Title Science and Technology Indonesia e-ISSN:2580-4391 p-ISSN:2580-4405 Vol. 8, No. 1, January 2023 Research Paper Photoprotective and Antioxidant Potential of Indonesia’s Klanceng Honey Beehive Waste Yuliana Purwaningsih1*, Ahmad Fuad Masduqi2, Erwin Indriyanti1, Mighfar Syukur1 1Bachelor Program of Pharmacy, Semarang Pharmaceutical College, Semarang, 50192, Indonesia2Vocational Program of Pharmacy, Semarang Pharmaceutical College, Semarang, 50192, Indonesia *Corresponding author: y14purwaningsih@gmail.com AbstractA byproduct of the honey processing called Klanceng honey beehive waste is said to still contain secondary metabolites that arebeneficial to health. The objective of this research was to assess the photoprotective properties of an ethanol extract, n-hexanefraction, and ethyl acetate fraction obtained from the waste of an Indonesian Klanceng honey beehive that originated in Magelang.The DPPH free radical technique was applied to quantify antioxidant properties, and the Mansur equation’s SPF value was used tocalculate photoprotective activity. The analysis of the data revealed that the IC50 values for the ethanol extract, n-hexane fraction,and ethyl acetate fraction were, respectively, 470.2935±0.9249, 207.1869± 2.6510, and 216.4892±0.8349. The ethanol extract,n-hexane fraction, and ethyl acetate fraction of the samples had SPF values of 3.872, 3.529, and 9.358, respectively. The ethylacetate fraction, as opposed to the ethanol extract and the n-hexane fraction, has greater potential as a photoprotective agent as aresult. KeywordsAntioxidant, Extract, Fraction, Honey Beehive, Photoprotective Received: 9 Oktober 2022, Accepted: 28 December 2022 https://doi.org/10.26554/sti.2023.8.1.137-143 1. INTRODUCTION The human skin serves as a protective border between the in- ternal and exterior environments, guarding against radiation, toxic substances, mechanical trauma, and microbial invasion. It has been abundantly obvious in recent years that the skin plays a significant role in the immune system. Environmental factors like ultraviolet (UV) radiation, oxidative sterss, haz- ardous and allergic substances, and mechanical damage, as well as intrinsic factors like genetic propensity, immunological and hormonal prestige, and stress, have an impact on the condition and function of the skin. The consequent abnormalities in the skin result in photoaging, inflammation, decreased immune responses, an imbalance of epidermal homeostasis, and other skin problems (Fernandez Garcia, 2014) . Photo-oxidative reactions caused by UV radiation expo- sure cause biomolecular damage, affecting the authenticity of skin cells and causing skin problems. Damage to these photo- oxidative contributes to the pathological process and is respon- sible for the oncet of many skin disorders. Continuous sun exposure and other environmental factors can result in the induction of oxidative stress, which has a high reactivity with genetic material, peptides, and fatty acids and frequently causes antioxidant significant damage (Vijayakumar et al., 2020) . Pho- toprotective agents shield the skin from the damage conse- quences of ultraviolet natural daylight (Latha et al., 2013) . Plant-derived phytochemical compounds have the potential to prevent molecular damage by capturing and attempting to destroy ROS raised by biological mechanisms, smog, inhaling, and medicines. Polyphenols are the most important natural product class in dermatology due to their absorption spectrum, which efficiently filters UV radiation, reducing the possibility of radiation penetrating deep into the skin layer (Vijayakumar et al., 2020) . Bee products are regarded as a source of natural antioxi- dant potential proficient in combating the effects of oxidative stress, which underlies the pathogenesis of several diseases (Kocot et al., 2018) . Beehive also contains various phenolic and flavonoid compounds that can be used as antioxidants (Pérez-Pérez et al., 2013) . Beehives provide protection for bee colonies from microbial species, mushrooms, infectious agents, and carnivores, as well as a location for honey pro- duction, bee pollen, and bee growth and development. The