Biological activities of Indonesian mangroves obtained by subcritical water extraction

Mangroves are still considered as underexploited marine resources in Indonesia, and functional materials from Indonesian mangroves are not characterized yet. In this study, different part of mangroves (Sonneratia alba leaves - SAL, Sonneratia alba roots - SAR, Sonneratia alba fruit - SAF, Rhizhopora mucronata - RMF, Rhizhopora lamarckii - RLF) were hydrolyzed using the subcritical water extraction (SCWE) system at 120 ºC. Mangroves hydrolysates (SAL, SAR, SAF, RMF and RLF) were further analyzed for total protein, sugar, reducing sugar, saponin and flavonoid content. The antioxidant capacity and functional material contents including total sugar, reducing sugar, protein, flavonoid and saponin contents. The highest flavonoid and saponin contents were obtained from RLF with the value of 20.13±0.17 mg/g and 31.83±0.18 mg/g, respectively. The antioxidant capacity and antibacterial properties of mangroves hydrolysates varied significantly based on the sample materials, with RLF showing the highest total antioxidant activity and antibacterial activity. The results of our study suggest that tropical mangroves especially R. lamarckii could be valorized efficiently, as a source of bioactive material using subcritical water extraction


Introduction
Mangroves are salt tolerant plant that can live in tropical and subtropical areas [1,2]. Mangroves term is a combination of the Portuegese word" mangue' and English word "grove". Mangrove ecosystem are among the most productive and important ecosystems because they provide goods and services to environment, other organisms and human society [3]. These salt tolerant plants have been used in traditional medicine practices for treatment of several diseases. Mangroves are recognized as rich sources of biologically active materials such as saponin, phenols, flavonoid, alkaloid and terpenoid. These bioactive materials can be obtained from the roots, leaves, barks, fruits and flowers of mangrove and can be used for treatment of many diseases [4].
In recent years, many studies reported that mangroves-derived bioactive compounds showed various pharmacological activities. As an example, Pcytotoxicity and antioxidant activity of Phoenix paludosa extracts have been reported [5]. Anti-inflammatory, anti-oxidant, anti-arthritic as well  [6,7]. However, most of those studies are using organic solvent extraction technique to obtain bioactive substances from mangroves [8]. The massive, wide-scale use of organic solvents by a diverse range of global industries represents a serious threat to the environment and human health. In order to minimizing solvent consumption, it is important to develop environmental friendly technology. Subcritical water extraction (SCW) has become an increasing alternative technology in the extraction of bioactive compounds from natural resources. SCW provides alternative to conventional extraction methods due to the reduced extraction time, efficient, lower extraction cost, and most importantly environmental friendly extraction technique.
In the present study we used different parts of mangroves i.e leaves, fruit and roots and analyze the effectiveness of green SCW technique to obtain bioactive materials from mangroves. Chemical characteristics of mangrove hydrolysates such as total protein, sugar, reducing sugar, saponin and flavonoid content of various mangroves hydrolysates were investigated. In addition, antioxidant capacity and antibacterial properties of mangroves hydrolysates were also demonstrated.

Sample preparation and subcritical water extraction
All mangroves samples (SAL, SAR, SAF, RMF, and RLF) were grind until become a fine powder. Mangroves powder were then transferred into the SCW reactor and mixed with distilled water (pH 7.2) at S/L ratios 1:50. The reactor was closed and heated up to 120 °C. Each hydrolysis process was run for 600 sec. Mangroves hydrolysates were then collected, filtered under a slight vacuum through a Buchi vacuum pump V100 with F1113 grade filter paper, pooled, and lyophilized using freeze dryer, hydrolysates were stored at − 20 °C until further analysis.

Yield, pH and colour characteristics of mangroves hydrolysates
The yield of mangroves hydrolysates was determined. The initial weight of mangroves (Wmi) was obtained from the sample weight before loading into the reactor. The final weight of mangroves hydrolysates was obtained from the final dry weight of hydrolysates after freeze dried (Wmf). The following formula was used for the calculation of yield:

Total flavonoid and saponin contents
Total flavonoid contents (TFC) and total saponin contents (TSC) were measured following the method described in our previous study [9].

Total protein, sugar and reducing sugar
Protein concentrations of mangroves hydrolysates were measured based on Lowry's methods. The samples were mixed with 5 mL of alkaline copper sulphate solutions (1% CuSO4.5H2O, 2% tartrate (KNaC4H4O6), 2% Na2CO3) at 1:100 ratio, and added with 0.5 mL of 1 N Folin-Ciocalteu reagent. Absorbance was measured at 660 nm, total content was expressed as mg BSA per gram dried sample (mg/g).The total sugar content (g glucose/100 g dried mass) of mangrove hydrolysates was determined using phenol sulfuric acid method. The reducing sugar content (mg glucose/g dried mass) of mangrove hydrolysate was measured using 3,5-dinitrosalicylic (DNS) colorimetric assay [9].

Total antioxidant activity
Mangroves hydrolysates (100 μL) was mixed with 3 mL of radical solution (0.6 M H2SO4, 28 mM Na3PO4, and 4 mM (NH4)6Mo7O24). The hydrolysates and radical solution mixture was incubated at 95 °C for 1.5 h. After incubation, the mixture was loaded into 96-well plates and the absorbance was measured at 695 nm using multimode microplate readers. All the measurements were made in triplicate, and methanol was used as the negative control. Ascorbic acid was used as the reference standard, and a standard curve was constructed. The results were expressed as mg ascorbic acid equivalent per g dry weight (mg AAE g−1 DW).

