selection of maceration solvent for natural pigment

Indones. J. Nat. Pigm., Vol. 02, No. 1 (2020), 8–12

Yuliati et al. (2020)

Content from this work may be used under the terms of the Creative Commons Attribution 4.0 licence 8

Selection of Maceration Solvent for Natural Pigment Extraction from Red Fruit (Pandanus conoideus Lam) Tantyo Ardy Bintoro Purnomoa, Yehezkiel Steven Kurniawanb, Ruth Febriana Kesumaa and Leny Yuliatia,b,*

a Department of Chemistry, Faculty of Science and Technology, Universitas Ma Chung, Malang 65151, East Java, Indonesia

b Ma Chung Research Center for Photosynthetic Pigment, Universitas Ma Chung, Malang 65151, East Java, Indonesia

*Corresponding Authors: [email protected] (Tlp. +62-341-550-171; Fax +62-341-550-175)

Article History: Received 28 February 2020, Revised 03 March 2020, Accepted 03 March 2020, Available Online 06 March 2020 Abstract Red fruit (Pandanus conoideus Lam) is rich with red-orange natural pigments, such as β-carotene. In this work, the solvent selection was investigated to extract the carotenoid pigment from the red fruit via a simple maceration technique. Three types of solvents were used in the maceration, which were distilled water, ethanol, and acetone. The obtained extracts were characterized using spectrophotometer ultraviolet-visible (UV-visible), Fourier transform infrared (FTIR) and spectrofluorometer. The different solvents gave different spectroscopic information, suggesting that the solvent selection influenced the type of the extracted compounds. Among the examined solvents, acetone was found to be the most effective one to extract the carotenoid pigment. The presence of the carotenoid pigment in acetone extract was confirmed by the appearance of the absorption peak at 476 nm on its UV-visible spectrum, while from its FTIR spectrum, the C-H sp3 functional group of carotenoid pigment was found at 2924 and 2854 cm-1. In addition, the emission peak of carotenoid pigment was found at 394 and 561 nm. This study confirmed that acetone performed as a better maceration solvent for carotenoid pigment as compared to the distilled water and ethanol, which would be strongly related to the non-polar property of the acetone.

© 2020 MRCPP Publishing. All rights reserved. http://doi.org/10.33479/ijnp.2020.02.1.8

Keywords: acetone, carotenoid pigment, extraction, maceration, red fruit

INTRODUCTIONIndonesia as a tropical country holds a mega and unique

diversity of the plant species [1]. One of the unique plant species is red fruit (Pandanus conoideus Lam), which has been reported for its wide biological activity such as antioxidant [2,3], antimicrobial [4], antitumor [5], antidiabetic [6,7], anti-inflammatory [8], and anticancer agents [9,10]. Due to the outstanding biological activity of the natural products in the red fruit extract [11], researchers are putting their efforts on the isolation and characterization of the contained natural products. It was found that the biological activity of red fruit is generated from several natural products, such as α-carotene, β-carotene, β-cryptoxanthin, α-tocopherol, and unsaturated fatty acids [9,12,13]. The composition of red fruit oil is listed in Table 1.

β-carotene, as one of the major natural pigments in the red fruit, is a red-orange natural pigment that belongs to the terpenoid group and derived from the biosynthesis pathway of

geranylgeranyl pyrophosphate [14]. β-carotene with a molecular formula of C40H56 contains forty carbon atoms, which is constructed from eight isoprene units. It is known for its useful application as the food colorant and vitamin A precursor, making the extraction of β-carotene from natural resources is pivotal in food and health sciences [15,16]. While several extraction methods have been developed, researches in this field are still required to obtain the optimum method for β-carotene extraction.

In this work, various solvents of distilled water, ethanol, and acetone were examined to extract carotenoid pigment from the red fruit by a simple maceration method. The extracts were then examined by various spectroscopic methods involving spectrophotometer ultraviolet-visible (UV-visible), Fourier transform infrared (FTIR) and spectrofluorometer. Both finding the suitable solvent for the carotenoid pigment

Crucial Solvent Selection: As compared to distilled water and ethanol, acetone was more suitable to be used as the solvent to extract carotenoid pigment from the red fruit by a maceration method. This would be due to the non-polar characteristic of acetone. The successful extraction was clarified from the spectroscopic studies by spectrophotometer ultraviolet-visible, Fourier transform infrared, and spectrofluorometer.

Maceration for 24 h




extraction from the red fruit and investigating the characteristics of the extracts are crucial to utilize the red fruit as the source of the carotenoid pigment.

