# Antioxidant and Anti-Inflammatory Activities of Phytochemicals fromRuellia tuberosa

Authors: Trinh Nhat Thi Pham; Tuan Trong Nguyen; Thuy Le Thi Nguyen; An Minh Nguyen Tran; Tuan Ngoc Nguyen; Danh Thanh Tong; Dung Tien Le
Journal: Journal of Chemistry
 (2022)

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## Abstract

In Vietnam, the aerial parts ofRuellia tuberosa L. are used to treat stress oxidation and inflammatory symptoms in diabetes mellitus. The present study was designed to determine the antioxidant and inflammatory inhibition activities of Ruellia tuberosa L. extract (RTE) and those of the isolated compounds by column chromatography. The crude ethanol extract and ethyl acetate fraction exhibited potent antioxidant activity in the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) assays with an IC50 of 25.18 and 14.71 (DPPH test) and 18.22 and 15.27 µg/ml (ABTS test), respectively. The RTE contained high concentrations of polyphenols (308.21 mg GAE/g) and moderate concentrations of flavonoids (97.80 mg QE/g). In the anti-inflammatory screening assay, the crude ethanol extract, ethyl acetate, and methanol fractions suppressed the release of IL-6 and nitric oxide production, but the production of IL-10 was not enhanced in LPS-induced RAW 264.7 cells. Three potential anti-inflammatory compounds as hispidulin (6), physalin E (7), and physalin D (8) along with five other compounds named myricitrin (1), afzelin (2), apigenin (3), taraxerol (4), and lupeol (5) were isolated and identified from the ethyl acetate fraction. Physalin D (8) exhibited a strong, dose-dependent anti-inflammatory activity by inhibiting the production of IL-6 proinflammatory cytokines; however, the IL-10 expression was independent of its concentration in macrophages at noncytotoxic concentrations ranging from 5 to 40 μg/mL. Based on the data obtained, compounds 6–8 sourced from Ruellia tuberosa L are potentially bioactive compounds for the treatment of inflammation symptoms in type 2 diabetes mellitus.


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## Body

## 1. Introduction

Diabetes mellitus is a chronic disease that is related to high plasma glucose because of a shortage of insulin production, insulin action, or both. The goal of type 2 diabetes treatment is to regulate blood glucose levels at normal or near-normal levels [1]. Recent studies have revealed a relationship between diabetes mellitus and secondary complications manifesting from chronic inflammation in people with diabetes. Therefore, in addition to lowering plasma glucose levels, hypoglycemic agents reduce inflammatory symptoms, thus leading to better outcomes [2]. Inflammation is considered an important etiological factor in the development of both types 1 and 2 diabetes mellitus [3]. Moreover, high blood glucose levels promote the autooxidation of glucose to form free radicals, which cause diabetes [4]. An excessive increase in reactive oxygen species or reactive nitrogen species accompanied by a decrease in antioxidants induces oxidative stress, which can lead to endothelial dysfunction, insulin resistance, and alterations in the number of reactive oxygen species and function of pancreatic beta cells [5]. Antioxidants can slow or inhibit cell damage due to their free radical scavenging activity [6]. Therefore, phytochemicals with hypoglycemic, anti-inflammatory, and antioxidant activities are suited for the treatment of diabetes.Traditional medicines, which utilize medicinal plants, are used by most of the global populace to treat various diseases.Ruellia tuberosa L., a Vietnamese medicinal plant, has been used for the treatment of diabetes mellitus as an anti-inflammatory and diuretic agent [7]. The aerial part of this plant exhibited significant hypoglycemic effects in alloxan-induced diabetic rats and rabbits [8, 9], and the leaf extract is a good inhibitor of the lipoxygenase enzyme [10]. Five flavonoids, including cirsimaritin, cirsimarin, cirsiliol 4′-glucoside, sorbifolin, and pedalitin, as well as betulin, vanillic acid, and indole-3-carboxaldehyde, were isolated from the ethyl acetate extract of R. tuberosa L. [11]. Seven compounds, which are apigenin and luteolin derivatives, were also isolated from R. tuberosa L. [12, 13]. As part of our continuing research on the biological activities and phytochemicals of medicinal plants [14–16], this study isolated potential anti-inflammatory compounds from R. tuberosa extract and evaluated their antioxidant and inflammatory inhibition activities.


