Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 4, 2021, Pages.

11631 - 11638
Received 05 March 2021; Accepted 01 April 2021.

The effect of Green Tea Extract, Black Tea exctract and Caffeine to the
Length of Femur on the DDY Strain Male Mice Which Given Calcium

Fransiska Ambarukmi Pontjosudargo1*, Hendri Priyadi2, Indarti Trimurtini3, Nasser I4,

Hasanah Y5
1,2,3,4,5
Universitas Jendral Achmad Yani, Cimahi, Indonesia
Email: ambarukmifransiska@gmail.com

ABSTRACT
The purpose of this study was to assess the effect of some kind of natural ingredients that are
commonly consumed to the length of the femur. The substances tested were green tea, black tea and
caffeine, in DDY strain male mice were given calcium. The target of this study was to prove
information about inhibition of bone growth from the length of green tea, black tea and caffeine. The
experimental laboratory study used 32 samples of mice devided into 8 groups. The subject was
treated to compare the effects of caffeine, green tea, and black tea on the growth of the femur length.
The data obtained were analyzed using one way ANOVA and Duncan's post hoc test, with a
confidence level of 95%. The results showed significant differences in bone length from each
treatment group with a value of p=0.000 (p<0.05). The longest bones were shown by a group
given only calcium (15.65 ± 0.26 mm), while the shortest was shown by a group given only
black tea (13.68 ± 0.13 mm). The groups, which were given green tea and caffeine, showed
significantly different bone lengths with negative control, but their effectiveness was not
comparable to those given only calcium. The group, which given black tea, showed the same
bone length as the negative control group although it was administered simultaneously with
calcium. The conclusion of this study, among the greens, the black and caffeine, the inhibitory
effect of long growth of the mice femur, that was given calcium, was shown by the group given
black tea.
Keywords: Black Tea, Green Tea, Caffeine, Femur Length, Calcium

1. Introduction
The bone, a dynamic organ, is a living structure composed of proteins and minerals that is
always undergoing improvement and provides the establishment of a structure. The bone is living
tissue that is hardest among other connective tissues in the body, consists of 50% water. The solid
part remainder cinsisting of various minerals, especially 76% of calcium salt and 33% of cellular
material. Bone continues to change its internal structure to reach the functional needs form through
thickening and expanding during remodeling of its growth. These changes occur through the activity
of osteoclast and osteoblast. This is influenced by several factors, mainly: minerals (calcium,
phosphorus and magnesium), vitamins (A, C and D), hormones (growth hormone), and stress when
received by the bone itself.1-3

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Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 4, 2021, Pages. 11631 - 11638
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Calcium has an important role in the formation of bones, which makes up its mass, strength
and hardness. Adequate Calcium intake has favorable effects on bone health such as higher bone
mineral accretion at early ages and prevention of osteoporosis. Almost 99% of calcium accounts as
the main component of the bone matrix as a constituent of the bones, as well as a backup when a
certain amount of calcium is needed by the body.1,2 Calcium absorption from an oral calcium source
is 20% - 40% after calcium administration, and the recommended levels of calcium is 400 – 1300
mg/day.4-6 Absorption of calcium in the intestinal mucosa is done by passive diffusion and active
transport. Calcium absorption is affected by several factors, such as the amount of calcium in the
diet, calcium needs, age, gender, use of certain medications, and the presence of other nutrients. In
addition, the absorption of calcium may have been hampered by the instability of the emotions, the
decrease in sports activity, laxatives or any substances that can cause diarrhea, the granting of
phosphorus and magnesium in large quantities, protein, sodium, fitat acid, fibers, drugs, caffeine,
tannin, and oxalic acid. The results of research conducted by Prabowo in 2011 indicated that a dose
of calcium of about 12.5 mg per day is the most effective intake for long bone growth of mice.1, 4-8
Based on the processing, tea (Camellia synthesis) divided into black tea (complete fermented
tea), green tea (unfermented tea), and oolong tea (partially fermented tea). Green and black tea
contain polyphenols and other components that may reduce the risk of developing chronic diseases
such as cancer, cardiovascular diseases, diabetes, promote weight loss and oral health and increase
bone mineral density. The compounds that can inhibit calcium are caffeine, tannin, and oxalic acid
contained in tea and coffee. Tannins are substances found in tea which can bind with calcium in the
intestine, therefore decreasing its absorption. Black tea has a high content of caffeine than green tea.
Black tea has the highest caffeine content compared to other teas. Caffeine is a chemical component
that has a negative effect against the calcium, but caffein effect on bone metabolism remains
controversial. Yoojin, et all in 2017 clearly showed the negative effects of caffeine on the long bones
and testes in rats. Huanxu, et all, in 2019, showed that treatment with caffeine did not adversely
affect organ weights, organ coefficients, femoral length, bone mineral density, biomechanical
properties, or bone microarchitecture in ovariectomized rats. The study demonstrated that caffeine
did not exert a damaging effect on the skeletal system of ovariectomized rats. This research was
conducted to find out the effects of black tea, green tea and caffeine in relation to long thigh bone
growth in mice.8-11

