Characteristics of analog coffee from the mangrove fruit Rhizophora mucronata origin Sangihe Islands

Coffee is a popular drink today. One of the coastal resources that can be processed into coffee-like products (analog coffee) is the mangrove fruits Rhizophora mucronata. The fruits of R. mucronata were reported to contain antioxidant substances that can support human health. This study aimed to characterize analog coffee products from the fruit of R. mucronata. The fruits were collected from Miulu village, Sangihe Islands, North Sulawesi. Mangrove fruits were soaked in a 2% lime solution for 72 hours and dried at 60 °C. Then it was roasted at 116-118 °C for 40 minutes, and the sample was ground until it resembled coffee grounds. The findings revealed that the water content of analog coffee was 4.14 ± 0.14%; ash 4.97 ± 0.02%; protein 14.38 ± 0.02%; fat 12.26 ± 0.08%; carbohydrate 63.67 ± 0.09%, energy 422.58 ± 1.01 kcal, caffeine 1.20 ± 0.00%, tannins 808.21 ± 2.02 mg/kg, and antioxidant capacity IC50 131.28 ± 0.44 μg/mL. Furthermore, the analysis of compounds using GC-MS from the coffee analog of R. mucronata fruits was dominated by cis-9-Hexadecenal (50.95%) and 9-Octadecenal, (Z)-(33.97%). These research findings provide that analog coffee from the R. mucronata fruits has the potential to become a coffee-like product as a functional drink.


Introduction
Mangroves are one of the potential coastal resources as a source of natural antioxidants [1].Mangroves are reported to provide many human benefits, ranging from ecosystem services to food and medicine [2].Mangroves can be used as coastal protection from abrasion, stabilize sedimentation, and as a filter for coastal waters [3,4].In addition, mangroves can also be used as a source of bioactives to produce pharmaceutical raw materials [5] and functional food [6].One of the mangroves reported as a functional food is Rhizophora mucronata [7,8].
Mangrove vegetation is widely distributed in the Sangihe Islands, North Sulawesi.In 2014, the mangrove area reported around 664 ha, specifically in Tabukan Tengah District was around 159 ha [15].Rhizophora mucronata is reported to be the dominant mangrove species in the Sangihe Islands [16,17].However, product diversification from R. mucronata fruits has not been reported.
Therefore, this study evaluates the potential of mangrove fruits from Sangihe Islands into a coffeelike powder product called analog coffee.Coffee products were chosen because coffee is one of the most popular beverage products today in Indonesia [18].Analog coffee from R. mucronata fruits was characterized by evaluating proximate, total tannin, caffeine level, and antioxidant capacity (IC50).In addition, the compounds contained in analog coffee were also identified by GC-MS.

Analog coffee procedure
The analog coffee of mangrove fruits was prepared following the instructions of Sukma and Spanton [19], which was modified using the whiting solution to soak the mangrove fruits.Mangrove fruits is separated between seeds and flesh.Then, soaked the mangrove fruits in clean water for 36 hours (changed the water every 6 hours).Afterward, the mangrove fruits were soaked in a 2% whiting solution (Ca(OH2) in 1000 mL water at room temperature for 72 hours.Then, the mangrove fruits were reduced to 2-3 cm and dried at 50-60 °C for 36 hours.The dried mangrove fruits were roasted for 40 minutes at 116-118 °C.The samples were mashed to a mesh size of 20-30.The appearance of analog coffee from R. mucronata fruits can be seen in Figure 1.

Proximate analysis
The analog coffee of mangrove fruits was analyzed proximately following the instructions of the Indonesian National Standard

Caffeine analysis
The caffeine content in analog coffee of mangrove fruits was carried out by isolation method with chloroform [23].Analog coffee samples of as much as 20 g and 90 mL of distilled water were refluxed for 30 minutes, then filtered by vacuum.After that, the analog coffee residue was refluxed and filtered.The filtrate obtained was then combined and 12.5 mL of Pb(CH3-COO)2 solution, boiled for 5 minutes, and filtered using a Buchner funnel with a layer of silica gel.The filtrate was extracted four times with 40 mL chloroform (MERCK).The chloroform phase was washed with KOH solution and distilled water.After that, evaporation was carried out to remove the chloroform solution in the filtrate.The caffeine extract was weighed and expressed in percent (%).

Gas Chromatography and Mass Spectrometry (GC-MS) analysis
Compounds in mangrove analog coffee powder were quantified using a GCMS (HP 6890) [24].GC-MS with an Agilent 19091S-433 HP-5MS column 30 m long and 250 µm in diameter.Helium was used as the carrier gas, with a flow rate of 1 mL/min and an adjustment temperature of 325°C.The starting temperature was 150 °C, with a rate of 1 °C/min.This process starts at 10 °C/min and then increases to 240 °C for 2 minutes.The total running time is 26 minutes.The scanning range is 50-550 amu.The structural determination was based on a fragmentation pattern analysis of the mass spectrum and a direct comparison of the mass spectrum with databases NIST (02.L and 17.L) and Wiley (275.L).

