Chemical Analysis of Iron, Vitamin C, Tannins and Total Flavonoids

in Ethanol Extract of Jamaican Cherries Fruits

(Muntingia calabura L) for Development of Antianemia

Noor Khalwati Afdhaliya

, Dono Indarto

2,3,4 b

and Brian Wasita

2,5 c

Postgraduate Student of Human Nutrition Sciences, Universitas Sebelas Maret, Surakarta, Indonesia

Postgraduate Program of Nutrition Sciences, Universitas Sebelas Maret, Surakarta, Indonesia

Department of Physiology, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia

Biomedical Laboratory, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia

Departement of Anatomical Pathology, Faculty of Medicine Universitas Sebelas Maret, Surakarta, Indonesia

Keywords: Vitamin C, Iron, Flavonoids, Tannins, Ethanol Extract and Jamaican Cherries.

Abstract: Various natural sources such as red guava, moringa leaves and snake fruit seeds have been developed for

anemia treatment. Fresh Jamaican cherries is known to have 178.96 mg / 100 g vitamin C of and 0.10 mg /

100g Fe. Flavonoids, especially chrysin and quercetin are bioactive compounds, which have an important

role in improvement of haemoglobin (Hb) levels. The aim of this study was to extract Jamaican cherries fruits

and to analyse that extract in terms of vitamin C and iron concentrations, total flavonoids and tannins. Ripe

fruits of Jamaican cherries were collected from Sleman, Yogyakarta and dried cherries were extracted using

the maceration method with ethanol solvent. Chemical analysis of vitamin C and flavonoids was carried out

at the Integrated Laboratory of Universitas Ahmad Dahlan while Fe and tannins concentrations were analysed

at the Food and Nutrition Research Centre, Universitas Gadjah Mada, Yogyakarta. We got 34% ethanol

extract of Jamaican cherries fruits. The highest chemical concentration was tannins (2.96 ± 0.01%), followed

by Fe (0.104 ± 1.05%), vitamin C (0.09 ± 0.01%), and total flavonoids (0.0012 ± 0.56%). In conclusion,

ethanol extract of Jamaican cherries fruits becomes a promising nutraceutical for anemia treatment in future.

1 INTRODUCTION

Iron deficiency anemia persists a nutritional health

problem in the world by which becomes one of the

main causes of Global Disability Adjusted Life Years

(DALY's) in adolescents (Vos et al, 2020). Iron

fortification and supplementation are recommended

to overcome this problem. However, some

adolescents with anemia have minor responses to

those treatments.

Medicinal plants are a very popular alternative

treatment in some countries. Data from WHO (2018)

have indicated that 85% African and Southeast Asian

countries, 63% western Pacific regions and 43%

eastern Mediterranean regions use traditional

medicine for treatment of human diseases (WHO,

https://orcid.org/0000-0003-4772-0577

https://orcid.org/0000-0001-7420-5816

https://orcid.org/0000-0002-5501-3541

2019). Iron deficiency anemia is one of human

diseases, which can be treated using natural sources

such as red guava, moringa leaves or snake fruit seeds

extract. However, research evidence is still required

to figure out efficacy and safety for long term use

(Yusnaini, 2016; Arini, 2018; Ristanti, 2019).

Jamaican cherries (Muntingia calabura L) for

example, it easily grows in any type of soil in

Indonesia and produces its fruits in all seasons

(Ningsih, 2016). Many people believe that the

Jamaican cherries have beneficial effects for human

health (Mahmood et al., 2014). Previous studies have

reported jamaican cherries extracts derived from

roots, leaves and barks as alternative treatments for

diabetes, microbial infection and inflammation.

Generally, 100g fresh fruits of Jamaican cherries

contain 178.96 mg vitamin C and 0.10 mg Fe

Afdhaliya, N., Indarto, D. and Wasita, B.

Chemical Analysis of Iron, Vitamin C, Tannins and Total Flavonoids in Ethanol Extract of Jamaican Cherries Fruits (Muntingia calabura L) for Development of Anti Anemia.

