INTRODUCTION:

Etlingera (Zingiberaceae) is one of the common plants in Indonesia used as traditional medicines¹, contains approximately 150–200 species of worldwide distribution and in Sulawesi (Indonesia) grows 48 species². Previous biological activities studies of Etlingera revealed interesting potencies.

Leaves and stems of E. brevilabrum exhibited anticholesterol activity³. E. littoralis rhizomes and E. maingayi leaves showed antibacterial potency⁴. Antibacterial and antioxidant activities also exhibited by the leaves of E. Fulgens⁵. Antioxidant was also showed by E. calophrys stems⁶, and E. pavieana rhizome displayed anti-inflammatory effect⁷. Moreover, essential oil of E.fenzlii was safe for repellent source⁸. In addition, the leaves and rhizomes of E.elatior performed antioxidant, antibacterial, and tyrosinase inhibitory activity^5,9-14, and also fruits of E.elatior has potency as hepatoprotector¹⁵. Another study showed that E.hemisphaerica demonstrated activity in lowering plasma glucose level and triglyceride¹⁶.^.

Chemical studies of Etlingera plants showed valuable secondary metabolites such as phenylpropanoids, flavonoids, phytosterols and other polyphenols. The leaves and rhizomes of E. brevilabrum and E. sphaerochepala var. grandiflora produced β-sitosterol and stigmasterol. The latter species also yielded a simple phenolic paeonol¹⁷. The stems of E. calophrys produced yakuchinone A, p-hydroxybenzoic acid and stigmasterol ⁶. Furthermore, 4-methoxycinnamyl alcohol, trans-4-methoxycinnamaldehyde, 4-methoxycinnamyl p-coumarate and p-coumaric acid were isolated from E. pavieana rhizomes⁷.

The leaves of E.elatior produced quinic acid-containing cinnamic acid derivatives, including 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, and 5-O-caffeoylquinic acid methyl ester¹⁸. The leaves also contained kaempferol-3-glucuronide, quercetin-3-glucuronide, and quercetin-3-rhamnoside⁹. In addition, 1,7-bis(4-hydroxyphenyl)-2,4,6-heptatrienone, demethoxycurcumin, 1,7-bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one, 16-hydroxylabda-8(17),11,13-trien-15,16-olide were isolated from rhizomes of E. elatior¹⁹.

According to the above information, the chemistry and pharmacology aspects of E.elatior fruits have not been reported their activities against radical scavenger and anti-diabetic in vivo. Hence, the present work will facilitate and report the isolation and identification of chemical compounds from the methanol extract of E.elatior fruits, as well as their radical scavenger activity and its activity as anti-diabetic in vivo.

MATERIALS AND METHODS:

General Procedures:

Instruments were used Perkin Elmer Spectrum One FT-IR spectrophotometer, and JEOL ECP 500 NMR spectrometer (500 MHz for ¹H and 125 MHz for ¹³C). Chromatography techniques including vacuum liquid chromatography (VLC) and radial chromatography (RC) were performed using Kieselgel 60 F₂₅₄ 0,25mm, silica gel 60 GF₂₅₄, and silica 60 G (Merck, Darmstadt, Germany). TLC plates were derivatised using a cerium sulphate reagent (Merck, Darmstadt, Germany). DPPH (2,2-diphenyl-1-picrylhydrazyl) was purchased from Merck (Darmstadt, Germany).

Sample:

Fruits of E.elatior were collected from the Wolasi Forest, South East Sulawesi, in May 2018 with No of Specimen EST04. The plant specimen was identified and stored in the Herbarium Bogoriense, Indonesia.

Animals and ethics:

Experimental animals used in the study were Wistar male rats. The animals were acclimatized normal for 1 week under controlled environmental. They were allowed free access to pellet chow and water ad libitum. The animals were used in this study were carried out in accordance with the Animal Ethics Committee of Halu Oleo University No. 248d/UN29.20/PPM/2019.