## 2. Materials and Methods

### 2.1. Chemicals

2,2-Diphenyl-1-picryl-hydrazyl-hydrate (DPPH, Cat. No. 257621), 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS, Cat. No. 10102946001), potassium persulfate (Cat. No. 216224), trifluoroacetic acid (Cat. No. 302031), ascorbic acid (Cat. No. 57803), gallic acid (Cat. No. G7384), quercetin (Cat. No. PHL89262), Trolox (Cat. No. 238813), and the Folin–Ciocalteu reagent (Cat. No. 47641) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Solvents including hexane (Cat. No. 1043742500), chloroform (Cat. No. 1024322500), ethyl acetate (Cat. No. 1007892500), ethanol (Cat. No. 1009744000), and methanol (Cat. No. 1060182500) were obtained from Merck (Darmstadt, Germany). Standard solutions were prepared using deionized water. Dulbecco’s modified Eagle’s medium (Cat. No. 11995040), 0.25% trypsin-EDTA (Cat. No. 25200072), penicillin-streptomycin (Cat. No. 15070063), sodium pyruvate (Cat. No. 11360070), and fetal bovine serum (Cat. No. A3840001) were purchased from Thermo Scientific (Waltham, MA, USA). Enzyme-linked immunosorbent assay reagents for IL-6 (Cat. No. 88-7064-88) and IL-10 (Cat. No. 88-7105-77) cytokine were obtained from eBioscience (San Diego, CA, USA). Lipopolysaccharide (Cat. No. L7770), dimethyl sulfoxide (Cat. No. 276855), Na2CO3 (Cat. No. 222321), AlCl3 (Cat. No. 563919), and phosphate buffered saline pH 7.4 (Cat. No. 806552) were purchased from Sigma-Aldrich Co. (Mumbai, India).


### 2.2. Plant Material

The aerial part ofRuellia tuberosa L. was collected in Tien Giang Province, Vietnam, in July 2017. A voucher specimen (D072017) was preserved at the Department of Pharmaceutical Biochemistry, Institute of Applied Materials Science (VAST, Ho Chi Minh City, Vietnam) by Dr. Luu Hong Truong.


### 2.3. Instruments

Melting points were determined using an IA9100 Digital Melting Point Apparatus (Cole-Parmer, United States). NMR spectra were recorded on a Bruker AM500 FTNMR spectrometer (Bruker, Karlsruhe, Germany) with an internal standard (TMS, Institute of Chemistry, VAST, Hanoi, Vietnam). Thin layer chromatography was performed on precoated silica gel 60 F254 (1.05554.0001, Merck, Darmstadt, Germany) and RP-18 F254S plates (1.15685.0001, Merck, Darmstadt, Germany). The zones on the TLC plates were detected by UV light or a solution of 10% (v/v) H2SO4 in EtOH solution. Column chromatography was performed on silica gel (240-430 mesh, Merck, Darmstadt, Germany), octadecylsilane (70-230 mesh, Merck, Darmstadt, Germany), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).


### 2.4. Extraction

The dried sample powder ofRuellia tuberosa L. (5.0 kg) was extracted with 96% ethanol at 30 C four times (4 × 30 L) and then filtered. The solvent was removed from the filtrate using a rotary evaporator, and a crude extract (RTE, 350 g) was obtained. The extract was successively partitioned into n-hexane (RTH), ethyl acetate (RTEA), and methanol (RTM) fractions using a flash silica gel column. The dried fractions (10 g) were stored in a refrigerator at 4 C to determine the antioxidant properties (total flavonoids, polyphenols, DPPH, and ABTS assays) and anti-inflammatory activities.