DOI: 10.5220/0010756700003235

In Proceedings of the 3rd International Conference on Social Determinants of Health (ICSDH 2021), pages 5-9

ISBN: 978-989-758-542-5

(Makahity et al., 2019). In addition, 1g dried fruits of

Jamaican cherries consists of flavonoids, which are

equal to 1.84-21.80 Myricetin Equivalents (ME)

(Nurholis & Saleh, 2019). Unripe and ripe fresh fruits

of Jamaican cherries also contain large amounts of

flavonoids such as rutin, myricetin, luteolin,

quercetin, apigenin, and kaempferol (Kubola et al.,

2011). Some studies have used flavonoids for anemia

treatment (Borawska et al., 2014) and a recent study

reported that administration of 50 mg / Kg BW

quercetin increases hemoglobin and serum iron levels

in mouse model with anemia (Mazhar et al., 2018).

Some previous studies related to extraction of

Jamaican cherries fruits have been done. For

example, Gomathi et al (2012) reported the extraction

of Jamaican cherries fruits using a mixture of

methanol, acetone and water. Meanwhile, Novita

(2016) extracted Jamaican cherries fruits with ethanol

or waters solvent. In addition, Subagya (2019)

extracted Jamaican cherries fruits with 70% ethanol

for anti-bacteria and Sujono et al (2021) reported the

extraction of Jamaican cherries fruits using a ethyl

acetate or methanol solvent for identification

phytochemical profile. From some research studies,

they did not measure Fe, total flavonoids and tannin

levels. In addition, only Novita’s study has measure

vitamin C level in the extract but it used the

iodometric titration method. Therefore, the aim of this

study was to extract Jamaican cherries fruits with

ethanol solvent and to analyse that extract in terms of

vitamin C, iron, total flavonoids and tannins

concentrations.

2 MATERIALS AND METHODS

2.1 Jamaican Cherries Extraction

Ripe Fruits of Jamaican cherries were collected from

street side in Ambarketawang, Sleman, Yogyakarta

for around 2 months. Washed Jamaican cherries were

then dried using cabinet dryer at 60

C for 26 hours

and then were grounded using a grinder with a 80

mesh sieve at Rumah Berkah Herbal Group in

Yogyakarta.

Jamaican cherries fruits extracted with the

existing maceration method by Ristanti (2019) for

extraction of snake fruit seeds. A total of 300g flour

of Jamaican cherries was extracted using the

maceration method with 1.5L 70 (volume/volume) %

ethanol (p.a merck, Germany) at room temperature

(28

C ± 2

C) for 3 days. Furthermore, the solution

was filtered using 40µm Whatman filter paper to

separate the residue and filtrate. Furthermore, the

residue was re-macerated using the same solvent with

1:3 ratios for 2 days at room temperature. The re-

suspended solution was filtered again using the 40 µm

Whatman filter paper to obtain the second filtrate.

The first filtrate was combined with the second

filtrate and then was concentrated with a rotary

vacuum evaporator (Rotavapor R215) at 60

C, 60

rpm and 175 mbar. Finally, Jamaican Cherries extract

was kept at refrigerator until further analysis.

2.2 Determination of Extract Yield

The yield of Jamaican cherries fruits extract was

calculated using the following formula:

% Yield

  

  

x 100%

2.3 Micronutrient Analysis

Measurement of vitamin C and total flavonoid levels

in Jamaican cherries fruit extract was carried out at

the Integrated Research Laboratory, Faculty of

Pharmacy, Universitas Ahmad Dahlan, Yogyakarta

while Fe and tannins levels were analysed at the

Laboratory of the Center for Food and Nutrition

Studies, Universitas Gadjah Mada. 100 mg Jamaican

cherries fruit extract was dissolved in distilled water

and were then analysed using the UV-Vis

spectrophotometer with 200 - 400 nm wavelength

(Makahity et al., 2019).

For measurement of Fe levels, 25 mg Jamaican

cherries fruits extract was dissolved in distilled water.

A total amount of 0.1 ml dissolved extract was

reacted with 1.1 ml of 100 ppm Na

, 1.10 ml of

1,000 ppm phenanthroline solution and 1.5 ml acetate

buffer pH 4.5. After that, 5 ml of acetone and distilled

water was added into the above solution and was

incubated at room temperature for 120 minutes. The

solution was analysed using the UV-Vis

spectrophotometer with 400 - 800 nm wavelength

(Makahity et al., 2019).

