173.devi karmilia a.characterization of bee pollen from ranca bungur, bogor

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CHARACTERIZATION OF BEE POLLEN FROM RANCA BUNGUR, BOGOR

Devi Kamilia A., Sukrasno and Irda Fidrianny

School of Pharmacy, Institut Teknologi Bandung

West Java, Indonesia

Abstract. Bee pollen is an agglomerate of pollen grains from various botanical sources, which are collected by honeybees as their food sources. Bee pollen contains nutrients, which is the reason why it is used by humans as food

supplement. The objective of this study was to characterize bee pollen that was obtained from a bee farm in Ranca Bungur, Bogor. The characterization include major composing pollen, carbohydrate, protein, and lipid contents, chromatographic pattern, and the main secondary metabolite. Total carbohydrates content was determined using orcinol-sulphuric acid assay. Total proteins content was determined using Kjeldahl method. Total lipids content was determined using Soxhlet extraction method with n-hexane as non polar solvent, the extract was determined in gravimetric as crude lipid. Bee pollen was extracted by maceration method with gradual solvent n-hexane, ethyl acetate, and ethanol. N-hexane extract was fractionated by column classic chromatography with n-hexane – ethyl acetate (9 : 1) as the eluent. The pure

isolate was characterized using spectrophotometer UV-visible and spectrophotometer infrared. Macroscopic and microscopic analysis showed the maize pollen as the major component of bee pollen from Ranca Bungur, Bogor. Carbohydrate, protein and lipid contents of the bee pollen were 56.14%, 25.82%, and 5.07% respectively. Total carbohydrate content of bee pollen was much higher than maize pollen, that were 56.14% and 34.7%. Carbohydrate components of bee pollen were fructose, glucose , sucrose and other oligosaccharides, whereas in maize pollen were glucose and fructose. Phytochemical screening showed positive result for flavonoid and steroid/triterpenoid. Based on TLC chromatogram with a specific reagent, UV and IR spectra showed that an isolate obtained from n-hexane extract was phenolic compound with hydroxyl, carbonyl, aliphatic and aromatic groups.

Keywords: bee pollen, maize pollen, honeybee, carbohydrate, protein, lipid

1 Introduction Apitherapy began in mainland China and the Middle East, especially Egypt.

Traditional medicine in China has thousands of years before the age of modern medicine began to rise in Europe. One form of modification of the popular acupuncture in the world today is by using a needle called honey bee stings bee acupuncture (bee sting prick). In Indonesia, bee therapy has been conducted since

Proceedings of the Third International Conference on Mathematics and Natural Sciences

(ICMNS 2010)

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the 1980s. Apitherapy is a therapy that uses bee products like honey, propolis, royal jelly, bee venom, and bee pollen. Apis mellifera is the kind of honey bee that commonly cultivated because of the ability to produce bee products in large numbers.

Bee pollen is an agglomerate of pollen grains from various botanical sources, which are collected by the bees and mixed with nectar and secretion from the hypopharyngeal glands, such as α and β-glycosidase enzymes. Pollen contains nutrients, such as carbohydrates, proteins, amino acids, lipids, vitamins and minerals, in addition to carotenoids, flavonoids and phytosterols, which is the

reason why it is used by humans as an alternative food source and/or food supplement [4].

Bee pollen is used by people as a food supplement and has been reported to have several pharmacological effects. However, there is insufficient knowledge about the relationship between chemical constituents with pharmacological effects are produced. In addition, the chemical content of bee pollen varies and depends on the condition and biodiversity in the area of cultivated honey bees. Therefore, this research aims to study the content of nutrients and secondary metabolites contained in bee pollen, both qualitatively and quantitatively. In addition, from this study are expected to know the nutritional quality of bee pollen in Indonesia through analysis of levels of nutrients such as carbohydrates, proteins, and lipids.

2 Materials and methods

Research methods include preparation of materials, inspection characteristics, determination of nutrient content, and isolation and identification of secondary metabolites.