### 2.5. Isolation

The remaining ethyl acetate fraction was chromatographed on a silica gel column and eluted with a mixture of hexane and ethyl acetate (0 : 1 - 1 : 0, v/v) to obtain six subfractions (E1–6). Fraction E1 was rechromatographed on a silica gel column using a mixture of hexane and ethyl acetate (from 30 : 1 to 10 : 1, v/v) as the eluent and this afforded five fractions (E1.1–5). Fraction E1.1 was loaded on a silica gel column, eluted with chloroform : methanol (95 : 5 to 90 : 10, v/v), purified via an ODS column, and eluted with methanol : water (3 : 1, v/v). This resulted in7 (22 mg) and 8 (15.8 mg). Fraction E1.4 was chromatographed on a silica gel column and eluted with chloroform : methanol (70 : 1 to 10 : 1, v/v), and this afforded five subfractions (E1.4.1–5). Fraction E1.4.2 was loaded on a silica gel column and eluted with chloroform : methanol (30 : 1, v/v). This afforded 4 (11.3 mg) and 6 (8.4 mg). Fraction E1.4.3 was purified using Sephadex LH20 (chloroform : methanol = 1 : 1, v/v) and then further chromatographed on an ODS column and eluted with methanol : water (1 : 1.5, v/v), and this yielded 5 (10.5 mg).Fraction E2 was applied to a flash silica gel column and eluted with hexane : acetone (10 : 1 to 3 : 1, v/v) to yield five fractions (E2.1–5). Subfraction E2.3 was subsequently loaded onto a silica gel column and eluted with a gradient of chloroform-isopropanol (5–30%, v/v), and this yielded compound3 (9.8 mg). Fraction E2.5 was further chromatographed on an ODS column with a mixture of methanol and water (1 : 3 to 1 : 1, v/v) and afforded three fractions (E2.5.1–3). Subfraction E2.5.2 was loaded on a Sephadex LH 20 column, eluted with MeOH, and then purified on an ODS column using acetonitrile-water (1 : 2.5, v/v) and yielded compounds 1 (22.1 mg) and 2 (14.3 mg).


### 2.6. Quantification of Total Polyphenols

The polyphenol content was measured using the Folin–Ciocalteu method [17]. Briefly, a known concentration (50 µg/mL) of the extracts (1 mL) was mixed with Folin–Ciocalteu reagent (1 mL) for 5 min. A saturated Na2CO3 solution (2 mL) was subsequently added to the mixture, and the new mixture was placed in the dark for 90 min at room temperature. The absorbance was measured using a UV spectrophotometer (Shimadzu, UV-1800) at 760 nm. The polyphenolic content was expressed as mg of gallic acid equivalents per gram of dry extract (mg GAE/g).


### 2.7. Determination of Total Flavonoids

The aluminum chloride colorimetric method was used to determine the total flavonoid content of the sample [18]. Quercetin was used to establish a standard calibration curve for the experiment. A solution of quercetin was prepared by dissolving quercetin (5.0 mg quercetin in 1.0 mL methanol) and then diluted with methanol to obtain known concentrations. Quercetin and the extract solution (0.6 mL) were mixed with 2% aluminum chloride (0.6 mL) and left to stand for 60 min at room temperature. The absorbance of the mixtures was measured against a blank at 420 nm using a UV-Vis spectrophotometer. The total flavonoids content was expressed as mg quercetin equivalent (QE)/g of dried extract (mg QE/g).


### 2.8. DPPH Radical Scavenging Activity

The antioxidant activity was evaluated by scavenging the DPPH free radicals [19]. The procedure was as follows. A sample of known concentration (0.5 mL) (methanol for blank) was diluted with methanol (3.5 mL). DPPH solution (0.5 mL, 1% in methanol) was added, and the solution was shaken and left to stand in the dark for 30 min. Finally, the absorbance was measured at 517 nm using a microplate reader. The IC50 value was calculated based on the scavenging activity of the samples against 50% of DPPH radicals.


### 2.9. ABTS Radical Scavenging Activity

The ABTS radical scavenging activities were determined using the following procedure [20]. The ABTS solution was prepared by mixing ABTS (7.4  mM) with potassium persulfate (2.6  mM) and diluted with phosphate buffer saline (pH 7.4) until the absorbance of the ABTS solution was 0.70 ± 0.01(a.u.) at 734 nm on a UV 1800 spectrophotometer (Shimadzu, Japan). Thereafter, diluted ABTS solution (950 μL) was allowed to react with the sample (50 μL) for 7 min in the dark, and the absorbance was measured at 734 nm. The ABTS radical scavenging activity was expressed as the ratio of the decrease in absorbance with and without the sample.


### 2.10. Cell Culture

RAW264.7 were cultured in DMEM (Gibco-BRL, Gaithersburg, MD, USA) with 10% heat-inactivated FBS (Gibco-BRL), sodium pyruvate (1 mmol/L), penicillin (50 IU/mL), streptomycin (50 µg/mL),β-mercaptoethanol (5 × 105 mol/L), sodium pyruvate (1 mM), nonessential amino acids, penicillin G (100 IU/mL), and streptomycin (100 µg/mL) [21].