Table 1. Natural products composition of red fruit oil [9]

Element Content per 100 g of red fruits oil

α-carotene 130 μg β-carotene 1980 μg

β-cryptoxanthin 1460 μg α-tocopherol 21.2 mg Linoleic acid 64.9 g

oleic acid 8.6 g EXPERIMENTAL General

The materials used in this study were red fruit juice (Pandanus conoideus Lam) obtained from Papua, distilled water, ethanol (C2H5OH, 99.5%, Merck EMPLURA), and acetone (C3H6O, 99%, Merck EMPLURA). The instruments used in this research were UV-visible spectrophotometer (V-760, JASCO), Fourier transform infrared spectrophotometer (FT/IR-6800, JASCO), and spectrofluorometer (FP-8500, JASCO).

Extraction of carotenoid pigments from red fruit through maceration technique

The extraction of carotenoid pigment from the red fruit was carried out by a maceration technique using three kinds of solvents, i.e. distilled water, ethanol, and acetone. The maceration process was carried out in a similar manner as previously described [17]. Briefly as much as five grams of red fruit juice was added into 50 mL of distilled water, ethanol, or acetone, separately. Then the solution was covered with aluminum foil at room temperature (25 °C) and left for 24 h in dark condition.

Characterizations of extracts

After 24 h, the extract was filtered to obtain a clear filtrate. The absorption spectrum of each filtrate was measured by the UV-Vis and FTIR spectrophotometers and their emission spectra were recorded with a spectrofluorometer. The obtained data were analyzed to find the best maceration solvent for carotenoid pigment extraction from the red fruit.

RESULTS AND DISCUSSION

The filtrate photograph of red fruit extracts obtained using three kinds of solvents is shown in Figure 1. It was obvious that different solvents gave different color appearances in the filtrates. Red fruit extracted using distilled water was obtained as a light yellow solution, while the ethanolic and acetone extracts were obtained as a red solution. From that appearance, it could be expected that both the ethanolic and acetone extracts contained a higher concentration of red natural pigments than the one extracted in the distilled water.

Each extract was then characterized by measuring its absorbance spectrum with a UV-Vis spectrophotometer, identifying its functional group with an FTIR spectrophotometer and recording its excitation and emission spectra with a spectrofluorometer. Recording of these spectra is important to support the successful extraction of carotenoid pigment from the red fruit as well as to determine the most suitable solvent for the extraction.

Figure 1. Photograph of red fruit extracts using (a) ethanol, (b) acetone, and (c) distilled water as the maceration solvents.

The UV-visible spectra of red fruit extract filtrates are shown in Figure 2. The distilled water extract gave the absorption peak below 300 nm at 266 nm and no peak was observed at visible region (l > 400 nm). This is in good agreement with the color appearance of the distilled water extract as shown in Figure 1 (c). In contrast, both the ethanolic extract and acetone extract gave absorption bands above the visible region, which were observed at maximum wavelength of 481 and 476 nm, respectively. These peaks could be assigned to the presence of carotenoid pigment [18,19].

It was demonstrated that distilled water could not be used to extract carotenoid pigment from the red fruit. This result is in accordance with the nature of carotenoid pigment as a non-polar compound so that it is impossible to be extracted into water [12]. On the other hand, ethanol and acetone could be used as the maceration solvent to extract the carotenoid pigment. However, the absorbance intensity of the extract in acetone was slightly higher than that in ethanol. In addition, the peak of carotenoid pigment in the visible region was red-shifted from 476 to 481 nm when the maceration solvent was changed from acetone to ethanol. This would be due to the positive solvatochromic effect, where increasing the solvent polarity would give the bathochromic (red) shift.

Figure 2. UV-visible spectra of red fruit extracts using distilled water, ethanol, and acetone as the maceration solvents.

The extracts were also characterized using fluorescence spectroscopy. The fluorescence spectra of the red fruits extracted in distilled water, ethanol and acetone are shown in Figures 3, 4 and 5, respectively. As shown in Figure 3 (a), the extract of red fruit using distilled water as the solvent gave the excitation peaks at 290 and 330 nm. Two different emission peaks were observed when two different excitation peaks were used as the monitored wavelength. The emission peak was observed at 360 nm when excited at 290 nm, while the peak was shifted to 430 nm when excited at 330 nm, as shown in

Ethanol Acetone Water

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Figure 3 (b). These different emission peaks observed with the different excitation energy would be the characteristic of the Raman scattering from the distilled water. Again, this result is in good agreement with the appearance and the UV-Vis data discussed above, showing the absence of the carotenoid pigments in the distilled water extract.