condition of beehives has a significant bearing on the quality of honey (Pérez-Pérez et al., 2013) . The content of compounds in https://crossmark.crossref.org/dialog/?doi=10.26554/sti.2023.8.1.137-143&domain=pdf https://doi.org/10.26554/sti.2023.8.1.137-143 Purwaningsih et. al. Science and Technology Indonesia, 8 (2023) 137-143 honey beehives serves as a protector and determinant of honey quality, including flavonoids, which are natural phenol com- pounds, and beeswax. The Trigona bee group is a non-rigid bee group that lives socially in a colony that usually tries to live in trunks, wood, bamboo, and soil. One bee colony has only one queen, hundreds of male bees, and thousands of worker bees; one species of Trigona sp. is T. Itama, which produces more raw propolis (Hrncir et al., 2016) . The content of propolis in the beehive has many benefits, such as drugs and cosmetics (Wagh, 2013) . Honey is a sticky substance made by stingless bees. Honey is well-known throughout the world for its excellent nutritional constituents that are advantageous to human health, and propolis is commonly referred to as "bee glue," which is a collective term for the resinous substance gathered by bees from diverse types of plants (Puspawati et al., 2019) . In Muntilan District, Magelang, Indonesia, many Klanceng honey bees, or Trigona sp., are cultivated. According to local honey breeders, taking Klanceng honey is extorting honey from the hive, which gives honey and Waste. It is estimated that honey beehive waste still contains phytochemicals that can be used to improve one’s health. The content of compounds in beehives functions as pro- tectors and determinants of honey quality, including phenolic compounds and flavonoids. Honey has lower antioxidant ac- tivity than propolis, which is due to the variety of phenolic compounds present in the extract (Mouhoubi Tafinine et al., 2016) . Notably, regardless of its composition, propolis ex- tract always contains protective effects. In both animal and cell culture studies, aqueous extracts of propolis have been shown to possess antioxidant capacity (Kocot et al., 2018) . Sev- eral studies have stated that propolis contains bioflavonoids (Sabir, 2005) . The phytochemical content of Lisotrigona ca- ciae propolis extract shows the presence of several compounds of flavonoids, xanthones, alkyl resorcinol, triterpenes, other phenol compounds, fatty acids, esters, and sugar (Georgieva et al., 2019) . Phenolic compounds and flavonoids have antiox- idant activity Sukweenadhi et al. (2020), antibacterial activity Yuan et al. (2021) , and anti-inflammatory activity Candiracci et al. (2012) , so it is thought that waste from a honey beehive in Muntilan, Magelang, may have these properties. Based on this description, the purpose of the research is to assess the photoprotective and antioxidant potential of an ethanol extract, n-hexane fraction, and ethyl acetate from honey beehive waste (Trigona sp.) that originated in Magelang, Indonesia. 2. EXPERIMENTAL SECTION 2.1 Materials The substances used in this study were 96% ethanol (Brat- aco), ethanol p.a. (Merck), Shinoda Reagent (Merck), FeCl3 (Merck), n-hexane (Brataco), ethyl acetate (Merck), HCl 2N, Mayer Reagent (Merck), Bourchard Reagent (Merck), Dra- gendroff Reagent (Merck), Na2CO3 (Merck), Folin-Ciocalteu (Merck), gallic acid (Sigma), Quercetin (Sigma), DPPH (Sigma), Ascorbic Acid (Merck), Anhydride acetate (Merck), Na acetate (Merck). The tools used in this study were a set of glass devices commonly used in laboratories, bransonic brand ultrasonic batch, UV-Vis Shimadzhu 1840 spectrophotometer, a vacuum evaporator, IR-ATR Agilent 630 spectrophotometer. 2.2 Methods 2.2.1 Sample The sample used was the waste of honey beehive Trigona sp. from Muntilan District, Magelang Regency, Central Java Province, Indonesia. Samples were sorted to separate rotten materials and impurities. The sample was reduced in size using a knife. 