### 2.11. Cell Viability Experiment

Cell viability was assessed using a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, CCK-8 (10 µL) was added, and the cells were incubated for 60 min in a Corning Costar 48 well plate. The absorbance of the mixture was measured at 450 nm. Cell viability was expressed as a percentage of the control (untreated) cells (100% survival) [21].


### 2.12. Quantification of Cytokines

RAW 264.7 cells were treated in Corning Costar 48 well plates and stimulated with LPS (1μg/mL) in the presence or absence of the extracts for 18 h. The cell culture supernatants were analyzed to produce proinflammatory cytokine interleukin 6 (IL-6) and the anti-inflammatory cytokine interleukin 10 (IL-10) using ELISA [21].


### 2.13. Measurement of NO Content

RAW 264.7 cells were seeded in a Corning Costar 48 well plate at a density of 106 cells/well for 16 h before treatment. The cells were treated with the test agent for 60 min and activated with LPS (1 µg/mL). The culture supernatants were obtained by centrifugation, and the NO concentrations were assessed by the Griess reaction using a NaNO2 standard curve at known concentrations [21].


### 2.14. Statistical Analyses

For statistical analysis, data obtained from independent experiments were expressed as mean ± SD and were analyzed using the Student'st-test with ANOVA for multiple comparisons. Differences were considered significant at p < 0.05.


## 2.1. Chemicals

2,2-Diphenyl-1-picryl-hydrazyl-hydrate (DPPH, Cat. No. 257621), 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS, Cat. No. 10102946001), potassium persulfate (Cat. No. 216224), trifluoroacetic acid (Cat. No. 302031), ascorbic acid (Cat. No. 57803), gallic acid (Cat. No. G7384), quercetin (Cat. No. PHL89262), Trolox (Cat. No. 238813), and the Folin–Ciocalteu reagent (Cat. No. 47641) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Solvents including hexane (Cat. No. 1043742500), chloroform (Cat. No. 1024322500), ethyl acetate (Cat. No. 1007892500), ethanol (Cat. No. 1009744000), and methanol (Cat. No. 1060182500) were obtained from Merck (Darmstadt, Germany). Standard solutions were prepared using deionized water. Dulbecco’s modified Eagle’s medium (Cat. No. 11995040), 0.25% trypsin-EDTA (Cat. No. 25200072), penicillin-streptomycin (Cat. No. 15070063), sodium pyruvate (Cat. No. 11360070), and fetal bovine serum (Cat. No. A3840001) were purchased from Thermo Scientific (Waltham, MA, USA). Enzyme-linked immunosorbent assay reagents for IL-6 (Cat. No. 88-7064-88) and IL-10 (Cat. No. 88-7105-77) cytokine were obtained from eBioscience (San Diego, CA, USA). Lipopolysaccharide (Cat. No. L7770), dimethyl sulfoxide (Cat. No. 276855), Na2CO3 (Cat. No. 222321), AlCl3 (Cat. No. 563919), and phosphate buffered saline pH 7.4 (Cat. No. 806552) were purchased from Sigma-Aldrich Co. (Mumbai, India).


## 2.2. Plant Material

The aerial part ofRuellia tuberosa L. was collected in Tien Giang Province, Vietnam, in July 2017. A voucher specimen (D072017) was preserved at the Department of Pharmaceutical Biochemistry, Institute of Applied Materials Science (VAST, Ho Chi Minh City, Vietnam) by Dr. Luu Hong Truong.


## 2.3. Instruments

Melting points were determined using an IA9100 Digital Melting Point Apparatus (Cole-Parmer, United States). NMR spectra were recorded on a Bruker AM500 FTNMR spectrometer (Bruker, Karlsruhe, Germany) with an internal standard (TMS, Institute of Chemistry, VAST, Hanoi, Vietnam). Thin layer chromatography was performed on precoated silica gel 60 F254 (1.05554.0001, Merck, Darmstadt, Germany) and RP-18 F254S plates (1.15685.0001, Merck, Darmstadt, Germany). The zones on the TLC plates were detected by UV light or a solution of 10% (v/v) H2SO4 in EtOH solution. Column chromatography was performed on silica gel (240-430 mesh, Merck, Darmstadt, Germany), octadecylsilane (70-230 mesh, Merck, Darmstadt, Germany), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).


