7, G5, G8, and G6 respectively in comparison to the
negative control group (G1). The improvement in hair den-
sity was significant (p < 0.0001) for all treated mice after
30days except with chamomile vehicle control group (G4).
It is noticeable that there was a significant enhancement
(p< 0.001) of hair density in mice treated with kombucha/
black tea with MSM (G7), kombucha/chamomile with MSM
(G8), and Kombucha black tea (G5) even when compared
with the group treated with minoxidil (G2).
Effect ofdifferent kombucha tea extracts onhair
length
The hair length analysis using caliper described in the
method section on days 15 and 30 are shown in Fig.2 and
supplementary Table2. The same trend of hair density can
be applied here, mice treated with kombucha/black tea
with MSM and without MSM (G7 and G5 respectively)
have gained the highest hair length. The results indicated
that there was a significant improvement (P < 0.0001) in
hair length in all groups except with (G4) on both days; 15
and 30. The length of hair was tripled for G7 (9.3 ± 0.87,
Table 1 Time taken (in days) to initiate hair growth after starting
respective treatments
GROUPS Number of days
for hair to initiate
growth
G 7 7
G 5 8
G 8 8
G 6 8
G 2 10
G 3 12
G 4 12
G 1 14

M.A.Alboreadi et al.
1 3
14.3 ± 0.77) compared with the length of hair in the negative
control group (G1) (3.7 ± 0.94, 5.9 ± 0.98) on the 15th and
30th days respectively, which shows that both kombucha
and MSM had a pronounced effect on the length of the hair.
Importantly, mice treated with black tea achieved a signifi-
cant hair length growth, which was not observed with group
treated with chamomile tea. It is also worth to mention that
the length of hair for the treated mice with different kombu-
cha extracts with or without MSM were significantly better
(p < 0.0001) than minoxidil-treated group.
Microbiological analysis andhair regrowth scoring
The microbiological analysis revealed that Zygosaccharomy-
ces bailii was the main fungus in the extract, while the acetic
acid producing bacteria was from the Acetobacter genus.
See supplementary resources. Differently, Fig.3 represents
the first day after removing hair from the dorsal part of the
mice for all groups (Fig.3: hair density score = 1). On day
15, we noticed that (G5 and G7) promoted the growth of
hair in the shaved area by almost 70%, while (G 2, G6 and
G8) by around 40% of the total denuded area with obvious
longer hair in groups (G5, G6, G7, and G8) Fig.4. However,
the vehicle control groups (G3, and G4) especially the cor-
responding negative control group (G1) showed less notice-
able, transient and irregular hair coats during this period.
The whole denuded skin in the mice had been fully covered
by hair when applied kombucha on day 30, however, groups
(G5, and G6) still had small area with no hair and/or hair
that is very short and not fully and densely covered Fig.5.
This obviously stipulates that kombucha extract with and
without MSM promoted the re-growth of hair, with better
result when combined with MSM.
Discussion
The results of this study demonstrated the potential effects
of kombucha extract with MSM in denuded mice as a hair
re-growth natural product. The presence of vitamins such
as vitamins B, and C, minerals and amino acids depends on
many factors such as symbiotic culture of yeast and bacteria,
Fig. 1 Hair density activity
based on the scoring system
adopted from six mice in the
treated mice group on day 15
and 30. G7 mice treated by
kombucha/ black tea with MSM
which have the best hair density
scores followed by kombucha/
black tea, kombucha chamo-
mile tea with MSM, kombucha
chamomile tea, and minoxidil.
Results are means ± SEM from
six mice in each group, on day
15 and day 30. *p < 0.05, **
p < 0.001, *** p < 0.0001 (p
value was calculated compared
to negative control group)
***
***
***
*** ***
*
***
*** ***
***
***
**
0
1
2
3
4
5
6
7
G7 G5 G8 G6 G2 G3 G4 G1
Hair Density Score
Aer 15 days
Aer 30 days
Fig. 2 Hair length in (mm) for
G7 mice treated by kombucha/
black tea with MSM which have
the best hair length results fol-
lowed by kombucha/ black tea,
kombucha chamomile tea with
MSM, kombucha chamomile
tea, and minoxidil. Results are
means ± SEM from six mice
in each group, on day 15 and
day 30. *p < 0.05, ** p < 0.001,
*** p < 0.0001 (p value was
calculated compared to negative
control group)
*** ***
*** ***
***
***
*
***
***
***
***
***
***
0
2
4
6
8
10
12
14
16
G7 G5 G8 G6 G2 G3 G4 G1
Hair Length (mm)
Aer 15 days
Aer 30 days

Evaluation ofhair growth properties ofTopical Kombucha tea extracts
1 3
sucrose content, temperature, and type of tea used in the fer-
mentation are all determinant factors for the components in
the final product. The fermented kombucha produces plenti-
ful of natural bioactive components, and their advantageous
and beneficial application could speed up hair growth and
regeneration. Kombucha is a symbiotic culture of number
of yeast and bacteria, such as Zygosaccharomyces bailii
thatproduces ethanol from the sugared tea (Karaman and
Sagdic 2019), in addition to the Acetobacter bacteria that
converts the ethanol to lactic and acetic acids (Martínez etal.
2018). Kombucha contains high levels of water-soluble vita-
mins, particularly; niacin, pyridoxine and ascorbic acid that
G7 G5 G8 G6
G2 G3 G4 G1
Fig. 3 A representative mouse of each group after on the 1st day of
treatment. Each mouse was treated with 3 puffs of its respective treat-
ments, G7: kombucah black tea and MSM, G5: kombucha black tea,
G8: kombucha chamomile tea and MSM, G6: kombucha chamomile
tea, G2: 2% minoxidil, G3: Black tea, G4: chamomile tea, G1: Nega-
tive control
G7 G5 G8 G6
G2 G3 G4 G1
Fig.4 A representative mouse of each group after 15 days of treat-
ment. Each mouse was treated with 3 puffs of its respective treat-
ments, G7: kombucah black tea and MSM, G5: kombucha black tea,
G8: kombucha chamomile tea and MSM, G6: kombucha chamomile
tea, G2: 2% minoxidil, G3: Black tea, G4: chamomile tea, G1: Nega-
tive control