2.4 Determination of Total Flavonoids

and Tannins

To determine total flavonoids, 25 mg Jamaican

cherries fruits extract was dissolved in 25 ml of 96%

ethanol. Then, 1 ml dissolved extract was reacted

with 3 ml of 96% ethanol, 0.2 ml AlCl

and 0.2 ml

sodium acetate. Furthermore, it was

spectrophotometrically measured at 430 nm

wavelength (Winahya et al., 2018). Meanwhile,

tannins measurement used 1 mg Jamaican cherries

ICSDH 2021 - International Conference on Social Determinants of Health

fruits extract, which was dissolved in distilled water

to reach 10 ml (100 ppm). After that, 9 ml the solution

was re-dissolved in distilled water until 10 ml (90

ppm). Next, the solution was mixed with 1 ml folin-

denis’ reagent for 3 minutes. The mixed solution was

then added with saturated Na

and was incubated

for 40 minutes. Furthermore, it was

spectrophotometrically measured at 650 nm

wavelength (Pratama et al., 2019).

3 RESULTS AND DISCUSSION

In the Table 1, we have extracted local Jamaican

cherries fruits with the existing maceration method

for extraction of snake fruit seeds (Ristanti, 2019).

Previously, we also calculated the yield value of

Jamaican cherries fruit flour to see the weight loss of

Jamaican cherries fruit due to the drying process. The

high water content of Jamaican cherries fruit a low

flour yield. The yield of this method extraction was

34 (weight/weight)%, which was slightly greater than

another previous study (32.68%).

Table 1: The Yield of Jamaican Cherries Extract.

Sample Yiel

(%)

Jamaican Cherries Fruits

Flou

0.14

Jamaican Cherries Fruits

Extract

The difference in the extract yield was caused by

maceration time and frequency. In our study, we

macerated Jamaican cherries fruits for 3 days while

Subagya study macerated them for 2 days. In

addition, the residue of Jamaican cherries was re-

macerated for 2 days. A research conducted by

Subagya, Jamaican cherries fruits extract was used to

inhibit Porphyromonas gingivalis proliferation

(Subagya, 2019) while our study used the ethanol

extract of jamaican Cherries for iron suplementation.

In this present study, we have firstly documented

the iron level in the ethanol extract of Jamaican

cherries fruits. Table 2. indicated micronutrients and

phtochemicals analysis in the ethanol extract of

Jamaican cherries fruits. The highest level of

chemical compounds in the extract was tannins (2.96

± 0.01%), followed by Fe (0.104 ± 1.05%), vitamin C

(0.09 ± 0.01%), and total flavonoids (0.0012 ±

0.56%). Because we used a polar solvent, it is not

surprising that our extract contains tannin. The result

of tannin analysis in our study is accordance to other

studies (Krishnaveni & Dhanalakshmi, 2014 ; Hadi &

Permatasari, 2019), but their studies only used a

qualitative method. Tannins are an important

chemical compound in the most plant kingdoms

because of their role for predator protection and

regulatory growth (Das et al., 2020).

There is a limited study that reported a

disadvantage effect of tannin for inhibition of Fe

absorption. Based on Greger et al (1988),

administration of tannic acid or black tea

consumption impairs iron bioavailability and status,

in anemia rats. In contrast, administration of

condensed tannins (proanthocyanidins) and Soybeen

seed ferritin (SSF) has slightly different effects of

ferritin and iron serum levels in rats with anemia,

compare to rats with anemia treated with SSF only

(Yun et al, 2011). In addition, epidemiological

studies and long-term trials show that individual iron

status is not affected by tannin consumption because

the human body has the adaptation mechanism such

as chloric acid in gaster and the present of vitamin C

from daily intake to minimize the anti-nutritional

effect of tannins (Delimont et al, 2017). Due to the

high present of vitamin C in our extract, we believe

that the anti-nutritional effect of tannin will be

neutralized by vitamin C although we need further

investigation.

Table 2: Results of Vitamin C, Fe, Total Flavonoids and

Tannins Levels of Ethanol Extract of Jamaican Cherries

Fruits.

