A New Flavanone as a Potent Antioxidant Isolated from Chromolaena odorata L. Leaves

Chromolaena odorata L. (Asteraceae) is one of the tropical plants which is widely used as traditional medicines for diabetes and soft tissue wounds treatment in some regions in East Indonesia. The present study was aimed at determining the bioactive compounds of C. odorata leaves. The methanol and ethyl acetate extracts of C. odorata leaves have the inhibitory activity against 2,2-diphenyl-1-picryl-hydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals as well as α-glucosidase rat intestine enzyme. A new flavanone was isolated from the methanol extract and elucidated as 5,3'-dihydroxy-7,6'-dimethoxyflavanone or, namely, odoratenin (1) together with two known compounds: isosakuranetin (2) and subscandenin (3). The antioxidant activity of odoratenin (1) exhibited very potent ABTS radical inhibitory activity with IC50 value of 23.74 μM which is lower than that of trolox (IC50 31.32 μM) as a positive control. The result showed that a new flavanone, odoratenin (1), should be potential as an antioxidant source.


Introduction
Antioxidant is a bioactive substance preventing the oxidation of the harmful chemicals. That oxidation is caused by free radicals that have unpaired electrons. So, those free radicals are very reactive to damage molecules in cell [1]. During the past decade, a lot of antioxidant products are consumed by people in the world as the synthetic drugs, supplements, or traditional medicines. The traditional medicines have been taken by people in the world derived from the natural sources like medicinal plants according to World Health Organization (WHO) data. 65% of population in India consume the medicinal plants as a primary health. The 40% of prescription drugs in China are also based on the component of medicinal plants. In addition, 70% of Canadians have also used the medicinal plants as both a health supplement and an alternative therapeutic product [2]. In Indonesia, the medicinal plants are recognized as jamu. Approximately 85% of jamu's ingredients are the extract of medicinal plants. Hence, a number of modern or synthetic medicines are made from the isolation of natural sources based on the traditional plant medicines [3]. One of the natural sources that has been used as a medicinal plant is Chromolaena odorata L.
C. odorata (Asteraceae) is one of the species of Chromolaena genus that has been identified by King and Robinson in 1970. C. odorata is recognized as siam weed. It is one of the invasive species with a rapid growth forming the thick bushes as high as about two meters. Besides, it spreads rapidly on the open areas such as grasslands, roadsides, forests, nature reserves, and wildlife sanctuaries [4]. Actually, C. odorata is used as a medicinal plant by people lived in the tropic and subtropic areas. For example, in Vietnam, this plant is used as a treatment of leech bites, soft tissue injuries, burns, and skin infections [5]. Furthermore, a leaf water extract is widely used as a diarrhea, malaria, and diabetes drug [6]. Additionally, this leaf is also used as the treatment of wounds because the leaf 's contents are protein, carbohydrate, and fiber source [7].
The previous studies have reported that most of the Chromolaena genus contains the flavonoids group. Based on a review information by Oliveira et al. (2017), they reported that about 40 flavonoids have been identified from this genus. One of species from this genus, C. hirsuta, has been reported 2 Evidence-Based Complementary and Alternative Medicine to contain quercetin and kaempferol derivatives which belong to flavonoids group [8]. Some researchers also reported that C. odorata contains the flavonoid compounds [9][10][11][12][13][14][15]. In addition, the qualitative phytochemical properties of C. odorata leaves extract also showed the presence of secondary metabolite compounds such as coumarins, flavonoids, tannins, and sterols [16]. Currently, this preceding research aims to isolate and identify other secondary metabolite compounds of C. odorata leaves. Furthermore, the antioxidant activity of the compounds will be assayed.

Extraction.
The dried leaves of C. odorata (30 g) were extracted with various solvents for the bioactivity preparation assay. The leaves were dried in room temperature. They were extracted by using n-hexane, dichloromethane, ethyl acetate, methanol, and water in 200 mL of solvent for each extract at room temperature for 24 hours. The solvent was removed from the extracts by rotary evaporator to obtain the five crude extracts.

Antioxidant
2.6.2. DPPH Radical Scavenging Assay. DPPH assay was performed based on the method published previously [30]. First, DPPH solution (6 × 10 −5 M) was separated by dissolving 2.37 mg of DPPH in 100 mL of methanol to obtain a working solution. Then, 1 mL the working solution was mixed with 33 L of samples (n-hexane, dichloromethane, ethyl acetate, methanol, and water extracts) at maximum dissolved concentration in methanol and mixed well. Finally, the mixed sample solution was incubated for 20 minutes at room temperature. Then, the absorbance (A s ) of the reaction mixture was measured by UV-Vis spectrophotometer at 517 nm. The mixed solution between methanol and the working solution was used as blank to give the blank absorbance (A b ). Gallic acid was used as a standard. The inhibitory activity was calculated by (1). The IC 50 value is expressed as a quantity of an extract inhibitory concentration against a half of DPPH radicals.