M.A.Alboreadi et al.
1 3
function as antioxidant which reduces the oxidative stress
and nourishes the dermal papilla cell thus reveals the ability
of kombucha extract to enhance the growth of hair (DiBaise
and Tarleton 2019). It was distinguished that the treatment
with kombucha extract in this study resulted in expanding
the length of the active phase “anagen phase” of hair cycle.
In addition, the capability of kombucha extract to promote
the growth of hair in denuded mice, was measured by dif-
ferent parameters, including hair initiation time, density and
length.
The microbiological analysis in our study indicated the
presence of Zygosaccharomyces bailii that produces organic
acids from fermentation of sucrose into glucose and fructose
and consequently itconverted to lactic and acetic acid (both
are alpha hydroxyl acid AHA) due to Acetobacter bacterial
activities (Spedding 2015). AHAs are suggested to act as
exfoliants which normalizes the keratinocyte accumulation
or keratinization process on hair follicle that enhances the
hair strand emerging through the scalp, which strengthen the
hair shaft and prevent it from breaking off at the skin level
(Rigdon and Packchanian 1974). Furthermore, AHAs are
also hypothesized to encourage angiogenesis and enhances
blood flow to the scalp therefore promoting the exchange of
nutrition in the follicle resulting in superior hair growth with
boosting in hair density (Woo etal. 2019).
Our results illustrate that kombucha fermented in black
tea gave better results in hair promoting effects than when
chamomile flowers with kombucha applied. This can be
attributed to the presence of polyphenols in black tea that
promoted hair growth such as caffeine and flavonoids. The
amount of black tea major polyphenols which are thearu-
bigins and theaflavins account for 12–18% and 3–6% of
dry weight of black tea respectively (Halder etal.2005).
Furthermore, the caffeine contents in black tea ranges
from 2.79 to 2.93% and this is because many factors con-
trol it such as water temperature and extraction method
(Horžić etal. 2009; Komes and Ganić 2009). It has been
reported that caffeine inhibits phosphodiesterase, that in
turn encourages cell proliferation in the hair matrix by
increasing the level of cAMP in the cell and thus stimulate
its metabolism, this would impede the negative effect of
dihydrotestosteroneDHT on hair follicle and promotes the
elongation of hair fiber (Bansal, Manchanda, and Pandey
2012; Fischer, Hipler, and Elsner 2007). Moreover, fla-
vanoids of black tea such as EGCG have high affinity for
estrogen alpha receptor and consequently they possess hair
growth promoting activity (Hou etal. 2013).
Lastly, the addition of methylsulfonylmethane contrib-
utes in promoting hair growth, which is an organic sulfur-
containing compound that occurs naturally in a variety of
plants and animals including humans. Due to the structural
similarity with DMSO, MSM is expected to act as a skin
penetration enhancer for many drugs by similar mecha-
nisms (Nishino etal. 2008). Additionally, some studies
proposed that MSM forms bonds with hair follicle in order
to delivering sulfur to the cortex layer of the hair, and
thus promoting the conversion of telogen to anagen phase
(Shanmugam etal. 2009).
G7 G5 G8 G6
G2 G3 G4 G1
Fig. 5 A representative mouse of each group after 30 days of treat-
ment. Each mouse was treated with 3 puffs of its respective treat-
ments, G7: kombucah black tea and MSM, G5: kombucha black tea,
G8: kombucha chamomile tea and MSM, G6: kombucha chamomile
tea, G2: 2% minoxidil, G3: Black tea, G4: chamomile tea, G1: Nega-
tive control

Evaluation ofhair growth properties ofTopical Kombucha tea extracts
1 3
Conclusion
From this work, it can be strongly proposed that kombucha
fermentation of black tea has bioactive components that pro-
mote, restore and enhance the growth of hair through elon-
gating the anagen phase of the mice model especially when
combined with a penetrating agent such as MSM. This study
recommends possible potential of kombucha extracts to be
used as natural alternative therapy for hair loss. This natural
alternative therapy is considered a cost-effective treatment
and definitely has fewer side effects compared to the more
expensive synthetic drugs.
Acknowledgements This work was funded by Al-Isra University,
Amman, Jordan.
Funding The project was fully funded by Al-Isra university, Amman,
Jordan, under grant No. 2019/2018/17-174
Compliance with ethical standards
Ethical statement All animal handling in the present study were con-
ducted according to the guidelines of Institutional Animal Care and
Use Committee (IACUC) and the research ethics committee of the
faculty of pharmacy, Isra University has approved it under approval
No. 55-5/2019-2020.
Conflicts of interest Mohammed Ayad Alboreadi has no conflict of
interest. Manal Mamdouh Al-Najdawi has no conflict of interest. Qais
Bashir Jarrar has no conflict of interest. Said Moshawih has no conflict
of interest.
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