2. Materials and Methods

This research used experimental trials with the comparative method. The objects used were
32 mice strains DDY aged from 8-12 weeks and divided into 8 groups, with the number of replication
of mice is 4 each group. P1, a negative control group, is given standard food; P2 is given calcium
12.5 mg/day/oral; P3 is given green tea extract 0.21 mg/day/oral; P4 is given black tea extract
0.21 mg/day/oral; P5 is given caffeine 0.52 mg/day/oral, P6 is given calcium 25 mg + green tea
extract 0.21 mg/day/oral; P7 is given calcium 25 mg + black tea extract 0.21 mg/day/oral; and P8
is given calcium 25 mg + caffeine 0.52 mg/day/oral. Mice were given preferential treatment during

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Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 4, 2021, Pages. 11631 - 11638
Received 05 March 2021; Accepted 01 April 2021.

the last 4 weeks, and then a limb dissection of the right femur was taken. Measuring the femur bone
was conducted by calculating the length of the femur bone from the Trochanter major until
the medial Condylus using a Vernier Caliper. The data was analized using One Way ANOVA, and
was continued with Duncan Test, with a degree of confidence of 95%. The animal care was
consistent with institutional guidelines, and the Padjadjaran University (Bandung, Indonesia)
committee approved all procedures involving animals.

3. Results and Discussion

After 4 weeks of treatment, the average length of the shortest femur was indicated by the group
given 0,21 mg black tea extract, and the average length of the longest femur was indicated by the
group given 12.5 mg dose of calcium. The groups given black tea extracts with or without calcium,
have a shorter femur length compared to the negative control group. The groups were given green tea
extract and caffeine with or without calcium, and the group that was only given calcium had a longer
femur than the negative control group (Table 1).

Tabel 1. The length of the Femur bones of Mice in week 4th (mm)
No Groups
I II III IV V VI VII VIII
1.1 13.7 15.6 14,4 13.5 14.2 15.5 13.4 14.6
1.2 14.1 15.9 14,7 13.8 14.3 15.7 13.7 14.3
1.3 13.8 15.8 14,5 13.7 14.1 15.6 13.8 14.2
1.4 13.9 15.3 14,5 13.7 14.2 15.6 14.1 14.4
Total 55.5 62.6 58.1 54.7 56.8 62.4 55.0 57.5
Average 13.88 15.65 14.53 13.68 14.20 15.60 13.75 14.38
SD 0.17 0.26 0.13 0.13 0.08 0.08 0.29 0.17
Group I: negative Control (standard pellets and water)
Group II: positive Control 1 (calcium 12.5 mg)
Group III: positive Control 2 (green tea extract 0.21mg)
Group IV: positive Control 3 (black tea extract 0.21mg)
Group V: positive Control (caffeine 0,52 mg )
Group VI: Treatment 1 (calcium 12.5 mg + green tea extract 0.21 mg)
Group VII: Treatment 2 (calcium 12.5 mg + black tea extract 0.21 mg)
Group VIII: Treatment 3 (calcium 12.5 mg + caffeine 0,52 mg)

Tabel 2. Analized by One-Way ANOVA

Sum of Mean
Squares df Square F Sig.
Between Groups 17.029 7 2.433 75.824 .000
Within Groups .770 24 .032 .770
Total 17.799 31 17.799