Extraction:

The dried powdered fruits of E.elatior (2.1kg) was macerated with methanol (MeOH, 3 x 5.0 L, 24 h each time) at room temperature and yielded a dried methanol extract as dark green gum (80g) for isolation and radical scavenger activity. Separately for anti-diabetic activity and hispathology of pancreatic organ, 1kg dried powdered fruits of E.elatior was macerated with ethanol (95% EtOH, 3 x 24 h each time) at room temperature and yielded 96.2g concentrated extract.

Isolation:

This extract was further fractionated using a silica gel VLC (10 x 5cm, 150g), eluted with n-hexane–ethylacetate (from 9:1 to 0:10) followed by MeOH, and gave 5 main fractions (F1-F5) with weight of 1.3, 4.1, 7.3, 6.2, and 29.6g, respectively. Main fraction F3 was re-fractionated using a silica gel VLC (10 x 5cm, 150 g) and gradiently eluted with n-hexane–ethylacetate (from 7:3 to 0:10) and MeOH as mobile phases, to yield subfractions F31 (0.1g), F32 (0.7g), F33 (0.7g), and F34 (4.4g). Subfraction F32 was chromatographed using a silica gel RC with chloroform–MeOH (95:5) and pure MeOH as mobile phases, to produce pure compound 1 (0.04g). Furthermore, subfraction F33 was further purified using the same method as compound 1 purification to get compound 2(0.08g). The main fraction F4 was subjected to a silica gel VLC (5 x 5cm, 75g), separated gradiently with n-hexane–ethyl acetate (from 6:4 to 0:10) followed by MeOH, and afforded subfractions F41 (0.3g), F42 (0.5g), F43 (1.0g), F44 (0.8 g), and F45 (1.4g). The sub-fraction F42 was also purified using silica gel RC, separated using a mixture of n-hexane–ethylacetate (75:25) and MeOH, and yielded pure compound 3 (0.05g). Moreover, compound 4(0.015g) was produced by purification of subfraction F44 using the same method as compound 3.

Radical scavenger activity:

The radical scavenging activity of the isolated compounds was adapted from Sahidin et al.[20], Ching et al.[21] and Babu et al. [22] with slight modification. The reduction of DPPH (1,1-diphenyl-2-picrylhydrazyl) radical was analyzed by using both qualitative and quantitative methods. One ml of 500µM (0.2mg/ml) DPPH in methanol was mixed with the same volumes as of the tested compounds at various concentrations. They were mixed well and kept in the dark for 30 min. The absorbance at λ 517nm was monitored in the presence of different concentrations of the samples. The blank experiment, i.e., with only solvent and DPPH (2ml of 500µM in methanol), was also carried out to determine the absorbance of DPPH before interacting with the compounds. The amount of sample, methanol extracts, compounds and standard (ascorbic acid) in mg/mL at which the absorbance at 517nm decreased to half of its initial value was used as the IC₅₀ of compounds. The analysis was done in triplicate for the standard and compounds²³.

Antidiabetic Activity:

The antidiabetic activity methods was adapted from Jain [24] and Sam et al. [25]. Total 30 rats were induced with 150mg/KgBW streptozotocin (STZ) intraperitoneally and experiencing diabetic syndrome (Elevated blood sugar). Animals were then divided into 6 groups randomly and treated for 7 days, by following:

Group I: Negative control (Na CMC 0.5%)

Group II: Normal control

Group III: Positive control (Glibenclamide 0.9 mg)

Group IV : ethanol extract of E. elatior 200 mg/kg BB (EE 200)

Group V: ethanol extract of E. elatior 300 mg/kg BB (EE 300)

Group VI: ethanol extract of E. elatior 400 mg/kg BB (EE 400)

At day-8, blood was collected from animals by tail vein sampling. Blood collected was put into EDTA-Tube then centrifuged at 3000rpm for 15 minutes. Afterward, 10µl plasma collected into tube and incubated at 25ºC for 10 minutes. Further, plasma was measured the absorbance under Spectrophotometer instrument. Data collected then analyzed statistically by using SPSS.

Observation of Pancreatic Hispatology:

Pancreatic hispatology was carried out to rats by performing surgery to obtain the pancreatic organ. Rats were anesthezed by using chloroform. Followed by fixation, dehydration, embedding and blocking, cutting, and staining with haemotoxylin-eosin (HE). Pancreatic organ then observed by using microscope with magnification 100x.