Chemical Analysis Mean ± SD (%)

Tannin 2.96 ± 0.01

Fe 0.104 ± 1.05

Vitamin C 0.09 ± 0.01

Total flavonoids 0.0012 ± 0.56

Our research finding showed that iron is the

second highest level of chemical compounds in the

ethanol extract of Jamaican cherries fruits. This result

of the present study is different from a previous study

by Makahity et al (2019). They reported that 100g

fresh Jamaican cherries fruits were collected from Air

Louw, Nusaniwe, Ambon contains 0.10 mg iron,

343,39 mg carbohydrates, 178,96 mg vitamin C and

1,48 β-carotene. Using the same extraction method,

Ristanti (2019) reported that ethanolic extract of

snake fruit seeds has higher iron level (211 mg/100g)

than simplicia of snake fruits seeds (26.88 mg/100 g).

Therefore, this shows that the extraction process

using 70% ethanol solvent can dissolve iron

effectively. It potentially becomes an important

source of iron for anemia treatment.

In contrast to tannin and iron levels, vitamin C in

the ethanol extract of Jamaican cherries fruits is lower

than the fresh Jamaican cherries fruit (178.96 mg /

Chemical Analysis of Iron, Vitamin C, Tannins and Total Flavonoids in Ethanol Extract of Jamaican Cherries Fruits (Muntingia calabura L)

for Development of Anti Anemia

100 g), reported by Makahity (2019). The decreased

level of Vitamin C in our extract is due to the drying

process that using a constant temperature (60

C) for

26 hours. The longer heating time is associated with

the higher degradation of vitamin C (Igwemmar et al.,

2013). Furthermore, our research finding of vitamin

C level in the Jamaican cherries fruits extract is higher

than that of Novita study (2016) which found that the

vitamin C level in her extract was 17.64 mg/100 g.

The discrepancy of different vitamin C levels may be

caused by the different analytical method. We used

Uv-Vis spectrophotometrical analysis while she used

the iodometric titration method, which is less

accurate.

The total flavonoids levels in Jamaican cherries

fruits have been reported by some researchers.

According to Senet et al (2017), they found that total

flavonoids level in fruits of Jamaican cherries ethanol

extract are 134.4 mg/100 g and 29.9 mg/100 g of

Jamaican cherries ethyl acetate extract, higher than

total flavonoids level in our study (12.25 ± 0.56

mg/L). The huge differences in flavonoid levels result

from the pre-extraction process. The fresh fruits of

Jamaican cherries were firstly soaked for 9 days and

directly macerated gradient maceration method using

n-hexane, ethyl acetate, and ethanol as solvents with

2 litres of solvent replacement every 3 days. The

longer time for soaking will get the higher amount of

phytochemicals during maceration process (Wijaya et

al., 2018).

Recently, the total flavonoids level is considered

as a new perspective for anemia treatment. Quercetin

and chrysin for instance, has been administered to

mouse model with anemia and normal rats.

Administration of 50 mg / Kg BW quercetin and 50

mg / Kg BW FeSO

in rat with anemia significantly

increase haemoglobin and serum iron levels for 30

days, compared to normal and anemic groups treated

with FeSO

only (Mazhar et al., 2018). In addition to

quercetin, administration of 100 mg/Kg BW chrysin

was reported to increase Hb levels in normal rats for

7 days, compared with the control group (Borawska

et al., 2014). However, the limitation of our study is

we did not quantify the quercetin and chrysin levels

in our extract.

4 CONCLUSION

Extraction of Jamaican cherries fruits using 70%

ethanol yields 34% extract. The ethanol extract of

Jamaican cherries fruits has 0.104% Fe level which

becomes a promising nutraceutical for anemia

treatment. However, further studies are required to

analyse quercetin and chrysin levels in ethanol extract

of Jamaican cherries fruits and to figure out the

synergetic effect of those flavonoids and Fe level to

increase haemoglobin level in anemia rats model.

ACKNOWLEDGEMENTS

We would like to thank all staff in the Integrated

Laboratory of Universitas Ahmad Dahlan for

extraction of Jamaican cherries fruits, testing vitamin

C and flavonoids levels. We also thanks to the

Laboratory of Food and Nutrition Study Center,

Universitas Gadjah Mada for testing tannin and

flavonids.

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Chemical Analysis of Iron, Vitamin C, Tannins and Total Flavonoids in Ethanol Extract of Jamaican Cherries Fruits (Muntingia calabura L)

for Development of Anti Anemia