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Table 2 showed the value of p ≤ 0.05, which indicated a significant difference between the
groups. There are at least two groups that have long femur differences which are significant. The
administration of the substance tested in the treatment group showed a significant influence.
Table 3 shows that there are four subsets for the eight treatment groups. Subset 1 was composed
of the group that was only given 0,21 mg black tea extract, the group was given 0.21 black tea
extracts combined with 12.5 mg of calcium, and negative control group. The third group was
classified under the same subset which means that among the treatment groups there were no
significant differences. That means administering 0.21 mg of black tea extract does not inhibit
the growth of long bones in the femur. Administration of calcium in the group given black tea
extract did not increase the femur length significantly compared to the negative control group.
Calcium administration may increase the femur length in the group given black tea extract,
but it was shorter significantly than the positive control group, which given only 12.5 mg of
calcium. This condition can occur because black tea contains several compounds that can inhibit
calcium absorption. Oxalic acid and tannins are substances which can bind with calcium,
therefore decreasing its absorption. Meanwhile, studies have shown that caffeine intake of up to
five or more cups of coffee per day is not a risk factor for increased bone mineral loss. Wendra in
2020 showed a significant influence of black tea extract administration dose 120mg/kg BW on
thick plate metaphysics (p=0.019). Administration of black tea extract dose 100mg/kg BW had a
significant effect on bone length growth (p=0,000). The addition of long and thick bone
metaphysical plates occurs due to the influence of catechins on black tea extract, which will
trigger osteoblastogenesis and prevent activation of osteoclasts. The bones will increase in length
on the metaphysical plate. In this study, the dose of black tea extract given on mice was 0.21 mg
equivalent to 80mg/Kg BW. The dosage used in this study was not effective in increasing femur
length.5,12

Tabel. 3 Post-Hoc Duncan Test

Subset
Group N
1 2 3 4
IV 4 13.6750
VII 4 13.7500
I 4 13.8750
V 4 14.2000
VIII 4 14.3750 14.3750
III 4 14.5250
VI 4 15.6000
II 4 15.6500
Sig. .148 .180 .248 .696
Group I: negative Control (standard pellets and water)
Group II: positive Control 1 (calcium 12.5 mg)
Group III: positive Control 2 (green tea extract 0.21mg)
Group IV: positive Control 3 (black tea extract 0.21mg)

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Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 4, 2021, Pages. 11631 - 11638
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Group V: positive Control (caffeine 0,52 mg )

Group VI: Treatment 1 (calcium 12.5 mg + green tea extract 0.21 mg)
Group VII: Treatment 2 (calcium 12.5 mg + black tea extract 0.21 mg)
Group VIII: Treatment 3 (calcium 12.5 mg + caffeine 0,52 mg)

Subset 2 consisted of groups, were only given 0.52 mg of caffeine, and the group has given
0.52 mg of caffeine combined with 12.5 mg of calcium. There was no significant difference
among the treatment groups in Subset 2. The Administration of 12.5 mg calcium and 0.52 mg
caffeine, resulting in a femur bone length similar to that of mice given only 0.52 mg of caffeine.
However, the groups of mice given 0.52 mg of caffeine with or without calcium 12.5 mg showed
femur length significantly longer than the negative control group. The study showed that caffeine
produces a significant increase in long femur growth when compared to a negative control group.
This suggests that caffeine does not influence the inhibition of growth in the long bones. The
group that was given caffeine combined with 12.5 mg of calcium resulted in a similar length
when compared to the group that was only given caffeine. Caffeine contains methyl-xantine
which is found in many types of coffee, tea and chocolate and is known to cause an increase in
calcium excretion through urine. 5 When calcium was given parallel with the caffeine did not
cause reduction of calcium. Calcium still absorbed and there is no interference of calcium
homeostasis in the body, so there is no disorder of long bone growth. Alshanbar et al, 2018
present the study that there is no significant correlation between bone mineral density and daily
caffeine consumption.13
In this study, the mice that were given caffeine did not provide an inhibitory influence and
it appears that the dosage given in this research did not cause disturbances in the balance of
calcium. The role of caffeine as a risk factor for bone loss is controversial. Moderate coffee
consumption has no effect on bone health.14 However, low calcium intake is clearly linked to
skeletal fragility, and it is likely that a high caffeine intake is often a marker for low calcium
intake.5 The negative effect of caffeine on calcium absorption is small enough to be fully offset
by as little as 1–2 tablespoons of milk. The observation implicating caffeine-containing
beverages as a risk factor for osteoporosis has been made in populations consuming substantially
less than optimal calcium intakes.15 Studies have shown that caffeine intake of up to five or more
cups of coffee per day is not a risk factor for increased bone mineral loss in postmenopausal
women. Even women with low daily calcium intakes did not show increased bone losses when
consuming this amount of caffeine. While high intakes of caffeine can cause urinary calcium
excretion, simply adding 1 - 2 tablespoons of reduced-fat milk to caffeinated coffee or tea will
negate this calcium loss.5
Subset 3 consisted groups that were given 0.52 mg of caffeine combined with 12.5 mg of
calcium and a group given 0.21 mg extracts of green tea. The administration of calcium in mice
that was given 0.52 mg of caffeine resulted femur length similar to mice who were given only
0.21 mg of green tea extract. In subset 4, there were groups that were given 0.21 mg extracts of
green tea combined with 12.5 mg of calcium and the group was only given 12.5 mg of calcium.