RESULTS:

Physicochemical property and spectroscopic data of the isolated compounds from E. elatior fruits:

Four compounds (1–4) were successfully isolated and identified from the methanol extract of E. elatior fruits. Those compound structures were determined based on their physicochemical property and spectroscopic spectra of FTIR and NMR. These values were also compared with those reported in the previous studies.

Quercetin (1), a yellow amorphous powder. Fourier Transforms Infrared (FTIR) spectrum (KBr), ʋ_max (cm^-1):3410, 3076, 1660, 1611, 1520, 1465 and 1169. Spectra of¹HNMR (500MHz, CDCl₃)δ_H (ppm): 12,18 (1H, s, OH), 9,73 (1H, br s, OH), 8,62 (1H, br s, OH), 8,40 (1H, br s, OH), 8,01 (1H, brs, OH), 7,84 (1H, d, 2,2, H-2’), 7,01 (1H, d, 8,6, H-5’), 7,71 (1H, dd, 8,6, 2,2, H-6’), 6,55 (1H, d, 1,7, H-8), and 6,28 (1H, d, 1,7, H-6). Spectra of ¹³CNMR (125MHz, CDCl₃) δ_C (ppm):177.3 (C-4), 165.7 (C-7), 162.5 (C-5), 158.2 (C-9), 148.7 (C-4’), 147.9 (C-2), 146.2 (C-3’), 137.2 (C-3), 124.1 (C-1’), 121.6 (C-6’), 116.2 (C-5’), 116.0 (C-2’), 104.4 (C-10), 99.3 (C-6), and 94.4 (C-8).

p-Coumaric acid (2); white amorphous powder. FTIR spectrum (KBr), ʋ_max (cm^-1): 3413, 3070, 2923, 1690, 1508, 1460, 1421, and 1125. Spectra of ¹HNMR (500MHz, CDCl₃)δ_H (ppm): 9.23 (1H, s,), 7.60 (1H, d, 15,9, H-7), 7.54 (2H, d, J = 8.6 Hz, H-2/H-6), 6.90 (2H, d, J = 8.4 Hz, H-3/H-5), 6.33 (1H, d, 15,9, H-8). Spectra of¹³CNMR (125MHz, CDCl₃) δ_C (ppm): 167.4 (C-9), 160.1 (C-4), 144.6 (C-7), 130.0 (C-2/C-6), 121.8 (C-1), 115.8 (C-3/C-5), and 114.9 (C-8).

Vanilic acid (3); a white powder. FTIR spectrum (KBr), ʋ_max (cm^-1): 3460, 3086, 2954, 1696, 1603, 1524, 1465, and 1206. Spectra of ¹HNMR (500MHz, CDCl₃)δ_H (ppm):10.87 (br, s), 8.40 (1H, s), 7.61 (1H, dd, 8.4, 1.9), 7.58 (1H, d, 1.,7), 6.93 (1H, d, 8.2), and 3.92 (3H, s).Spectra of ¹³CNMR (125MHz, CDCl₃) δ_C (ppm): 166.6 (C-7), 151.2 (C-4), 147.2 (C-3), 124.0 (C-6), 122.0 (C-1), 112.6 (C-5) and 55.4 (C-8).

p-Hydroxybenzoic acid (4); white amorphous powder. FTIR spectrum (KBr), ʋ_max (cm^-1): 3348 (OH), 3079 (Csp2-H), 1692 (C=O), 1610, 1595, 1449 (aromatic) and 1244 (C-O-C). Spectra of ¹HNMR (500MHz, CDCl₃)δ_H (ppm): 9.43 (1H, s), 7.91 (2H, d, J = 8.6 Hz, H-2/H-6), 6.92 (2H, d, J = 8.4 Hz, H-3/H-5). Spectra of¹³CNMR (125MHz, CDCl₃) δ_C (ppm): 166.7 (C-7), 161.8 (C-4), 131.8 (C-2/C-6), 121.8 (C-1), and 115.1 (C-3/C-5).

