Bioprospecting of katang-katang leaves (Ipomoea pes-caprae) from Sumba Island, East Nusa Tenggara: antimicrobial, antioxidant and secondary metabolites content

Ipomoea pes-caprae (Katang-katang) is one of the vegetations that grows in coastal area. Previous studies have proven that I. pes-caprae showed antibacterial, anti-inflammatory, and antioxidant properties. This research was conducted to characterize secondary metabolites content, antimicrobial activity and antioxidant activity of I. pes-caprae crude extract from Sumba, East Nusa Tenggara. The extraction was carried out using consecutive extraction method using n-hexane, ethyl acetate, and methanol with agitation (115 r.p.m for 24 hours). Phytochemical test and thin layer chromatography were performed to analyse the secondary metabolites in I. pes-caprae. In addition, total phenolic content (TPC) was calculated using Folin-Ciocalteu method. The antimicrobial activity was conducted using paper disc diffusion method against Bacillus cereus, Salmonella typhi, Escherichia coli, Micrococcus luteus, Candida albicans, and C. tropicalis. Phytochemical test and TLC analysis showed that n-hexane extract contained flavonoid, tannin, and steroid; ethyl acetate extract contained alkaloid, flavonoid, and terpenoid; while methanol extract contained alkaloid, tannin, and steroid. The antimicrobial activity gave negative results against all microbial pathogen. The ethyl acetate and methanol extracts were noted as a strong antioxidant agent with IC50 values of 65.7 ppm and 81.8 ppm. Moreover, TPC in each extract was 3.7 mg GAE/g in n-hexane extract, 6.4 mg GAE/g in ethyl acetate extract and 8.9 mg GAE/g in methanol extract.


Introduction
The early exploration of bioactive compounds from marine environment in Indonesia successfully discovered various potential sources especially from the invertebrates such as sponge, soft coral, tunicates, etc [1][2][3] [4].On the other hand, seaweed and mangroves have been widely studied compare to other plants [5][6] [7] [8].Therefore, studies of bioactive compounds from other coastal plants is very interesting to be conducted.
Ipomoea pes-caprae or 'Katang-katang' is one of vegetations that grows as halophyte [9].This plant is commonly found in coastal zones including tropical and subtropical areas.Local people in several countries including Brazil, Mexico, Thailand, India, French Guiana, Papua New Guinea, Australia, Nigeria, Bahamas and Indonesia utilize this plant as a medicinal plant to treat several diseases [9] [10].In addition, I. pes-caprae has been applied to treat inflammation, gastrointestinal disorders, skin infection and inflammation caused by jellyfish [11] [12].In addition, Marie et al [13], stated that most of the reports focused on the bioactivity in the laves.
Previous studies have reported that I. pes-caprae exhibited antibacterial, anti-inflammatory and antioxidant properties.For instance, Kumar et al. [14] reported the antioxidant ability of I. pes-caprae extract against DPPH free radicals.In addition, the antimicrobial activity of I. pes-caprae was also noted to combat Staphylococcus epidermidis, S. aureus, Salmonella typhi, E. coli, and Candida albicans [15] [16].Accordingly, I. pes-caprae is known to poses various bioactive groups, such as alkaloid, norisoprenoids, phenols, terpenoids, steroids and glycosides [12] [16].Unfortunately, study of this plant is dominated from western part of Indonesia.On the other hand, research on secondary metabolites of this plant from the eastern part of Indonesia is barely studied.Thus, this study was carried out to understand the antimicrobial, antioxidant, and the bioactive profiles of I. pes-caprae leaves from Sumba Island, East Nusa Tenggara, Indonesia.

Materials and method
2.1 Sample collection I. pes-caprae was collected from a sandy beach in Tanjung Karoka, Sumba Island, East Nusa Tenggara.The sample was put in a sterilized zip-lock plastic and then transferred to Natural Product Laboratory, Diponegoro University for further steps.Several environment parameters such as substrate temperature, substrate drought level and light intensity were measured in situ using digital soil analyser tester.

Extraction
Extraction was carried out according to Frederick et al. [17].In total 90 g of sample was resize using mechanical grinder, then the sample was extracted using consecutive maceration method with n-hexane, ethyl acetate, and methanol, while the ratio of sample and solvent (1:10).Maceration was conducted with agitation (115 r.p.m for 24 h) at 27 o C. The filtrate was filtered using filter paper and evaporated using a rotary evaporator.

Metabolites profiling
2.3.1 Phytochemical Test.Phytochemical test was performed according to Sibero et al. [18] and Tepal [19].Several bioactive groups were targeted in this study using procedures as mention in Table 1.

Bioactive Groups Procedures
Alkaloid Crude extract of I. pes-caprae was dissolved in 1 mL of 2 N H 2 SO 4 and then filtered using filter paper.The filtrate was added into a test tube then added a few drops of Dragendorff reagent.Yellowish to orangish precipitate showed the positive result of alkaloid in I. pes-caprae extract.

Flavonoid
Crude extract of I. pes-caprae was dissolved in 10 mL aquadest.Five drops of concentrated hydrochloric acid (HCl) were added to I. pes-caprae extract.
The appearance of red colour indicates the presence of flavonoids.