The materials used are derived from a bee farm in the area Ranca Bungur, Bogor on February 2010. Bee pollen was collected by pollen traps placed at the entrance of beehives. Maize pollen which allegedly is the main source of bee pollen on the bee farm is also collected.

Examination includes examining the characteristics of macroscopic, microscopic, water content, loss on drying, water-soluble extract levels, ethanol-soluble extract levels, and total ash content. Microscopic examination conducted by the color of pellets of the results of macroscopic examination.

Determination of nutritional undertaken include: carbohydrates, protein, and lipid.

Determination of total carbohydrate content was done by colorimetric method using orsinol-sulfuric acid reagent. Determination of total protein content was done by Kjeldahl method. Determination of total lipid content was done by Soxhlet equipment using the solvent n-hexane and the weight of extract was determined in gravimetric as crude lipid.

Characterization Of Bee Pollen From Ranca Bungur, Bogor

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Isolation of secondary metabolites was done by extracting with maceration method using solvents n-hexane, ethyl acetate, and ethanol. The extraction was done on bee pollen and maize pollen. Monitoring extract performed with thin layer chromatography (TLC). Monitoring extracts were made to each extract to see the patterns of chromatograms on bee pollen and maize pollen. N-hexane extract of bee pollen was fractionated with classical column chromatography using eluent n-hexane - ethyl acetate (9 : 1). Monitoring of fractions was done by TLC method. Pure isolates were characterized by using a UV-visible spectrophotometer and infrared spectrophotometer. The identification was done by using specific

reagents for flavonoid, phenolic, terpenoid, and steroid/triterpenoid.

3 Results and discussion

Bee pollen was collected by honey bees from plants, i.e. pollen from the flowers. Therefore, bee pollen can be categorized as plant exudates. Bee pollen was collected in the honey bee farms in the area Ranca Bungur, Bogor, Indonesia in February 2010. The collection of bee pollen was done by installing a pollen trap at the entrance of hives that when worker bees were collecting bee pollen about to enter the hive, bee pollen was carried in baskets corbiculae pollen found on his hind legs will fall. Bee pollen stored in containers that are stored in the bottom of the appliance trap pollen. Bee pollen was collected in the form of pellets that vary in color. Bee pollen dried with a cold and are not exposed to direct sunlight, then stored in the freezer, to keep the bee pollen remain durable.


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Figure 1 Pollen trap placed at the entrance of beehive and bee pollen collected in the form of pellet.


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By macroscopic, bee pollen form was pellets that vary in colors: dark yellow, white, brown, orange, and yellow. Then, the microscopic examination of bee pollen pellets was done based on these colors.

(i) (ii) (iii)

(iv) (v)

Figure 2

Result of macroscopic examination of bee pollen by color of pellets: (i) dark yellow, (ii) white, (iii) orange, (iv) yellow, and (v) brown.


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(i) (ii) (iii)

(iv) (v)

Figure 3

Result of microscopic examination of bee pollen by color of pellets: (i) dark yellow, (ii) white, (iii) orange, (iv) yellow, and (v) brown.

From the image can be seen that honey bees collect pollen from various plants. However, the majority of bee pollen pellets was dark yellow that after identified microscopically containing maize pollen.

Figure 4 Result of microscopic examination of the maize pollen.


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Table 1 Quality Characteristics of Bee Pollen

Characteristics Result (%)

Water content 10.00 Water-soluble extract levels 36.55 Ethanol-soluble extract levels 15.95 Total ash content 1.88 Loss on drying 14.47

Table 2 Nutritional Contents of Bee Pollen

Nutritional content Bee pollen (%) Maize pollen (%)

Carbohydrate 56.14 ± 0.07 34.70 ± 0.09

Crude protein 25.82 ± 0.34 -

Lipid 5.07 ± 0.12 -

1 2 3 4 5 6 7 8 9

Figure 5 Paper chromatogram of water extract of bee pollen and water extract of maize pollen, mobile phase n-butanol - pyridine - water (10 : 3 : 3), diphenylamine - aniline - phosphoric acid spray reagent, (1) arabinose, (2) fructose, ( 3) galactose, (4) glucose, (5) maltose, (6) rhamnose, (7) sucrose, (8) water extract of maize pollen, (9) water extract of bee pollen