Data of DPPH test of extract and compounds from E.elatior fruits

The IC₅₀ of methanol extract, isolated compounds and ascorbic acid displayed in Table 1.

Table 1. Radical scavenger potency of methanol extract and compounds from E.elatior fruits

No	Sample (s)	IC₅₀ (g/mL)
1	Methanol Extract	390.0 ± 2.42
2	Quercetin	30.98 ± 0.84
3	p-Coumaric acid	148.6 ± 0.88
4	Vanilic acid	243.0 ± 1.60
5	p-Hydroxybenzoic acid	309.0 ± 1.94
6	Ascorbic acid	25.18 ± 0.46

Data of Plasma Glucose Level of Animals:

The ethanol extract of E.elatior in various concentration, negative control, positive control and plasma glucose level are shown in Table 2.

Table 2. Measurement of Plasma Glucose Level of Animals

Group

Mean Plasma Glucose Level (mg/dL)

EE 200

EE 300

EE 400

Negative Control

Positive Control

156^a;b

109.4^a;b

95.1^a;b

388.4

134.6^a

^a: significant difference compared to negative control (p<0.05); ^b: there is no significant difference compared to positive control

Histopathology of Pancreatic Organ

Figure 1. Histopathology of Pancreatic Organ (a) Negative Control (NaCMC) (b) Normal Control (c) Positive Control (d) EE200 (e) EE300 (f) EE400

DISCUSSION:

Extraction and purification of E.elatior fruits using chromatography methods produced four isolates (1-4). Compound structure of isolates were determined by comparison of FTIR, ¹H and ¹³C NMR spectra the isolates with the same data from literatures. The results can be concluded that Isolate 1 is quercetin ²⁶, Isolate 2 is identical with p-coumaric acid ²⁷, Isolate 3 is similar with vanilic acid ²⁸ and Isolate 4 is p-hydroxybenzoic acid ²⁹. All of the isolates are aromatic compounds and quercetin is a flavonoid. The compounds are displayed at Figure 2.

Figure 2. Compound structures from E. elatior fruits

The four phenolic compounds are firstly isolated and identified from E. elatior fruits. Based on references, quercetin is also firstly isolated and identified form E.elatior. Although the presence of quercetin has been identified using UHPLC from E. elatior flowers without isolating process³⁰. In addition, leaves of E. elatior produced quercetin-3-glucuronide, quercetin-3-glucoside, and quercetin-3-rhamnoside⁹. Moreover, vanilic acid was identified from leaves and rhizomes of E. philippineni ³¹ and p-coumaric acid has been isolated and identified from ethylacetate fraction of E. pavieana rhizome⁷. Meanwhile, p-hydroxybenzoic acid has reported from E. calophrys stems⁶. The invention of these compounds increases the diversity of phenolic compounds from the Etlingera plant.

Biological activities of extract and isolated compounds of E.elatior fruits are evaluated against radical scavenger potency using DPPH radicals. The results showed at Table 1. In the evaluating, qualitative experiments showed that the methanol extracts and all isolated compounds bleached the purple DPPH color to pale yellow when the TLC plate on which they were sprayed by 0.2% of DPPH in methanol. It is indicated that those samples have potency as radical scavengers. Generally, phenolic compounds have good antioxidant activity^32-35. In the quantitative DPPH radical scavenger assay, the extracts and the compounds of E. elatior fruits were able to neutralize the DPPH free radicals but less active than positive control (ascorbic acid). The radical scavenging activities of phenolic acids depend on the number of hydroxyl moieties attached to the aromatic ring of the benzoic or cinnamic acid molecule^36-37. Vanilic acid, p-hydroxybenzoic acid and p-coumaric acid are phenolic compounds that have only one hydroxyl moieties attached to the aromatic ring. The difference of radical scavenger ability is caused by other units in their molecular skeleton. In vanilic acid, there is a methoxy unit in C-3, whereas p-coumaric acid has addition of one vinyl unit thus extending the double bond conjugation. It caused the radical scavenger ability of p-coumaric acid >vanilic acid >p-hydroxybenzoic acid. Furthermore, quercetin is a flavonoid compound, has 4 hydroxyl (OH) attached to the aromatic ring and a hydroxyl at C-3, so the ability of quercetin as radical scavenger is more active than benzoic acid derivatives. The high ability of quercetin as radical scavenger is also caused by the presence of OH in C-4’³⁸. Number of aromatic compounds which are identified at methanol extract of E. elatior fruits, support radical scavenger potency of the extract to be developed as antioxidant and others herbals ³⁹.

