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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: [email protected]

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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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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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

Squares df

Mean

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

Group N Subset

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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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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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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References

[1] Setiawati R, Rahardjo P. 2018. Bone Development And Growth. IntechOpen. DOI:

http://dx.doi.org/10.5772/intechopen.82452

[2] Guyton H. 2020. Hormon Paratiroid, Kalsitonin< Metabolisme Kalsium dan Fosfat, Vitamin D, tulang dan gigi. 13 ed. jakarta: Buku Kedoteran EGC

[3] Gerard J. Tortora, Bryan H. Derrickson. 2017. Tortora’s Principle of Fundamental Anatomy and Physiology. 15th ed. Vol. 1. New Yort: John & Wiley & Sons Inc.

[4] G Cormick, Jose M. Belizan. 2019. Calcium Intake and Health. Nutrients 2019, 11, 1606 [5] Houtkooper L, Farrel V, Mullins V. 2017. Calcium supplement guidlines. The University

of Arizona College of Agriculture And Life Sciences Tucson, Arizona Cooperative Extension;

[6] Yukari Ueda, Zenei Taira. 2013. Effect of Anions or Food on absolute bioavailability of Calcium From Calcium Salt in mice by pharmacokinetics. Journal of Experimental Pharmacology 2013:5 65-71

[7] Ian R Reid, Mark J Bolland. 2020. Calcium and/or Vitamin D Supplementation for the Prevention of Fragility Fractures: Who Needs It? Nutrients 2020, 12, 1011

[8] Prabowo, RA. 2011. Pengaruh Pemberian Kalsium Terhadap Pertumbuhan Panjang Os.

Femur Pada Mencit (Mus Musculus) Jantan Galur Swiiss Webster. Fakultas Kedokteran Unjani.

[9] Kodagoda K.H.G.K., Wickramasinghe I. 2017. Health benefits of Green and Black Tea: A Review. International Journal of Advanced Engineering Research and Science (IJAERS) Vol-4, Issue-7, p 107-12

[10] Vishnoi H, Ramesh B. Bodla and Ravi Kant. 2018. Green Tea (Camellia Sinensis) And Its Antioxidant Property: A Review. IJPSR, 2018; Vol. 9(5): 1723-36

[11] Yoojin Kwak, Hyeonhae Choi and Jaesook Roh. 2017. The Effects of Caffeine on the Long Bones and Testes in Immature and Young Adult Rats. Toxicol. Res. Vol. 33, No. 2, pp.157- 164

[12] Wendra, Mone ZJ, Fenandy AL. 2020. Pemberian Ekstrak Etanol Teh Hitam Meningkatkan Pertumbuhan Lempeng Metafisis Dan Panjang Os Femur Mencit. Medika Kartika Vol. 2

[13] Alshanbar MF, Alsofyani EM, Almalki DM, Alswat KA. 2018. Caffeine Effect on Bone Mineral Density: A Cross-sectional Study. Journal of Clinical and Diagnostic Research Vol-12(2): p 14-17

[14] Xua H, Titi Liua, Lihong Hua, Jin Lia, Chunxia Gana, Jing Xua, et al. 2019. Effect of caffeine on ovariectomy-induced osteoporosis in rats. Biomedicine & Pharmacotherapy 112: 108650

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[15] Abdulaziz Al-Othman, Sara Al-Musharaf, Nasser M Al-Daghri, Sobhy Yakout, Khalid M Alkharfy, Yousef Al-Saleh, et al. 2012. Tea and coffee consumption in relation to vitamin D and calcium levels in Saudi adolescents. Nutrition Journal 2012, 11:56 .

[16] Prasetyaningrum N, Soemardini, Fadillah MN. 2018. Efek Ekstrak Daun Teh Hijau (Camellia Sinensis) Terhadap Sel Osteoklas Tulang Alveolar Tikus Putih (Rattus Norvegicus). E-Prodenta Journal of Dentistry 2(1): p 130-139.

[17] Chiodini I, Bolland MJ. 2018. Calcium supplementation in osteoporosis: useful or harmful?. European Journal of Endocrinologyn178: p D13–D25.

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