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Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 4, 2021, Pages. 11631 - 11638
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Administration of 12.5 mg of calcium with or without green tea extract produced the longest
femur and the most significant difference in femur length than any other group. It can conclude
that giving black tea extract either with or without calcium results in the shortest femur among
all groups. Administration of caffeine and green tea extract with or without calcium increased
femur growth, but not comparable to the length of the femur growth that resulted from groups
that are only given calcium. Green tea extract can increase the length of the thigh bone in mice,
even more, if given the extra calcium intake, although the length of the bone that is generated
does not exceed the length of the femur mice that only given calcium. This can be explained
because green tea less caffeine content and contain no brominated compounds oxalic absorbs
calcium.5 Tea extracts are source of polyphenols, which are antioxidant components. Green tea
phenolic compounds are predominately composed of catechin derivatives, although other
compounds such as flavonols and phenolic acids are also present in lower proportion. The main
catechin compounds found in green tea are Epigallocatechin Gallate (EGCG), Epigallocatechin
(EGC), Epicatechin Gallate (ECG) and Epicatechin (EC) and other compounds. Green tea Green
tea contains more catechins than black tea or oolong tea, appeared to benefit bone health more
than other kinds of tea (black tea) which may be due to decreased oxidative stress, increased
activity of antioxidant enzymes, and decreased expression of pro-inflammatory mediator. An
interesting study showed that the tea drinkers had significantly greater mean bone mineral
density (BMD) measurements, independent of smoking status, use of hormone-replacement
therapy, coffee drinking, and whether milk was added to tea. Older women who drank tea had
higher BMD measurements than did older women who did not drink tea. Green tea leaf extract has
an effect on decreasing the number of alveolar bone osteoclast cells of rats with lipopolysaccharide-
induced where the effective dose of green tea leaf extract is 150mg/100grBB.10,16
The longest femur bone found in the group that given calcium 12.5 mg. This is in
accordance with the benefits of calcium can increase the growth of long bones. Calcium is an
important component in bone growth and is a mineral that is found in large quantities in the
human body. Ninety-nine percent of all the calcium in the body is found in the bones and teeth.
Additional calcium in mice can be directly synthesized by osteoblasts for the formation of matrix
without disturbing the balance of calcium. Calcium supplement have positive effects on bone
mineral density.5,8,16,17

4. Conclusions
The Black tea extract of 0.21 mg inhibits thigh-length bone growth in male DDY rat strains
but not significantly. The Green tea extract 0.21 and caffeine 0.52 mg affects the addition of long
femurs in male DDY rat strains significantly, but not comparable to the calcium effect of 12.5
mg in affecting femur growth

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Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 4, 2021, Pages. 11631 - 11638
Received 05 March 2021; Accepted 01 April 2021.

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Received 05 March 2021; Accepted 01 April 2021.

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