Based on result of plasma glucose level of animals (Table 2; Figure 2), ethanol extract of E. elatior at concentration 200mg/KgBw (EE 200); 300mg/KgBw (EE 300); and 400mg/KgBw (EE 400) were able to decreased the plasma glucose level in animals with hyperglycemic condition (p<0.05). A dose-dependent in decreasing plasma glucose level was observed in this study. Extract with various concentrations gave the significant difference in decreasing plasma glucose levels at animals compared to negative control. EE 400 was most effective in decreasing plasma glucose levels than EE300, positive control (glibenclamide), and EE200 respectively.

Figure 2. Profile of Plasma glucose level (pre-induced, post-induced STZ, and post-treatment

Flavonoid contained in E. elatior is most likely to decrease the plasma glucose level. Quercetin is a flavonoid mostly isolated from E. elatior. Quercetin increases the replication of beta-cell DNA, thus increasing the production of insulin, as well as inhibits α-glucosidase activity thus delayed increases of plasma glucose plasma after a meal^40-41.

Results in observation of pancreatic organ showed damage of pancreatic beta cell (β-cell) induced-streptozotocin (STZ) characterized by necrosis (Figure 1). Negative control (Figure 1(a)) was indicating necrosis, normal control (Figure 1(b)) showed no necrosis due to not induced by STZ and no treated utterly, and positive control (Figure 1(c)) gave an improvement at β-cell that treated by glibenclamide. On the other hand, extract of E. elatior gave an improvement and protective effect to pancreatic β-cell. Despite EE200 were still indicating some necrosis (Figure 1(d)), EE300 and EE400 were not indicating necrosis at pancreatic β-cell.

STZ induces the necrosis and degeneration of pancreatic β-cell. The necrosis at the β-cell lead host unable to produce insulin thus plasma glucose level will be elevating (hyperglycemia). The hyperglycemia is worsen the damage of β-cell by increasing the reactive oxygen species (ROS) via glucose-metabolism pathway, thus increase the oxidative stress lead worsen the condition of beta cell⁴².

Wualae gave protective effect to pancreatic beta cell, which was not showing the necrosis at EE300 (Figure 1(e)) and EE400 (Figure 1(f)). Flavanoid compound contained in Wualae like quercetin is showing radical scavenger activity thus decrease the stress oxidative, prevent free radicals and prevent diabetagonic agent induced damage to β-cell. Quercetin demonstrated the antidiabetic effect by its mechanism as antioxidant by protecting against oxidant damage to β-cell^43-44.

CONCLUSION:

Four aromatic compounds, firstly isolated and identified from methanol extract of E.elatior fruits are quercetin, p-coumaric acid, vanilic acid, and p-hydroxybenzoic acid. All isolated compounds and the methanol extract have potency to be developed as radical scavenger herbals or drugs. Quercetin is the most active isolated compound as radical scavenger. The flavonoids contained in Wualae showed activity as anti-diabetic by decreasing plasma glucose level, as well showing protective effect to pancreatic β-cell at concentration 300 and 400 mg/KgBw.

ACKNOWLEDGEMENT:

We would like to thanks to the Ministry of Research, Technology and Higher Education of Republic of Indonesia for supporting research financial by Hibah Penelitian Dasar Scheme 2019 with Contract no: 519a/UN29.20/PPM/2019.

CONFLICT OF INTEREST:

No conflict of interest associated with this work.

CONTRIBUTION OF AUTHORS:

The authors declare that this work was done by authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by them.

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Received on 23.11.2020 Modified on 09.06.2021

Research J. Pharm. and Tech. 2022; 15(5):2141-2146.

DOI: 10.52711/0974-360X.2022.00355