ABTS Radical Cation Scavenging
Assay. Free radical scavenging by ABTS radical was based on the method described previously by us [30]. First, ABTS solution (7 mM) was prepared by dissolving 19.2 mg of ABTS in 5 mL of water and, then, 140 mM K 2 S 2 O 8 in 88 L of water. Those two solutions were mixed and incubated for 12-16 hours to obtain ABTS radical cation solution which is a dark blue solution. It was added with ± 274 mL of ethanol to give an absorbance of 0.7 ± 0.02 units at 734 nm for making a working solution. 1 mL of working solution was mixed with 10 L of samples (nhexane, dichloromethane, ethyl acetate, methanol, and water extracts) at maximum dissolved concentration in DMSO and mixed well. Finally, the mixed sample solution was incubated for four minutes at room temperature; then, the absorbance (A s ) of the reaction mixture was measured by UV-Vis spectrophotometers at 734 nm. The mixed solution between DMSO and the working solution was used as blank to give the blank absorbance (A b ). Trolox was used as a standard. The inhibitory activity was calculated by (1). The IC 50 value was expressed as a quantity of an extract inhibitory concentration against a half of ABTS radicals.

2.7.
-Glucosidase Inhibitory Activity Assay. The glucosidase inhibitory assay was performed based on the procedure from Ayinampudi et al., (2012) with some modifications [31]. First, rat intestinal acetone powder (1 g) was suspended in 30 mL of normal saline. This suspended solution was sonicated for five minutes at 4 ∘ C. After centrifugation (12,000 rpm, 30 minutes, 4 ∘ C), the resulting supernatant was used for the assay. Briefly, a mixture of 10 L samples, 30 L of 0.1 M phosphate buffer (pH 6.9), 20 L of 10 mM maltose, 80 L glucose kit, and 20 L of enzyme supernatant were incubated in 96-well plates at 37 ∘ C for 10 minutes. Acarbose was used as a standard. The absorbance was recorded at 490 nm by microplate reader (Biotek ELx800UV). The inhibitory activity was determined from the formula as follows: where A blank = A enzyme reaction − A blank of enzyme reaction and A sample = A sample reaction − A blank of sample reaction .

Extraction.
The five crude extracts from C. odorata leaves have been obtained. The methanol extract has the highest yield of all extracts. From 30 g dried leaves in 200 mL of each solvent, the yields of the five extracts were obtained such as 4.33% yield of n-hexane, 6.77% yield of dichloromethane, 7.33% yield of ethyl acetate, 10.00% yield of methanol, and 7.33% yield of water extract.

Total Phenolic Content.
The total phenolic content of different extracts of C. odorata leaves was determined by using FCR according to the procedure of Qassabi et al. (2018) with slight modifications. The tested extracts are n-hexane, dichloromethane, ethyl acetate, methanol, and water extracts at concentration 61.91 g/mL. The evaluated result of total phenolic content of each extract is showed in Table 1. Gallic acid was used as a standard for calibration curve to determine the amount of total phenolic content. Based on study, the total phenolic content of different extracts varied from 14.65 to 104.08 gGAE/mg of extract. The ethyl acetate extract is the highest amount of total phenolic content of all the extracts with value of 104.08 gGAE/mg of ethyl acetate extract.

DPPH Radical Scavenging
Activity. DPPH radical scavenging activity of the five extracts and gallic acid as a standard are presented in Figure 1 and summarized in Table 1   isolated from C. odorata, and trolox as a standard are presented in Figure 2 and summarized in Table 1 3.5. -Glucosidase Inhibitory Activity. Rat intestinal acetone powder was used in this assay system. The five extracts, compounds isolated from C. odorata, and acarbose as a standard are showed in Figure 3 and summarized in Table 1. In this assay system, the ethyl acetate extract was found to be slightly more active than that of the methanol extract. In contrast, the n-hexane, dichloromethane, water, and the compounds (1-2) had a weak effect on the enzyme activity. The ethyl acetate extract presented the inhibitory activity with an IC 50 value of 779.54 g/mL. Acarbose, which is known as a potent -glucosidase inhibitor, was used as a standard and showed an IC 50 of 7.67 g/mL in our assay system.  Figure 4 and summarized in