Anti-bacterial activity
Antibacterial activity of mangroves hydrolysates were tested against gram positive bacteria (Bacillus subtilis) and gram negative bacteria (Escherichia coli) by agar diffusion method [10]. Twenty μL of mangroves hydrolysates (500 μL) were poured into antibiotic disk in agar plate containing bacteria. The agar plates were incubated at 37 ºC for 24 h. The anti-bacterial activity was expressed as clearing zone around antibiotic disk (mm).

Absorption spectra and MRPs of mangroves extracts
Mangroves extracts obtained from SCW process were analyzed for the UV-absorption spectra. As shown in Figure 1, mangroves extract obtained from SCW process showed high absorbance value ranging from 220 to 270 nm. These peaks might correlate to the absorption spectra of some aromatic compounds. Many studies have reported the presence of aromatic compounds in mangroves. As an example, Li et al. have reported five new aromatic compounds from Bruguiera gymnorrhiza [11]. Mangroves represent a unique marine ecosystem and have to adapt with drastic environmental conditions (especially salt, temperature and nutrient supply) due to the tidal changes. To adapt these conditions, mangroves synthesize various secondary metabolites and therefore being a rich source for natural products.  were ranged from 6.69±0.07 to17.11±0.52, with the highest value obtained from S. alba roots followed by R. lamarckii fruit. MRPs also determines flavour and aroma during cooking process; and it is used to make food tastier [12]. In addition, MRPs also reported for their antioxidant properties and their ability to retard lipid oxidation.  [13,14]. Reducing sugar content was found highest in SAR (126.29±11.95 mg/g) followed by RLF (121.40±8.80 mg/g) and RMF (98.79±5.10 mg/g). Low amount of reduced sugar observed in mangroves extracts may be due to the degradation of sugar into other products, including aldehydes and ketones, from which organic acids could be produced [9]. The comparative evaluation of protein contents from SAL, SAR, SAF, RMF and RLF were carried out. The highest protein content was found in RLF (119.97±0.34 mg/g) whereas the lowest was obtained from SAL (33.10±0.20 mg/g) (Figure 3). Protein content of R. lamarckii fruit was up to two times as compared to S. alba. The results in the present study revealed that the protein content in fruits of R. lamarckii was higher than Xylocarpus granatum fruits (4.49±0.17 mg/g), Bruguiera gymnorrhiza (1.09±0.72 mg/g), and S. alba (0.93±0.02 mg/g) collected from Rawa Aopa Watumohai National Park, Southeast Sulawesi Sites [14]. These results indicated that SCW is a suitable and potential method to obtain protein, peptides, and amino acids from mangroves.  Table 2 and following this order: RLF> RMF> SAF> SAR> SAL. The flavonoid contents found in this study were higher compared to the previous study carried by Syahidah et al. (2019). In their study, they extracted Rhizhopora sp with acetone and methanol and reported that flavonoid contents were 2.641 mg/g, 1.998 mg/g, respectively [15]. In flavonoid and saponin extraction, solvent and temperature were the most important factors for extraction. At elevated temperature, the solubility of flavonoid and saponin increases because of the breaking of chemical bonds, mass transfer rates, and molecular diffusion was enhanced which indicated that SCW enabled the recovery of flavonoid and saponin from mangroves. Previous studies reported that, flavonoids and saponin show diverse biological activities including anti-cancer, antioxidant, anti-allergic, anti-inflammatory, and anti-microbial activity. In addition, flavonoids have play an important role in protecting DNA from damage, protecting product from degradation; therefore, flavonoids have been used in the production of cosmetics, pharmaceutical and also food products [16].

Antioxidant and antibacterial activity of mangroves extracts
The antioxidant activities of mangroves extracts were determined using total antioxidant assay. The total antioxidant results were represented as mg ascorbic acid equivalent per g dry weight (mg AAE g−1 DW). As shown in Table 3, all mangrove species exhibited promising antioxidant activity. The highest antioxidant content was obtained from RLF (3.35 ±0.11 (AAE mg/g) followed by RMF (3.18±0.08 AAE mg/g) and SAF (2.60±0.22 g AAE/g). Natural antioxidants are presented in various part of plant, such as leaves, fruit, bark, flowers, and seeds. Interestingly, all mangrove fruit extract showed more potent antioxidant activity as compared to leaves and roots. The results of antibacterial activity of mangroves extract are presented in Table 2. All mangroves extract showed antibacterial activity in Escherichia coli but only R. lamarckii fruit extract showed antibacterial activity against Bacillus subtilis. The highest inhibition zone against E.coli was observed in RLF (8.80±0.00 mm) followed by RMF (7.85±0.21 mm) and SAF (7.68±2.30 mm). Mangroves has been known as treasure house of therapeutic compounds which affected by many factors such as the mangroves species, mangrove parts, habitat and the season of sample collection, different growth stages of plant and experimental methods [17]. In addition, the biological (antioxidant and antibacterial) activity of mangroves showed in this study was positively correlated with total flavonoid and saponin contents.

Conclusions
The findings of this study demonstrate that SCW are potential method to be used a s eco-friendly technology to obtain functional materials (including proten, sugar, flavonoid and saponin) from S. alba, R. mucronata and R. lamarckii. The R. lamarckii fruit extracts showed higher protein, flavonoid, saponin and also better antioxidant and antibacterial activities. Therefore, mangroves especially R. lamarckii could be used in the traditional medicines, and develop in foodas well as pharmaceutical industries.