Figure 4 shows the three-and two-dimensional fluorescence spectra when ethanol was employed as the maceration solvent. Different from the one extracted by distilled water, the red fruit extract using ethanol solvent gave two excitation peaks at 266 and 294 nm. Only one emission peak was observed around 343–344 nm when using these two excitation wavelengths. These peaks could be assigned to α-tocopherol [20]. However, it is noted here that as compared to the absorption data from the UV-visible spectrophotometer, the fluorescence spectra do not show any excitation and emission peaks in the visible area. This result shows that the ethanol could extract the α-tocopherol better than the carotenoid pigment, which made it

not that suitable to be used to extract the carotenoid pigment. The fluorescence spectra of the red fruit extracts using

acetone are shown in Figure 5. It was revealed that two excitation peaks at 334 and 350 nm were detected and both excitation peaks corresponded to emission peaks at 394 and 561 nm. However both emission peaks were generated in different peak intensities when the excitation peaks are changed from 334 to 350 nm. When excited at 334 nm, the intensity of the emission peak at 394 nm was higher than the emission peak at 561 nm. In contrast, when the extract was excited at 350 nm, the intensity of the emission peak at 394 nm was lower than the emission peak at 561 nm. While the reason for such different intensities is still unclear, the presence of the emission peak in the visible region could be assigned to the presence of carotenoid pigment. Based on the fluorescence data, it could be suggested that acetone would be the most suitable solvent for the extraction of carotenoid pigment from the red fruit.

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Figure 3. (a) Three-dimensional and (b) two-dimensional fluorescence spectra of red fruit extract using distilled water as the maceration solvent.

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Figure 4. (a) Three-dimensional and (b) two-dimensional fluorescence spectra of red fruit extract using ethanol as the maceration solvent.




Further characterization was carried out by the FTIR spectrophotometer only for the red fruit extract using acetone as the solvent. As shown in Figure 6, the presence of acetone in the red fruit extract could be confirmed from the carbonyl vibration of acetone at 1712 cm-1 [21]. However, red fruit oil was also reported to show such absorption peak in the region of 1700–1800 cm-1, which was assigned to the carotenoid pigment [22]. Additionally, the alkane groups (C-H sp3) of carotenoid pigment were confirmed by the absorption peaks at 2854 and 2924 cm-1. All these peaks showed the characters of the carotenoid pigment, in which the representative structure of carotenoid pigment is also shown as an inset in Figure 6. This study demonstrated that carotenoid pigment was successfully extracted from the red fruit by using acetone as the maceration solvent.

Figure 6. FTIR spectrum of red fruit extract using acetone as the maceration solvent. CONCLUSION Among distilled water, ethanol, and acetone, acetone was found as the best solvent for the extraction of carotenoid pigment through a maceration technique for 24 h. Compared to distilled water and ethanolic extract, acetone extract gave the highest intensity of the absorption peak at 476 nm. The acetone extract also gives the emission peaks at 394 and 561 nm, which corresponded to the presence of carotenoid pigment. Furthermore, the FTIR spectrum revealed the C-H sp3 functional groups of carotenoid pigments as observed at 2924 and 2854 cm-1. These findings demonstrated that the carotenoid

pigments could be extracted from red fruit through a simple maceration technique. Acknowledgement

This work was financially supported by the Directorate General of Strengthening Research and Development, Ministry of Research, Technology and Higher Education of the Republic of Indonesia via the Higher Education Excellent Applied Research Scheme (PTUPT 2019, No. 041/SP2H/LT/MULTI/L7/2019 and No. 005/MACHUNG/LPPM/SP2H-LIT-MULTI/III/2019).

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Figure 5. (a) Three-dimensional and (b) two-dimensional fluorescence spectra of red fruit extract using acetone as the maceration solvent.

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Abstrak Buah merah (Pandanus conoideus Lam) kaya dengan pigmen alami berwarna merah-oranye, seperti β-karoten. Pada penelitian ini, pemilihan pelarut dipelajari untuk mengekstrak pigmen karotenoid dari buah merah melalui teknik maserasi yang sederhana. Tiga jenis pelarut digunakan dalam maserasi, yaitu air suling, etanol, dan aseton. Ekstrak yang diperoleh dikarakterisasi menggunakan spektrofotometer ultraviolet-tampak (UV-tampak), inframerah transformasi Fourier (FTIR), dan spektrofluorometer. Pelarut yang berbeda memberikan informasi spektroskopi yang berbeda, menunjukkan bahwa pemilihan pelarut mempengaruhi tipe senyawa yang terekstrak. Diantara pelarut-pelarut yang digunakan, aseton ditemukan sebagai pelarut yang paling efektif untuk mengekstraksi pigmen karotenoid. Keberadaan pigmen karotenoid pada ekstrak aseton dikonfirmasi dengan munculnya puncak absorpsi pada 476 nm di spektrum UV-tampak, sedangkan dari spektrum FTIR didapatkan gugus fungsi C-H sp3 pada 2924 and 2854 cm-1. Selain itu puncak emisi dari pigmen karotenoid ditemukan pada 394 dan 561 nm. Studi ini mengonfirmasikan bahwa aseton bekerja lebih baik sebagai pelarut pada proses maserasi untuk pigmen karotenoid dibandingkan dengan pelarut air suling dan etanol, dimana hal ini sangat berkaitan dengan sifat non-polar aseton. Kata kunci: aseton, pigmen karotenoid, buah merah, ekstraksi, maserasi

selection of maceration solvent for natural pigment

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