4.1.
Antioxidant Activities of C. odorata. C. odorata is a species of the genus Chromolaena which is one of the largest genera of the family Eupatorieae (Asteraceae) [8]. In Indonesia, C. odorata, known as sungga-sungga, was collected from Ambon, Maluku, East Indonesia. This plant is a popular folk medicine widely used as alternative herbal treatment for diabetes and soft tissue wounds in East Indonesia. Besides, in Vietnam, this plant is used as a treatment of leech bites, soft tissue injuries, burns, and skin infections [5]. Furthermore, a leaf water extract is widely used as a diarrhea, malaria, and diabetes drug [6]. In the past 40 years, this plant has been reported in phytochemical studies in the United States [14,15]. Recently, C. odorata was described for its beneficial attributes in some Asia-Africa countries, especially the pharmacological effects of this plant. The specific reported attributes of C. odorata include being antibacterial [17], antifungal [18,19], anti-inflammatory [20,21], anticancer [11,13,22], antiplasmodial [9], antidiabetic [23,24], and antioxidant [6,[25][26][27][28]. However, the antioxidant activity of the isolated compound from C. odorata has never been reported. This present study demonstrated the antioxidant activity of the isolated compound from C. odorata for the first time. Related to this study, the antioxidant effect from this plant has been done by two radical scavenging assays supported with the total phenolic content data. As we know, there are a lot of free radical types caused of reactive oxygen species (ROS) [32]. They are the dangerous free radicals against the human body. These free radicals come from either the body itself or the external factors. The free radicals are by products  of energy production by mitochondria which are energyproducing cells as adenosine triphosphate (ATP), while the external factors come from the pollutions, ultraviolet radiation, diet, or lifestyle. Furthermore, the free radicals have an unpaired electron so this condition makes them to be reactive with other molecules around them [33]. Then, the molecules in cells are attacked by free radicals. Normally, the body's antioxidant defence system can block the free radicals before they become harmful to the body. Unfortunately, because of the old age or a lot of toxin that accumulate inside, the defence system works slowly and then the free radicals start to cause cell damage. The cell damage caused by free radicals is called 8 Evidence-Based Complementary and Alternative Medicine oxidative stress. On the long term, the danger of free radicals inside is related to aging and chronic diseases such as cancer, diabetes, and neurodegenerative and cardiovascular diseases. In the body, free radicals are superoxide anion (SOA) from 2.5% oxygen (O 2 ). The using of O 2 in the body as a distributor of energy products changes because of free radicals of SOA. Because of that, the body is protected from those free radicals by its antioxidant defence system with these following steps [34]. First, SOA is neutralized by the antioxidant enzyme superoxide dismutase (SOD) changed as hydrogen peroxide (H 2 O 2 ). H 2 O 2 is a weak free radical which is used as an immune compound to inhibit the pathogen bacteria or to treat the broken cell tissue. However, the large amounts of H 2 O 2 will be toxic for the body. So, there is the second step from the body's defence system that helped with glutathione peroxidase (GPO) enzyme. Two GPOs covert H 2 O 2 into two water molecules (H 2 O). Certainly, H 2 O is safer than that of H 2 O 2 . Those two steps are very important to protect the cell body. Unfortunately, there is not enough amount of SOD and GPO in the body. So, the amount of free radicals of SOA and H 2 O 2 will increase in the cell. The SOA and H 2 O 2 might not be worse. But in excess, they will react with each other into more dangerous free radicals, namely, hydroxyl radicals (•OH). Hence, an antioxidant is needed as a resistance support from the outside of the body's defence system [35].
Studies are in our laboratory to identify the antioxidant compound present in C. odorata. The determination of the antioxidant effect was assayed by using DPPH and ABTS radicals. As we described previously, there are a lot of free radical types caused by ROS including DPPH and ABTS radicals. DPPH radicals are expressed as the free radical with high reactivity at room temperature. The high reactivity is caused by delocalization of electrons around the molecules. The mechanism of radical scavenging is hydrogen donors. When the DPPH radical is reacted with a substance that donates a hydrogen atom, DPPH radical is reduced into a nonradical DPPH. In the assay, this reaction is characterized by decolorization of the solution. It changes its colour solution from purple to yellow. At room temperature, ABTS radical is more stable and has higher reactivity than that of DPPH. The ABTS radicals are expressed as cation radical with high reactivity ability [36]. The radical cation is formed from the oxidation reaction between ABTS and buffer solution especially using K 2 S 2 O 8 . Furthermore, the mechanism of radical scavenging as well as DPPH's mechanism is hydrogen donors [35]. Thus, the antioxidant activity for both of the two assays is evaluated by using UV-Vis Genesys Thermo Scientific spectrophotometer.
According to this study, there is a linear relationship between the antioxidant activity and total phenolic content. This evidence means that the higher the total phenolic content, the higher the antioxidant activity. Among the five tested extracts, the ethyl acetate extract exhibited the highest antioxidant activity against either DPPH or ABTS because of the high amount of total phenolic content. Not only the ethyl acetate extract, but also the dichloromethane, methanol, and water extracts, showed fine antioxidant activity as well as the ethanol and chloroform extracts reported previously [27,28]. However, there was only weak activity in the nhexane extract. These results suggest the presence of phenolic compounds could be major contributor to antioxidant activity. The phenolic compounds including xanthone [37] or stilbene [38] have been reported as a potent antioxidant activity. Based on this study, the phenolic compounds of C. odorata could be extracted by the polar and semipolar solvents very well. When the methanol extract was fractionated and elucidated, the major secondary metabolite came from flavanone compounds which is one of the phenolic groups. The finding of a new flavanone, odoratenin (1), indicates the presence of two hydroxyls and two methoxyl group. They might be donated and the hydrogen atom also supported the electron conjugation system from the phenolic ring for stabilizing the free radicals. Interestingly, the new compound odoratenin (1) has higher antioxidant activity than that of trolox as a standard. The present study and these results reveal odoratenin (1) isolated from C. odorata as a potent antioxidant source.