## 2.4. Extraction

The dried sample powder ofRuellia tuberosa L. (5.0 kg) was extracted with 96% ethanol at 30 C four times (4 × 30 L) and then filtered. The solvent was removed from the filtrate using a rotary evaporator, and a crude extract (RTE, 350 g) was obtained. The extract was successively partitioned into n-hexane (RTH), ethyl acetate (RTEA), and methanol (RTM) fractions using a flash silica gel column. The dried fractions (10 g) were stored in a refrigerator at 4 C to determine the antioxidant properties (total flavonoids, polyphenols, DPPH, and ABTS assays) and anti-inflammatory activities.


## 2.5. Isolation

The remaining ethyl acetate fraction was chromatographed on a silica gel column and eluted with a mixture of hexane and ethyl acetate (0 : 1 - 1 : 0, v/v) to obtain six subfractions (E1–6). Fraction E1 was rechromatographed on a silica gel column using a mixture of hexane and ethyl acetate (from 30 : 1 to 10 : 1, v/v) as the eluent and this afforded five fractions (E1.1–5). Fraction E1.1 was loaded on a silica gel column, eluted with chloroform : methanol (95 : 5 to 90 : 10, v/v), purified via an ODS column, and eluted with methanol : water (3 : 1, v/v). This resulted in7 (22 mg) and 8 (15.8 mg). Fraction E1.4 was chromatographed on a silica gel column and eluted with chloroform : methanol (70 : 1 to 10 : 1, v/v), and this afforded five subfractions (E1.4.1–5). Fraction E1.4.2 was loaded on a silica gel column and eluted with chloroform : methanol (30 : 1, v/v). This afforded 4 (11.3 mg) and 6 (8.4 mg). Fraction E1.4.3 was purified using Sephadex LH20 (chloroform : methanol = 1 : 1, v/v) and then further chromatographed on an ODS column and eluted with methanol : water (1 : 1.5, v/v), and this yielded 5 (10.5 mg).Fraction E2 was applied to a flash silica gel column and eluted with hexane : acetone (10 : 1 to 3 : 1, v/v) to yield five fractions (E2.1–5). Subfraction E2.3 was subsequently loaded onto a silica gel column and eluted with a gradient of chloroform-isopropanol (5–30%, v/v), and this yielded compound3 (9.8 mg). Fraction E2.5 was further chromatographed on an ODS column with a mixture of methanol and water (1 : 3 to 1 : 1, v/v) and afforded three fractions (E2.5.1–3). Subfraction E2.5.2 was loaded on a Sephadex LH 20 column, eluted with MeOH, and then purified on an ODS column using acetonitrile-water (1 : 2.5, v/v) and yielded compounds 1 (22.1 mg) and 2 (14.3 mg).


## 2.6. Quantification of Total Polyphenols

The polyphenol content was measured using the Folin–Ciocalteu method [17]. Briefly, a known concentration (50 µg/mL) of the extracts (1 mL) was mixed with Folin–Ciocalteu reagent (1 mL) for 5 min. A saturated Na2CO3 solution (2 mL) was subsequently added to the mixture, and the new mixture was placed in the dark for 90 min at room temperature. The absorbance was measured using a UV spectrophotometer (Shimadzu, UV-1800) at 760 nm. The polyphenolic content was expressed as mg of gallic acid equivalents per gram of dry extract (mg GAE/g).


## 2.7. Determination of Total Flavonoids

The aluminum chloride colorimetric method was used to determine the total flavonoid content of the sample [18]. Quercetin was used to establish a standard calibration curve for the experiment. A solution of quercetin was prepared by dissolving quercetin (5.0 mg quercetin in 1.0 mL methanol) and then diluted with methanol to obtain known concentrations. Quercetin and the extract solution (0.6 mL) were mixed with 2% aluminum chloride (0.6 mL) and left to stand for 60 min at room temperature. The absorbance of the mixtures was measured against a blank at 420 nm using a UV-Vis spectrophotometer. The total flavonoids content was expressed as mg quercetin equivalent (QE)/g of dried extract (mg QE/g).