2.2.2 Extraction and Fractination A total of 100 g of samples were macerated in the sonicator for 1 hour with 96% ethanol and allowed to stand for 24 hours with a sample and solvent ratio of 1:10. The extract was fil- tered and separated into filtrates and residues. The residue was remacerated in the same manner. Maceration was performed three times. In a vacuum evaporator set to 50°C, the filtrate was evaporated until a thick extract was obtained. Ten grams of thick extract were fractinated in hexane, ethyl acetate, and ethanol. Using an evaporator, the fractionation results were evaporated to obtain a concentrated n-hexane fraction, an ethyl acetate fraction, and an ethanol extract. 2.2.3 Phytochemical Screening The chemical content of the sample was confirmed using color reagents. Flavonoid, tannin, saponin, and terpenoid tests were all performed. A shinoda reaction was used for the flavonoid test, which included extract and filtate plus HCl 2 N, mag- nesium powder, and amyl alcohol. If the amyl alcohol layer is red, the flavonoid sample is positive (Nurhasnawati et al., 2019) . The tannin test was performed with a FeCl3 solution that yielded a positive blackish-green color. The alkaloid test was performed using three methods: the dragendroff reagent, the Mayer, and the Bouchard tests (Nurhasnawati et al., 2019) . The saponin test was performed by first adding distilled water to the sample and managing it, then adding HCl 2 N and shak- ing it to determine the presence of stable foam-positive saponin (Nurhasnawati et al., 2019) . The terpenoid test was performed by dissolving a number of samples in ether, which was then evaporated and anhydride acetic acid added. The existence of terpenoids was indicated by the presence of a red or green color in this test (Nurhasnawati et al., 2019) . An ATR-FTIR spec- trophotometer was used to identify the functional groups of ethanol extract, n-hexane fractions, and ethyl acetate fractions. The sample was placed on the prism, and the % transmittance on the wave number 4000-400 cm−1 was measured (Liu et al., 2006) . 2.2.4 Total Phenolic Content (TPC) Using Visible Spec- trophotometry This method based on Mathur and Vijayvergia (2017) with little modification. The TPC test was performed by pipetting a 1 mL sample (500 – 900 mg/L) solution and 4 mL of Folin- Ciocalteu (F-C) reagent. This mixture was kept for 4 minutes © 2023 The Authors. Page 138 of 143 Purwaningsih et. al. Science and Technology Indonesia, 8 (2023) 137-143 before adding 0.4 mL of 7% Na2CO3 solution and incubating for 120 minutes with distilled water up to 10 ml.. The mix- ture is measured at its maximum wavelength of 775 nm. The standard solution used was gallic acid with a concentration of 60–100 mg/L in the 10 range. 2.2.5 Total Flavonoid Content (TFC) Using a Visible Spec- trophotometry This test was carried out by the colorimetric method using AlCl3 based on Tristantini and Amalia (2019) with modifica- tion. As many as 1 mL of samples plus 0.1 mL of 10% AlCl3 solution and 0.1 mL of 1 M sodium acetate solution was ad- hered with aquadest to a volume of up to 10 mL and incubated for 30 minutes. The mixture is measured at a maximum wave- length of 436.5 nm. 2.2.6 AntioxidantActivityTestUsingDPPH(2,2-Diphenyl- 1-Picrylhydrazyl) With minor modifications, this antioxidant test method is based on Sukweenadhi et al. (2020) . Allowing 3 mL of DPPH solu- tion 0.4 mM plus 1 mL of samples to stand for 30 minutes. The wavelength of the solution was 517 nm. Ascorbic acid was used as a positive control, and DPPH in ethanol was used as a blank. Using equation 1, the absorbance obtained from the instrument was used to calculate the percentage inhibition(Syarifah et al., 2021) . %inhibition = Ablank-Asample Ablank (1) 2.2.7 SPF Test Using UV Spectrophotometry The SPF value is calculated using a modified method based on Sabir (2005) . UV spectrophotometry is used to measure absorbance at 290-320 nm in the 5 nm range for samples with concentrations ranging from 100 to 500 ppm (in ethanol sol- vents). The absorbance is calculated using the Mansur equation (2) to yield the SPF value. SPF = CF × 320∑︁ 290 EE(_) × I × A(_) (2) Where, CF = correction factor (10), EE (_ ) = erythmogenic effect of radiation with wavelength _ , Abs (_ ) = spectrophoto- metric absorbance values at wavelength _ . Table 1 shows the values of the EE (_ ) x I constants (Khan, 2018) . 