Tannins analysis
The tannin content of mangrove fruit analog coffee powder was determined using tannic acid equivalence [23].An analog coffee powder sample (0.5 g) was extracted for 20 hours at room temperature with 300 mL diethyl ether.The residue was boiled for 2 hours in 100 mL of distilled water, cooled, and filtered.In a volumetric flask, the extract was adjusted to a volume of 100 mL.The tannin content of the extract was determined colorimetrically using the Folin-Denis reagent and measured using a UV-vis spectrophotometer (Shimadzu) at an absorbance of 760 nm, with the tannic acid solution serving as the standard solution.The tannin content was given in mg/kg.

Antioxidant capacity analysis
The DPPH method was used to assess the antioxidant capacity of mangrove fruit analog coffee [5,27].After dissolving the analog coffee sample in ethanol (200 g/mL), a series of extract concentrations 20 to 100 µg/mL were made.A total of 2 mL of each concentration was added 50 µM DPPH, then stored for 30 minutes at room temperature (25-27 °C) in a dark room.The UV-VIS spectrophotometer (Shimadzu) was then used to measure it at 517 nm.The 50% inhibition concentration (IC50) was calculated using the regression equation between the sample concentration and the antioxidant capacity percentage value.The IC50 value was given in µg/mL.The antioxidant capacity is calculated using the following formula:

Results and discussion
The Rhizophora mucronata fruits dried, roasting and mashing are then characterized to determine analog coffee products' proximate and caffeine content.The analysis of proximate and caffeine content results compared with SNI Coffee Powder No. 01-3542-2004 [28].Table 1 shows the results of the proximate and caffeine content tests.
Table 1 shows the water content of R. mucronata analog coffee products that meet the Indonesian National Standard for ground coffee.The moisture content of dry food is important to evaluate, to prevent the growth of pathogenic microbes such as fungi and molds so that the product is more resistant during storage [29].Previous research has found that the water content of Rhizophora sp.ranges between 1.12 and 2.01%.The analog coffee prototype shows the ash content meet the SNI standard on ground coffee.However, ash content indicates the presence of minerals in a food ingredient [30].Ash content of Rhizophora sp. was reported to be between 1.13 -4.2% [14].Previous studies also reported that R. mucronata fruits contain minerals Na, K, Ca, Fe, Mn, Cu, and Zn [31].In addition, R. mucronata analog coffee also contains protein and fat because fresh Rhizophora fruits are also reported to have protein and fat in minor amounts [32].
Table 1 also shows the highest composition of R. mucronata fruits analog coffee, namely carbohydrates, thus providing a relatively high amount of energy in analog coffee products [33].The dominant carbohydrate content of mangrove fruits is included in non-reducing sugar [31,34], so it will give the impression of a sweet taste in analog coffee products.Caffeine is one of the parameters evaluated in a ground coffee product.Caffeine levels in analog coffee products are still within the Indonesia National Standard for powder coffee.Caffeine is a substance found in some plants' leaves and fruit [35].Caffeine is a substance that stimulates increased activity in the human brain and nervous system [36,37].Caffeine also increases the circulation of chemicals such as cortisol and adrenaline in the body [38].
The profile of compounds contained in analog coffee from R. mucronata fruits was identified by GC-MS.The profile of compounds contained in analog coffee of R. mucronata fruits is presented in Table 2.
Oleic acid is also reported as a potent antioxidant [52] and promotes adaptability to oxidative stress [53].
Furthermore, for each compound detected by GC-MS, computational predictions of absorption, distribution, metabolism, and excretion-toxicity (ADMET) characteristics were calculated.The ADMET analysis was used to predict the safety and effectiveness of analog coffee products when consumed by humans [59,60,61].If ADMET's predictive value is close to one, it is said to be good.Table 3 shows the ADMET profile of the analog coffee compound of the mangrove fruits R. mucronata.
The six compounds identified by GC-MS are predicted to be well absorbed by the human intestine, as shown in Table 3.The human intestine's absorption property refers to the process by which the compound is predicted to be absorbed by the digestive system into the bloodstream of the human body [62].Caco-2 permeability predicts human intestinal permeability and is used to study drug efflux [63].P-glycoprotein is involved in the transport of compounds in many organs.According to Table 3, the six compounds are predicted to be negative as P-glycoprotein substrates and inhibitors.According to this prediction, the six compounds have low bioavailability and must be dissolved first if taken orally [64,65].
Table 3 also shows that the six compounds can diffuse well in maintaining normal central nervous system hemostasis.The Blood-Brain Barrier (BBB) is a semipermeable property of endothelial cells that restricts and prevents solutes in the circulating blood from crossing non-selectively into the extracellular fluid of the central nervous system, where neurons are located [66].Table 3 shows the six compounds predicted to be localized in the plasma membrane and mitochondria.CYP450 (Cytochrome P450) plays a central role in cellular metabolism through enzymes in the liver and maintaining cellular homeostasis [67].Table 3 shows the six compounds predicted to be negative as CYP450 substrates and inhibition.This prediction indicates that the six compounds can be metabolized in the human liver so that it is predicted not to burden liver performance.However, oleic acid is predicted to act positively on CYP450 2C9 substrate, which indicates oleic acid will undergo chemical reactions or metabolism with CYP450 2C9 enzymes to produce products or metabolites that are different from the original substrate [59].Table 3 also shows that the six compounds are predicted to be negative for the CYP inhibitory promiscuity property.This prediction indicates that the six compounds are predicted not to bind or reduce the activity of several CYP450 isoform enzymes (reducing the activity of the CYP450 isoform enzyme can reduce the metabolic activity of the compound) [68].
Table 3 shows the six compounds predicted to have no hepatotoxicity, respiratory toxicity, reproductive toxicity, and mitochondrial toxicity.Although oleic acid is predicted to be mitochondrial toxicity, oleic acid compounds at the subcellular level are predicted to be localized to the plasma membrane.
The toxicity of orally consumed compounds is classified into six categories based on the LD50 range, according to the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS) 2015.Class I is fatal (LD50 ≤ 5 mg/kg), Class II is fatal (LD50 5-50 mg/kg), Class III is toxic (LD50 50-300 mg/kg), Class IV is dangerous (LD50 300-2000 mg/kg), Class V is potentially hazardous (LD50 2000-5000 mg/kg), and Class VI is non-toxic (LD50 > 5000 mg/kg) [5].Table 3 shows that only oleic acid is classified as class II toxicity, but the amount of oleic acid is small in coffee analog of R. mucronata fruits.
In addition, the analog coffee of mangrove fruits was also evaluated for its tannin content and antioxidant capacity.The results of previous studies reported that mangrove fruits is high in tannin content and high in antioxidants [1].Evaluation of total tannins is equivalent to tannic acid.At the same time, the antioxidant capacity is the ability of analog coffee to inhibit DPPH radicals by 50% (IC50).Table 4 shows the results of the total tannins and antioxidant capacity evaluation.4 shows that the analog coffee of R. mucronata fruits is still quite high.However, tannins in mangroves are polyphenolic and flavonoids compounds, which contribute to their antioxidant and antimicrobial properties [69].Acceptable The daily intake of tannins in foods requires a safe limit of 560 mg/kg body weight/day [70].Blois antioxidant activity is classified into four categories: very strong (IC50≤50 µg/mL), strong (50<IC50≤100 µg/mL), moderate (100<IC50≤150 µg/mL), weak (150<IC50≤200 µg/mL), and very weak (IC50>200 µg/mL) [71].Table 4 shows that the antioxidant capacity of R. mucronata fruits analog coffee is moderate.Even though analog coffee has been roasted at high temperatures, its antioxidant properties persist.This result is thought to be related to the compounds in analog coffee, which are included in volatile compounds generally resistant to heat.The characteristics of R. mucronata fruits analog coffee containing water, ash, and caffeine that meet the Indonesia National Standar for ground coffee.The compunds identified in analog coffee are predicted to be absorbed, distributed, metabolized, have antioxidant properties, and have safe toxicity for the human body.

Conclusion
These research findings provide that analog coffee from the R. mucronata fruits origin Sangihe Islands has the potential to become a coffee-like product as a functional drink with antioxidant properties.The compounds detected by GC-MS in analog coffee are derivatives of fatty compounds with antioxidant properties.The prototype of analog coffee from Rhizophora mucronata fruits needs to be reduced in tannin levels to be safer for human consumption.

Figure 2 .
Figure 2. The structure of identified compounds in the analog coffee of Rhizophora mucronata fruits via GC-MS.

Table 1 .
Characteristics of analog coffee from Rhizophora mucronata fruits.

Table 2 .
Chemical profiles of analog coffee from Rhizophora mucronata fruits by GCMS.

Table 3 .
ADMET profile of analog coffee from Rhizophora mucronata fruits.

Table 4 .
Tannins total and antioxidant capacity of analog coffee from Rhizophora mucronata fruits.