## 2.8. DPPH Radical Scavenging Activity

The antioxidant activity was evaluated by scavenging the DPPH free radicals [19]. The procedure was as follows. A sample of known concentration (0.5 mL) (methanol for blank) was diluted with methanol (3.5 mL). DPPH solution (0.5 mL, 1% in methanol) was added, and the solution was shaken and left to stand in the dark for 30 min. Finally, the absorbance was measured at 517 nm using a microplate reader. The IC50 value was calculated based on the scavenging activity of the samples against 50% of DPPH radicals.


## 2.9. ABTS Radical Scavenging Activity

The ABTS radical scavenging activities were determined using the following procedure [20]. The ABTS solution was prepared by mixing ABTS (7.4  mM) with potassium persulfate (2.6  mM) and diluted with phosphate buffer saline (pH 7.4) until the absorbance of the ABTS solution was 0.70 ± 0.01(a.u.) at 734 nm on a UV 1800 spectrophotometer (Shimadzu, Japan). Thereafter, diluted ABTS solution (950 μL) was allowed to react with the sample (50 μL) for 7 min in the dark, and the absorbance was measured at 734 nm. The ABTS radical scavenging activity was expressed as the ratio of the decrease in absorbance with and without the sample.


## 2.10. Cell Culture

RAW264.7 were cultured in DMEM (Gibco-BRL, Gaithersburg, MD, USA) with 10% heat-inactivated FBS (Gibco-BRL), sodium pyruvate (1 mmol/L), penicillin (50 IU/mL), streptomycin (50 µg/mL),β-mercaptoethanol (5 × 105 mol/L), sodium pyruvate (1 mM), nonessential amino acids, penicillin G (100 IU/mL), and streptomycin (100 µg/mL) [21].


## 2.11. Cell Viability Experiment

Cell viability was assessed using a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, CCK-8 (10 µL) was added, and the cells were incubated for 60 min in a Corning Costar 48 well plate. The absorbance of the mixture was measured at 450 nm. Cell viability was expressed as a percentage of the control (untreated) cells (100% survival) [21].


## 2.12. Quantification of Cytokines

RAW 264.7 cells were treated in Corning Costar 48 well plates and stimulated with LPS (1μg/mL) in the presence or absence of the extracts for 18 h. The cell culture supernatants were analyzed to produce proinflammatory cytokine interleukin 6 (IL-6) and the anti-inflammatory cytokine interleukin 10 (IL-10) using ELISA [21].


## 2.13. Measurement of NO Content

RAW 264.7 cells were seeded in a Corning Costar 48 well plate at a density of 106 cells/well for 16 h before treatment. The cells were treated with the test agent for 60 min and activated with LPS (1 µg/mL). The culture supernatants were obtained by centrifugation, and the NO concentrations were assessed by the Griess reaction using a NaNO2 standard curve at known concentrations [21].


## 2.14. Statistical Analyses

For statistical analysis, data obtained from independent experiments were expressed as mean ± SD and were analyzed using the Student'st-test with ANOVA for multiple comparisons. Differences were considered significant at p < 0.05.


## 3. Results and Discussion

### 3.1. Antioxidant Activities

The antioxidant capacities of three fractions (n-hexane, ethyl acetate, and methanol) and the crude ethanol extract are listed in Table 1. Aside from the crude ethanol extract, the ethyl acetate fraction exhibited stronger antioxidant activity than the other fractions, exhibiting an IC50 value of 12.29 and 14.70 μg/mL in the DPPH and ABTS experiments, respectively. The antioxidant activity conforms with the high concentration of the total flavonoids (TFs) and polyphenols (TPs) of the ethyl acetate fraction (56.07 mg QE/100 g and 259.12 mg GAE/100 g). Free radicals and oxidants are known to enact a significant dual role as both toxic and beneficial reagents. The imbalance between free radicals and antioxidants (in favor of radicals) in living humans is called oxidative stress, and it causes degenerative diseases such as atherosclerosis, cancer, arthritis, diabetes, and aging [22–24]. Excessive generation of free radicals can trigger inflammation in type 2 diabetes mellitus [25]. In addition, the oxidative stress and inflammation can cause development of diabetic complications [26]. Therefore, antioxidants and anti-inflammatory compounds from plants may play a major role in preventing and treating these complications [27]. Many studies have showed that flavonoids and phenolics appeared to be the good antioxidants [28–31]. In addition to the antioxidant effects, they also exhibited anti-inflammatory activity [32, 33]. The results of this study indicate that the crude ethanol extract and ethyl acetate fraction are potential sources of antioxidant compounds and Ruellia tuberosa may help prevent type 2 diabetes mellitus.Table 1
The antioxidant activity, total polyphenols (TPs), and total flavonoids (TFs) content of crude ethanol extract and its derived fractions.
SampleIC50 (μg/mL)Content (mg/100 g dry sample)DPPHABTS•+PolyphenolsFlavonoidsCrude ethanol extract20.65 ± 3.12∗13.93 ± 2.18∗308.21 ± 4.72c97.8 ± 10.25an-Hexane fraction96.82 ± 5.65∗71.82 ± 5.39∗21.45 ± 2.05a15.75 ± 1.91cEthyl acetate fraction12.69 ± 2.35∗14.70 ± 2.25∗259.12 ± 1.42b56.07 ± 2.23bMethanol fraction62.93 ± 4.17∗49.34 ± 3.62∗158.41 ± 9.47d71.82 ± 3.76cVitamin C (positive control)3.66 ± 0.28—Trolox (positive control)—2.90 ± 0.03Values represent the mean of three replicates (n = 3) ± SD. Mean values followed by the different superscripts (a–d) in a column are significantly different (p < 0.05).∗p < 0.05 vs. positive control.