3. RESULT AND DISCUSSION Remaceration is used to extract Klanceng honey beehive waste for 1 hour at room temperature, aided by ultrasonic waves. In secondary metabolite extraction, the sonification method is an ecofriendly extraction method that is used to remedy the flaws of the maceration method. Sonication methods can be com- pleted in much less than an hour, and the impact of damage throughout the phase is negligible or can be lessened (Puspawati et al., 2019) . Ultrasonic radiation with a frequency greater than 20 kHz makes it easier to extract compounds both natural in- gredient and inorganic from porous support utilizing liquid solvents. The sound wave of sonicator creates cavity bubbles close the sample tissue, which disintegrate cell walls and release cell contents, including secondary metabolites (Khoddami et al., 2013) . The use of an ultrasonic wave to aid extraction rises compound solubility in the solvent, resulting in a reduction in the volume of solvent required (Alara et al., 2021) . The mac- eration results are evaporated in a vacuum rotary evaporator to produce a concentrated brown color extract with a honey aroma, yielding a 36.09 ± 1.69% yield. The use of a vacuum evaporator can lower the temperature of the solvent because the pressure in the flasks decreases, making the solvent more volatile. Concentrated extracts are fractionated using n-hexane sol- vents to obtain nonpolar fractions; the residues are then frac- tionated with ethyl acetate solvents to obtain semipolar frac- tions. Water fractions are ethyl acetate fractionation residues. Each fraction is evaporator to obtain a thick fraction of each fraction of the n-hexane fraction; the ethyl acetate fraction and the water fraction are 49.52 ±8.58%, 37.54 ±3.40%, and 2.15 ± 0.26%, respectively. The secondary metabolite content of the sample was ascer- tained using phytochemical screening (Gurning et al., 2021) . Phytochemical analysis reveals that alkaloid compounds, flav- onoids, tannins, and terpenoids are present in ethanol ex- tract, n-hexane fractions, and ethyl acetate fractions (Table 2). The FTIR spectrophotometer results of the ethanol ex- tract, n-hexane fraction, and the ethyl acetate fraction show the same spectrum pattern (Figure 1). The three samples have the same spectrum pattern based on the IR spectrum results. The n-hexane fraction exhibits a lower -OH absorption at wave number 3300 cm−1 than the ethanol extract and ethyl acetate fraction. The presence of -OH functional groups in a sample identifies the existence of phenol or alcohol groups (Revathi and Rigley, 2019) . The aliphatic CH- alkyl group in the sam- ple was demonstrated by wave number 2800-2900 cm−1. The availability of -C=C- aromatic groups was indicated by the wave number 1700 cm−1, while the presence of carbonyl groups C=O was noted by the wave number 1350-1500 cm−1. The absorption of the C-OH group from alcohol was implied by the wave number at 1025 cm−1 (Stuart, 2004) . According to this analysis, the samples contained polyphenolic compounds. Plants produce polyphenols in reaction to external and physiological pressures including pathogenic and insect attacks, UV exposure, and wounds (Khoddami et al., 2013) . The ba- sic structure of polyphenols is the benzene ring with hydroxy groups. The phenolic level of the sample is determined by measuring the absorbance at a wavelength of 775 nm using the Folin-Ciocalteu method. The standard used is 60-100 ppm galic acid with a 10 ppm range. The galic acid standard linear regression equation is y = 0.0082x - 0.231, R2 = 0.9970. The total amount of phenolic is given in milligrams of galic acid © 2023 The Authors. Page 139 of 143 Purwaningsih et. al. Science and Technology Indonesia, 8 (2023) 137-143 Table 1. The Result of Phytochemical Screening compound group Etanol extract Ethyl Acetate Fraction n-hexane Fraction Alkaloids + + + Flavonoids + + + Saponin - - - Tannin + + + terpenoid + + + Figure 1. ATR-FTIR Spectrum of Sample Figure 2. Total Phenolic Content of Sample per 100 g of sample. Galic acid is used as a standard because its availability and stability (Wabaidur et al., 2020) According to Figure 