From the results of determination of total carbohydrate content showed that bee pollen contains more carbohydrate than the pollen of maize. This showed the addition of carbohydrates in bee pollen. Carbohydrates were likely derived from the


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nectar which is also carried by honey bees. Next step was the identification of the sugar compounds found in bee pollen and maize pollen by paper chromatography using the comparison compounds are sugar derivatives of arabinose, fructose, galactose, glucose, rhamnose, maltose, and sucrose. Mobile phase used was n-butanol - pyridine - water (10 : 3 : 3) and reagent used were diphenylamine - aniline - phosphoric acid. Extracts tested were water extract of bee pollen and water extract of maize pollen.

From the results of paper chromatography can be seen that the extract of bee pollen contains more sugar. Also, it can be seen that the carbohydrate component

in bee pollen include fructose, glucose, sucrose and other oligosaccharides, whereas in maize pollen contained only glucose and fructose, with fructose as the main component.

Isolation of secondary metabolites involves several stages, at the beginning of phytochemical screening experiments performed to determine the class of compounds contained in bee pollen. The phytochemical screening showed that bee pollen contains flavonoids and steroid/triterpenoid.

Table 3 Phytochemical Screening of Bee Pollen

Golongan senyawa Hasil

Flavonoid + - - - - +

Tannin Alkaloid Quinone Saponin Steroid/Triterpenoid

Bee pollen and maize pollen was extracted by maceration method gradually in 3 solvent with increasing polarity. After extraction, monitoring of the extract performed by thin layer chromatography (TLC) with the stationary phase of silica

gel GF254. Mobile phase used for each extract n-hexane, ethyl acetate, and ethanol in succession was toluene - ethyl acetate (9 : 1), ethyl acetate - acetone (9 : 1), and ethyl acetate - acetone - acetic acid glacial (7 : 2 : 1). Spray reagent used was UV λ 254 nm, UV λ 366 nm, and 10% H2SO4 in methanol as a universal reagent.

In this monitoring extracts carried out a comparison between the chromatogram patterns of bee pollen extract and extracts of maize pollen. From the monitoring results obtained that there is no significant difference between the content of compounds in bee pollen and maize pollen. In n-hexane extract contained some brownish spots which would then be isolated and characterized, and was expected to be a marker compound bee pollen.


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Figure 6 Thin layer chromatogram of n-hexane, ethyl acetate, and ethanol extracts at stationary phase silica gel GF254, (1) n-hexane extract

with mobile phase toluene - ethyl acetate (9 : 1), (2) ethyl acetate extract with mobile phase ethyl acetate - acetone (9 : 1), and (3) ethanol extract with mobile phase ethyl acetate - acetone - glacial acetic acid (7 : 2 : 1), (i) UV λ 254 nm, (ii) UV λ 366 nm, and (iii) 10% H2SO4 in methanol visual, (a) bee pollen, (b) maize pollen.

From the results of monitoring extract of ethanol can be seen there is a difference between the compound extract of bee pollen and maize pollen. In the extract of bee pollen contained blue spots under UV light at λ 366 nm Rf 0.7, while the extract of maize pollen is not there. After addition of 10% H2SO4 reagent in methanol contained brownish spotting at Rf 0 to 0.4 which allegedly is sugar derived compounds.

In this research study focused on secondary metabolites spots with Rf 0.8 of n-hexane extract. Furthermore, n-hexane extract was fractionated by classical column chromatography using silica gel 60 as stationary phase and eluent n-hexane - ethyl acetate (9 : 1). Fractions were collected by volume, 10 mL and obtained 33 fractions. Then the monitoring fractions of bee pollen was done to the fraction numbers 5, 10, 15, 20, 25, and 30 that conducted in TLC with the stationary phase silica gel GF254. Mobile phase used was n-hexane - ethyl acetate (9 : 1). From the monitoring results obtained fractions isolated compounds will be present in early fractions, namely fraction 5. Furthermore, the monitoring was done back in fractions of fractions 1, 2, 3, 4, 5, and 6 with the same chromatographic system.