### 3.2. Effect of RTFs on Cell Viability

To examine the effect ofRuellia tuberosa fractions (RTFs) on inflammation in LPS-induced RAW 264.7 cells, ELISA and nitric oxide (NO) quantitation were performed with and without these fractions. The cytotoxic effect of the fractions and dexamethasone (Dex) on the cells were examined using the Cell Counting Kit-8 (CCK-8) assay. The cells were treated with various RTFs at different concentrations (5–40 µg/mL) or Dex (100 nmol/L) for the indicated times. The response of cell viability was represented by the cell viability index, in which the number of RAW264.7 cells cultured at 0 h was used as a reference point. Interestingly, the number of cells in all treatments indicated the proliferation of RAW264.7 cells after 18 h of exposure. Collectively, these results support the safety of all the fractions at the concentrations examined in RAW264.7 cells. Thus, the concentrations of RTFs in the 5–40 µg/mL range and Dex (100 nmol/L) were used for subsequent inflammatory assays (Figure 1).Figure 1
Effect of (a) extract, (b) compounds 1–4, and (c) compounds 5–8 on RAW 264.7 macrophage cell viability. Cells were incubated for 24 h with the indicated concentrations (5, 10, 20, 30, and 40μg/mL). Data are expressed as mean ± S.D. of three independent experiments. RTH: n-hexane fraction; RTEA: ethyl acetate fraction. RTM: methanol fraction; RTE: crude ethanol extract.
(a)(b)(c)


### 3.3. Effect of RTFs on NO Production in LPS-Induced RAW 264.7 Cells

To assess the inflammatory ability of all fractions, LPS-induced inflation in macrophages was used as anin vitro model in this study. Treatment with LPS (1 µg/mL) alone induced a nonsignificant increase in the viability of RAW264.7 cells compared with noninduced RAW264.7 cells. Thus, LPS (1 µg/mL) was used to activate RAW264.7 cells in this study. The RAW 264.7 cells were treated with various concentrations (5–40 μg/mL) of RTFs, and inflammation was stimulated by LPS. The data indicated that NO generation in LPS-induced cells was significantly inhibited in a dose-dependent manner by the ethanol extract and the different fractions compared with LPS control cells (p < 0.05) (Figure 2). At a concentration of 40 µg/mL, the ethyl acetate and methanol fractions decreased the NO production by 45.63% and 48.76% (p < 0.05), respectively, compared with that of the LPS-treated cells. The positive control, Dex (100 nmol/L), decreased NO production by 51.83% (p < 0.05). The results indicated that the two potentially effective extracts were the ethyl acetate and methanol fractions.Figure 2
Effects of (a) extract and (b) compounds1–4 and (c) 5–8 on the production of NO in LPS-induced RAW264.7 macrophages. Cells were pretreated for 2 h with the indicated concentrations (5, 10, 20, 30, and 40 μg/mL) and then stimulated with LPS (1 μg/mL) for another 24 h. Data are expressed as mean ± S.D. of three independent experiments. ∗p <  0.05 versus the LPS group; #p <  0.05 versus the positive group. RTH: (n)-hexane fraction; RTEA: ethyl acetate fraction. RTM: methanol fraction; RTE: crude ethanol extract.
(a)(b)(c)