-Glucosidase
Inhibitory Activity of C. odorata. Diabetes mellitus is a metabolic disorder caused by a lack of insulin [39]. Insulin helps the blood glucose level to be a normal circumstance and not turn into hyperglycemia or hypoglycaemia. According to the type of an abnormal insulin, diabetes mellitus is divided into two types [40]. The first type is known as insulin dependent diabetes mellitus (IDDM) caused by a genetic factor such as the destruction of pancreatic -cells which produce insulin and type 2 is non-insulin dependent diabetes (NIDDM) caused by a wrong lifestyle especially on diet. This study focuses on the effective treatment for type 2. As we know, carbohydrates are the major components of our daily foods, for instance, polysaccharides or disaccharides. After carbohydrates intake, the amount of polysaccharides is transformed into monosaccharides as known as the simple sugars, and then they are transferred through the bloodstream system for energy [41]. However, before they are transferred, they are absorbed on the intestine. In the small intestinal tissue, there is a catalyse of the cleavage of polysaccharides to glucose, namely, -glucosidase. It makes the total of glucose too large. Certainly, the increasing of glucose level as known as hyperglycemia in the blood is not good enough for health [42,43]. Related to hyperglycemia,glucosidase inhibitor is recommended as antidiabetic [44]. In this assay system, the rat intestinal acetone powder has been used as enzyme to determine the antidiabetic inhibitory activity of C. odorata extracts. The tested extracts might inhibit or compete with maltose as a substrate. Based on our work, there is a linear correlation between -glucosidase inhibition and antioxidant activity. The ethyl acetate and methanol extracts performed a fine inhibitory activity. This result as well as the antioxidant activity screening previously reported that these two extracts have a good radical scavenging activity also against both DPPH and ABTS radicals. Furthermore, the isolated compound from C. odorata reported a significantly higher -glucosidase inhibitory activity than that of deoxynojirimycin and acarbose as the standards in previous research [24]. From this study, it should be noted that the -glucosidase inhibitory effect of the ethyl acetate showing that the vicinal-geminal protons were attached to C-3. Furthermore, five aromatic protons were placed at C-6, C-8, C-2' , C-4' , and C-5' . They have hydrogen-to-carbon correlations between H-6/C-4, C-7, C-9, C-10; H-8/C-6, C-7, C-9, C-10; H-2'/C-2, C-1' , C-4'; H-4'/C-1'; H-5'/C-2, C-2' , C-3' , C-6' which were also confirmed by HMQC spectrum as Figure 4(b). Accordingly, both hydroxyl and methoxy substituents were assigned as 3'-hydroxy [9,13] and 8methoxy [14] at C-3' and C-8. Interestingly, although more than 79 flavonoid compounds have been isolated from the genus Chromolaena [8], the methoxy substituent at C-6' has not been reported yet before. Accordingly, the compound of (1) is a new flavanone named odoratenin (1) as in Figure 5 (1).
Isosakuranetin (2) is a white needles solid powder with a melting point of 173-174 ∘ C. The elucidation process of isosakuranetin (2) was determined as these following steps. First, the FT-IR data showed the strong intensity of peaks as follows, ] maks (KBr): 3504,2955,1639,1599,1518,1492,1302,1253,1163, and 833 cm −1 . The peaks of 3504 cm −1 with medium intensity and 1639 cm −1 with strong intensity revealed the presence of a hydroxyl (-OH) group chelated with carbonyl group (-C=O). In addition, the peaks of 1518, 1492, and 833 cm −1 with medium to weak intensities indicated the presence of a conjugated aromatic group. Furthermore, this information was confirmed by data 1 H and 13 C-NMR (CDCl 3 , 400 MHz) presented in Table 2. Based on the presented data, there are a number of detected