2 and Table 3, the total phenolic con- tent of the ethanol extract, as well as the n-hexane and ethyl acetate fractions, increases with increasing sample concentra- tion. This image shows that the ethyl acetate fraction has a higher total phenolic level than the ethanol extract and the n-hexane fraction. The total flavonoid content (Figure 3) was defined utilising visible spectrophotometry and AlCl3 as a reagent. The con- struction of a complex among AlCl3 compounds and quercetin compounds is the essence of calculating amount of flavonoid using colorimetry method (Meilawati et al., 2021) . The total Figure 3. Total Flavonoids Contents of Sample quantity of flavonoid was described in milligram equivalent of quercetin/100 g sample. Measurement of quercetin resulted in a linear regression equation y=0.0072x+0.0038, r=0.9998. The antioxidant activity of the ethanol extract, the n-hexane fraction, and the ethyl acetate fraction was tested using the DPPH radical. This method was chosen because DPPH is a stable free radical so it is easy to do. This test was executed by visible spectrophotometry at a wavelength of 517 nm with an operating time of 30 minutes (Gurning et al., 2021; Sembiring et al., 2018). The decrease in DPPH concentration was pro- portional to the increase in antioxidant concentration. IC50 is the effective concentration that induces a 50% reduction in the previous DPPH concentration (Molole et al., 2022) . The IC50 value for the ascorbic acid standard used in this study was 8.875 ppm, indicating very strong antioxidant activity. Ascorbic acid easily and frequently catches superoxide anion species such as superoxide and hydroperoxyl radicals, oxidants, ozone, peroxynitrite, nitrogen dioxide, nitrous oxide radicals, and hypochlorous acid, thereby preventing oxidative stress to other substrates (Birangane et al., 2011) . The sunscreen activity of the sample was carried out by UV spectrophotometry. This measurement is made at a wavelength of 290–320 nm. The SPF value is estimated based on the Mansur equation. Table 3 and Figure 5 illustrate that the greater the sample concentration, the higher the SPF value. The SPF value of the ethyl acetate fraction is greater than that of the extract and greater than that of the n-hexane fraction. The © 2023 The Authors. Page 140 of 143 Purwaningsih et. al. Science and Technology Indonesia, 8 (2023) 137-143 Table 2. Total Phenolic Content (TFC) of Sample Concentration (mg/L) Ethanol Extract Ethyl Acetate Fraction n-hexane Fraction 500 0.6227±0.0004 0.7685±0.0014 0.5683±0.0000 600 0.6641±0.0006 0.8307±0.0004 0.5723±0.0022 700 0.7640±0.0006 0.9803±0.0005 0.6466±0.0013 800 0.8788±0.0012 1.0877±0.0007 0.8109±0.0007 900 0.9042±0.0007 1.2389±0.0015 0.8304±0.0006 Table 3. SPF Value of Sample Concentration of Extract Protection n-hexane Fraction Protection Ethyl Acetate Fraction Protection Sample (ppm) SPF Value Category SPF value Category SPF Value Category 100 1.424 Low 1.991 Low 5.010 Low 200 1.870 Low 2.466 Low 6.535 Low 300 2.700 Low 3.389 Low 6.898 Low 400 3.872 Low 3.529 Low 9.358 Low 500 5.832 Low 4.464 Low 11.898 Low Figure 4. Antioxidants Test of the Sample Using DPPH concentration of 500 ppm of the extract, n-hexane fraction, and ethyl acetate fraction was able to provide low protection against UV B rays, but in this concentration, ethyl acetate had a higher SPF value than the extract and n-hexane fraction. According to Geoffrey et al. (2019), protection from UV rays is graded based on the SPF value: low (SPF 2-15), medium (SPF 15-30), high (SPF 30-50), and highest (SPF>50). Honey and propolis contain active compounds involve polyphenol, flavonoids, carotenoids, and avitamin C (Mouhoubi Tafinine et al., 2016) . 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Page 143 of 143 INTRODUCTION EXPERIMENTAL SECTION Materials Methods Sample Extraction and Fractination Phytochemical Screening Total Phenolic Content (TPC) Using Visible Spectrophotometry Total Flavonoid Content (TFC) Using a Visible Spectrophotometry Antioxidant Activity Test Using DPPH (2,2- Diphenyl-1-Picrylhydrazyl) SPF Test Using UV Spectrophotometry RESULT AND DISCUSSION CONCLUSION ACKNOWLEDGMENT