Tannin
Five mL of the I. pes-caprae extract was placed in a test tube and then 2 mL of 5% FeCl 3 reagent were added.A greenish-black precipitate indicates the presence of tannins.

Steroid/Terpenoid
Two mg crude extract of I. pes-caprae was mixed with 3-5 drops of acetic anhydride, then boiled and cooled.When it's already cooled, 8 drops of concentrated H 2 SO 4 were added from the sides of the test tube.The positive result was showed by appearance of green coloration.

Saponin
Two mg crude extract of I. pes-caprae was diluted in 5 mL aquadest, heated then shaken vertically until the foam was formed, and kept for 15 min.HCl 2 N was added to check the foam stability, the positive result was showed by the presence of stable foam for the last step.

TLC profiling.
Thin-layer chromatography (TLC) was carried out for the further bioactive compounds profiling with silica gel 60 (Merck, F254) as the stationary phase while chloroform and ethyl acetate as mobile phase with ratio 9:1.FeCl 3 , vanillin in H 2 SO 4 then heated at 60-80 o C, and DPPH reagents were sprayed onto the TLC plate for spot visualization [7][20][21] [22].

Total Phenolic Content (TPC).
Total phenolic content (TPC) was calculated using Folin-Ciocalteu method according to Sami et al. [23].Before the calculation, a standard solution of gallic acid (Merck) and the crude extract of I. pes-caprae were prepared.For the standard solution, 5 mg gallic acid was dissolved into 5 mL aquabidest.The fractions of the standard solution were suspended into several concentrations such as 4 ppm, 6 ppm, 8 ppm, 10 ppm and 12 ppm.One mL of Folin-Ciocalciteu reagent and 1 mL of 1% Na 2 CO 3 were added to each solution.Each concentration was dissolved in aquabidest until the volume reached 5 mL.The mixture was homogenized and incubated for 30 minutes and the absorbance was determined using spectrophotometer (UV VIS Shimadzu 1800) with 680 nm absorbance light.Total Phenolic contents was expressed as mg GAE (Gallic Acid Equivalents).[25].The pathogenic bacteria were inoculated onto Mueller-Hinton agar (MHA, HiMedia) while the fungi were revived onto Potato Dextrose Agar (PDA, HiMedia) using a streak method and incubated for 24 hours at 37 o C. On the screening day, the pathogens were suspended into Nutrient Broth (NB, Himedia) with 0,5 McFarland (HiMedia).The microbes were inoculated onto MHA and PDA using sterilized cotton swabs.The crude extracts were diluted using dimethyl sulfoxide (DMSO) to obtain 2000 ppm concentration.Each crude extract was injected into a paper disc and placed on inoculated media and incubated for 24 hours at 32 o C. DMSO was used as negative control while amoxicillin 0,1 mg/mL was used as positive control.The presence of clear zone after incubated period indicated the antimicrobial activity of I. pes-caprae extract.