chemical shifts as 14 protons and 16 carbons. The 14 proton signals including the singlet signal of H 12.04 ppm with integration in the downfield area indicated the presence of one proton as 5-OH. This proton is deshielded because it bonds with O atom which has more electrons directly to be a hydroxyl group and also as a typical signal at the same time. The typical signal means a hydroxyl proton chelated with a carbonyl group. So, the proton has a far chemical shift. Next, a strong singlet signal of H 3.83 ppm with three integration processes in the upfield area showed three protons as 4'-OCH 3 . They are shielded because they do not bond with O atom directly. So, these three protons have near chemical shifts and also they are suspected strongly to be protons from the methoxy group. Furthermore, three signals with doublet multiplicity at H 5.36 (H-2), 2.78 (H-3a), and 3.09 ppm (H-3b) coupling with the vicinal-geminal proton system indicated the presence of a pyran group [45]. This proton system indicated strongly that compound (2) is one of the flavanone groups which is similar to a previous known compound reported by Suksamrarn et al. (2004) [13]. In addition, the doublet signals of H 7.37 (H-2'/6') and 6.95 ppm (H-3'/5') coupling with each other with double intensity showed four proton signals indicating the protons of an aromatic group. Based on our results, compound (2) has a flavanone skeleton with an ABC ring system substituted with the methoxy and hydroxyl groups as Figure 5 (2). Furthermore, two signal doublets of H 5.97 and 5.99 ppm also coupling with each other are strongly suspected as two signals of aromatic potons as H-6 and H-8 on ring A. The determination of the structure of compound (2) is confirmed with 13 C-NMR data. Based on our 13 C-NMR data, there are 16 carbons including a carbon carbonyl group which is strongly expected as a position of C-4 ( C 196.18 ppm), one chiral carbon assumed as a position of C-2 ( C 79.10 ppm), one carbon methoxy at position of C-4' ( C 55.48 ppm), and the aromatic carbon expected as the position of C-2'/6' ( C 114.33 ppm) and C-3'/5' ( C 127.85 ppm) with double intensity, respectively. And the other aromatic carbons including C 95.54, 96.76, 103.20, 130.36, 160.14, 163.36, 164.53, and 164.59 ppm. Based on this elucidation study, compound (2) is an isosakuranetin (2) as in Figure 5 (2) which was also isolated by Suksamrarn et al. (2004).
Subscandenin (3) is yellow needles solid powder with a melting point of 174-175 ∘ C. Compound (3) is strongly expected as subscandenin which is one of the derivatives from flavanone compounds with a skeleton similar to compounds (1-2). The structure is confirmed by the interpretation of 1 H, 13 C-NMR, and HMBC data (CDCl 3 , 400 MHz) presented in Table 2. The results of the NMR characterization showed characters that are similar to the NMR characterization of compounds (1-2). Based on the presented data, there are a number of detected chemical shifts as 16 protons and 17 carbons. This total of protons and carbons is equal to the total of compound (1). The 1 H NMR spectrum (Table 2)

Conclusion
C. odorata, collected from East Indonesia, contributes to drug discovery and healthcare. This is the first report on the antioxidant activity of a new flavanone isolated from the C. odorata leaves methanol extract. Among the tested five extracts, the ethyl acetate extract exhibited the highest inhibitory effect against ABTS radical and -glucosidase rat intestinal enzyme. Further investigations will focus on the identification of the other active flavanone compounds responsible for the antioxidant as well as -glucosidase inhibitory activity of C. odorata leaves ethyl acetate extract.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
All authors declare that there are no conflicts of interest regarding the publication of this article.