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Figure 7 Thin layer chromatogram monitoring of the fractions with stationary phase silica gel GF254, and mobile phase n-hexane - ethyl acetate (9 : 1), with 10% H2SO4 reagent in methanol under UV λ 366 nm, (a) bee pollen.

From the monitoring results to 1-6 fraction was obtained that the isolated compound will be contained in fraction 3. Furthermore, the purity test was done to

determine the purity of the compound. The purity of two-dimensional TLC performed with the stationary phase of silica gel GF254. Mobile phase used was n-hexane - ethyl acetate (9 : 1) and toluene - ethyl acetate (7 : 3).

Figure 8 Two-dimensional thin-layer chromatogram fraction 3, with stationary phase silica gel GF254, mobile phase n-hexane - ethyl

acetate (9 : 1) and toluene - ethyl acetate (7 : 3), with 10% H2SO4 reagent in methanol under light UV λ 366 nm.


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From the purity test results can be seen that the fractionation was done purely so that further characterization. From UV-visible light spectrum can be seen that there are compounds give maximum absorbance at a wavelength of 235 nm and 321 nm.

Figure 9 The spectrum of UV-visible spectrophotometry isolates in the n-hexane solvent.

From the infrared spectrum can be seen that there are compounds present in the hydroxy group at wave numbers 3363 cm-1 which shows the OH stretch. At wave numbers 2923 and 2857 cm-1 show CH stretch, which means there is an aliphatic

group. At wave numbers 1735 cm-1 shows a stretching C = O, which means there are carbonyl groups. At wave numbers 1643 cm-1 shows a stretching C = C, which may indicate the existence of an aliphatic or aromatic group. At wave numbers 1461 and 1376 cm-1 shows the CH bending and the wave numbers of 721 cm-1 shows the bending C = C, which also can indicate the existence of aromatic group.


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Figure 10 The spectrum of infrared spectrophotometry isolates in KBr pellets.

In addition, the isolate was characterized by a variety of specific reagents such as Liebermann-Burchard for steroid/triterpenoid, anisaldehid for terpenoid, sitroborat and AlCl3 for flavonoid, and FeCl3 for phenol. The isolate gave a positive reaction with FeCl3 reagent. From the results of this characterization can be concluded that the isolate was a phenolic compounds.

4 Conclusion

Bee pollen was used as the test material in this research the main source of pollen from maize plants. Bee pollen contains several nutrients i.e. carbohydrate, protein and lipid in the number row is 56.14 ± 0.07%, 25.82 ± 0.34%, and 5.07 ± 0.12%. Total carbohydrate content in the bee pollen is higher than the levels of

carbohydrate in maize pollen. Carbohydrate component contained in bee pollen include fructose, glucose, sucrose and other oligosaccharides, whereas in maize pollen contained only glucose and fructose. The phytochemical screening showed that bee pollen contains flavonoid and steroid/triterpenoid compounds. Isolates obtained from n-hexane extract of bee pollen was thought to phenolic compounds that contain hydroxy groups, carbonyl, aromatic and aliphatic, possibly a conjugate of hydroxy cinnamate.

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Material, World Health Organization, Geneva, 10, 28. Details of author(s)

DEVI KAMILIA A School of Pharmacy, Institut Teknologi Bandung West Java, Indonesia e-mail: [email protected] SUKRASNO School of Pharmacy, Institut Teknologi Bandung West Java, Indonesia

IRDA FIDRIANNY School of Pharmacy, Institut Teknologi Bandung West Java, Indonesia

mailto:[email protected]

173.devi karmilia a.characterization of bee pollen from ranca bungur, bogor

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