Result and Discussion
This research is the first study about bioactive compounds from coastal organisms in Tanjung Karoka beach, Sumba Island, East Nusa Tenggara, Indonesia.After measuring the environmental parameters, we found that the temperature of substrate was 33 o C, drought level of substrate was dry and sunlight intensity was low.According to Zhang et al. (2018) [26], I pes-caprae usually grows in a hightemperature such as 33-35 o C, dry and sandy substrate.This research shows that Tanjung Karoka beach has a suitable environmental condition for I. pes-caprae to grow.The maceration method was carried out because of this procedure is widely applied to obtain almost all phytochemical, relatively easy to conduct, and time efficient [27].This method was chosen based on the consideration the sample characteristics and the presence of thermolabile compounds which are easily damaged in high temperature.In this study, we conducted consecutive maceration method to obtain crude extracts with different polarity as many as possible.Consecutive maceration method can obtain more compounds compare to single maceration method because consecutive maceration method was carried out using 3 solvents with different polarity while single extraction method was carried out using only one solvent [28] [29].
Following the extraction, we conducted bioactive groups profiling using phytochemical test (Table 2).It is highlighted that our sample contained four out of five targeted bioactive groups, such as alkaloid, flavonoid, tannin and steroid/terpenoid.In comparison to this study, Andayani and Nugrahani [30] reported similar bioactive groups from I. pes-caprae, except saponin.In addition, Venkateasan et al. [15] has proven that I. pes-caprae extract contained nine bioactive group compounds including flavonoid flavanol, phenolics, tannin, amino acid, steroid, terpenoid, protein and carbohydrates.One of the factors that caused the differences of bioactive content is the bioactive only produced by plants to protect them against pathogenic attacks and environmental stresses.Thus, the bioactive compounds that produced by a plant will be differences compare to others according to their environmental stresses.Moreover, the phytochemical test based on solution has several weakness therefore a metabolite analysis using TLC plate was conducted.2 shows the result of metabolite profiling using TLC plate.The UV visualization indicated several different colours in I. pes-caprae extracts from different solvents.It is suggested that the solvent extracted compounds with different polarity from the samples.The function of vanillin + H 2 SO 4 was for detected terpenoid compound, while FeCl 3 for detected phenolic compound [21].The visualization using vanillin solution indicated that n-hexane could extracted terpene derivative compounds (7 spots), rather than ethyl acetate (4 spots) and methanol (2 spots).A similar result was also showed for phenolic detection using FeCl 3 solution.Extract from n-hexane had 5 spots, while ethyl acetate and methanol had 4 spots.Moreover, in this study we also applied DPPH reagent to visualize some spots with antioxidant properties.Interestingly, some potential spots which indicated by the presence of white or yellowish colour were appeared after the reagent spraying [25].The n-hexane extract had 2 yellow spots, whereas ethyl acetate and methanol extract demonstrated 3 yellow spots.Furthermore, the antioxidant activity of these extract was continued to determine the IC 50 values.
The result of antioxidant assay and total phenolic content (TPC) in I. pes-caprae showed that methanol extract has the highest IC 50 value compared to ethyl acetate and n-hexane extracts (Table 4).According to Purwanto et al. [30], antioxidant activity is categorized very strong with the IC 50 value (<50 ppm), strong with (50-100 ppm), medium with (100-150 ppm), weak with (150-200 ppm), and very weak with (>200 ppm).Table 7 shows that the ethyl acetate and methanol extract of I. pes-caprae had strong antioxidant activity with IC 50 values of 65.7 ppm and 81.8 ppm.Unfortunately, the n-hexane extract was not tested due to the limited amount.Respectively, this result was also supported by Andayani and Nugrahani [30] who studied the antioxidant activity of I. pes-caprae ethanol extract with IC 50 values of 46,774 ppm.Furthermore, Pothula and Kanikaram [31] has reported that I. pes-caprae methanol extract has antioxidant activity with the value of IC 50 was 15 µg/mL.TPC in each extract was 3.7 mg GAE/g in n-hexane extract, 6.4 mg GAE/g in ethyl acetate extract and 8.9 mg GAE/g in methanol extract.The highest TPC content was noted in methanol extract followed by ethyl acetate and n-hexane.TPC analysis were commonly conducted to determine the phenolic compound in the particular natural substances.The phenolic compounds have an outstanding capability as an antioxidant agent [34].The utilization of Folin-Ciocialteu reagent was based on its ability of phospomolybdic-phosphotungstic acid to reduce by phenolic with an alkaline condition during the oxidation process then became blue molybdenum-tungsten complex [23].Some of phenolic compounds has identified as semi-polar to polar, and methanol has a remarkable capability to extract various compounds with wide range of polarity [35] [36].Therefore, methanol extract was allegedly contained more bioactive with antioxidant potential.Previous study has reported there is a correlation between the result of TPC and antioxidant activity.For instance, Widyastuti et al. (2020) [35] has reported the methanol extract of curcuma has a high TPC value and strong antioxidant activity.This research shows the methanol extract of I. pes-caprae has the higher TPC value and the lower IC 50 value compared to ethyl acetate and n-hexane extract of I. pes-caprae.
Regretfully, all extracts demonstrated negative result in the antimicrobial assay (Figure 3).On the opposite, previous studies have reported that I. pes-caprae extract showed potential as antimicrobial agents against Staphylococcus sp., Enterococcus faccalis and B. subtilis [9] [32].All of the extract gave negative result caused of the low concentration and the tested pathogens were clinical multi-drug resistant (MDR) strains.MDR is referred to microbes that having resistance to several classes of antibiotics [33].Therefore, we suggested that these extracts were not potential as an anti-MDR agent.

Conclusion
This study conducted the experiments of I. pes-caprae or also known by locals as Katang-katang from Sumba Island, East Nusa Tenggara.I. pes-caprae was carried out using methanol, ethyl acetate and nhexane.Phytochemical test and TLC analysis showed that n-hexane extract contained flavonoid, tannin, and steroid; ethyl acetate extract contained alkaloid, flavonoid, and terpenoid; while methanol extract contained alkaloid, tannin, and steroid.The result of TPC test was that each extract was 3.7 mg GAE/g in n-hexane extract, 6.4 mg GAE/g in ethyl acetate extract and 8.9 mg GAE/g in methanol extract.The ethyl acetate and methanol extracts were noted as a strong antioxidant agent with IC50 values of 65.7 ppm and 81.8 ppm.Unfortunately, all of the extract none of this extract have a potential of antimicrobial and antifungi agents.

3
indicates the presence of the compound; -: indicates the absence of the compound.
In total, 150 µL of each concentration and 150 µL of DPPH stock were transferred into vials then mixed gently.The mixture was incubated in a dark condition for 30 mins 27 o C. Afterward, the absorbance was measured at 517 nm using spectrophotometry UV VIS Shimadzu 1800.The radical scavenging activity (%RSA) was calculated with the following formula.Then to obtain the IC50 values, a linear regression was applied by correlated the concentration of the extract used with the value of %RSA.

Table 3 .
Metabolites profile of I. pes-caprae leaves extract using TLC analysis.: indicates the the presence of the same Rf; -: indicates the the absence of the same Rf.

Table 3 and
Figure