### 3.4. Effects of RTFs on Cytokines IL-6 and IL-10 Production in RAW 264.7 Cells

After 18 h of incubating with LPS (1μg/mL), RAW264.7 cells were cultured with various concentrations (5, 10, 20, 30, and 40 μg/mL) of RTFs for 45 min following the determination by the ELISA assay of proinflammatory cytokines (IL-6) and anti-inflammatory cytokines (IL-10) in macrophage cells according to the manufacturer’s protocol. Both IL-6 and lL-10 were strongly expressed in LPS-activated RAW264.7 cells (Figures 3(a) and 3(b)). The expression levels of IL-6 and IL-10 in the native control RAW264.7 cells were 0.03 ± 0.0027 ng/mL and 0.078 ± 0.0048 ng/ml, respectively, which increased significantly to 1.20 ± 0.0148 ng/mL (p < 0.05) and 0.97 ± 0.0186 ng/ml (p < 0.05) in the LPS-induced RAW264.7 cells, respectively. Interestingly, coculture with RTFs suppressed the expression of IL-6 but did not increase IL-10 at all the tested concentrations. Many proinflammatory cytokines play an important role in the inflammatory response and have been shown to promote the risk of type 2 diabetes mellitus [34, 35]. Moreover, high content of anti-inflammatory and proinflammatory cytokines such as IL-6 and IL-10 is found in the plasma of type 2 diabetic patients [36, 37]. The glucose homeostasis is regulated by IL6 cytokine [38]. The studies showed that patients with type 2 diabetes had a higher level of IL-6 compared with nondiabetic persons [39–41]. Meanwhile, IL-10 cytokine possesses both immunosuppressive and antiangiogenic function [42]. The level of IL-10 in people with impaired glucose tolerance or type 2 diabetes was lower than that in healthy control [43]. The present results show that RTFs possess a good anti-inflammatory activity, so they might efficiently prevent or slow down type 2 diabetes.Figure 3
Effects on the secretion of IL-6 and IL-10 of the (a, b) extract and (c, d) compounds 6–8 in LPS-induced RAW 264.7 macrophages. Cells were pretreated for 2 h with indicated concentrations (5, 10, 20, 30, and 40 μg/mL) and then stimulated with LPS (1 μg/mL) for another 24 h. Data are expressed as mean ± S.D. of three independent experiments. ∗p < 0.05 versus the LPS group; #p < 0.05 versus the positive group. RTH: (n)-hexane fraction; RTEA: ethyl acetate fraction; RTM: methanol fraction; RTE: crude ethanol extract.
(a)(b)(c)(d)The level expression of IL-6 was reduced to 5.99 and 5.72 times (p < 0.05) after a 45 min incubation with the methanol and ethyl acetate fractions at 30 µg/mL compared with the LPS-treated control cells, respectively. The inhibition of the IL-6 production by these two fractions (at a concentration of 30 µg/mL) was equivalent to that of the positive control (p < 0.05). The hexane fraction and ethanol extract reduced the release of IL-6 by three to four times compared with the LPS-activated cells. For IL-10, the RTFs significantly maintained the same level of IL-10 in the LPS-induced cells, although the concentration of RTFs increased from 5 µg/mL to 40 µg/mL.Collectively, the obtained data indicated that all the extracts and fractions can suppress the inflammation-associated expression of IL-6 in the LPS-stimulated RAW 264.7 cells. However, the tendency of IL-6 was upregulated with an increase in the RTF concentration (>5µg/mL), whereas the IL-10 expression was independent of the RTF concentration. Specifically, the strongest inflammatory inhibition was observed in the methanol and ethyl acetate fractions. This suggests that there may have been active compounds present in the those fractions that exhibited counteracting effects [44–46].


### 3.5. Isolation of Potentially Anti-Inflammatory Compounds

The anti-inflammatory results indicated that ethyl acetate and methanol were the potential fractions that would contain the anti-inflammatory compounds. These fractions exhibited the highest activity in the downregulation of the proinflammatory cytokine IL-6 secretion and NO production. Moreover, the ethyl acetate fraction contained the highest concentration of phenolic compounds and exhibited the strongest radical scavenging activities in the DPPH and ABTS assays compared with the other fractions. Therefore, the ethyl acetate fraction was chosen for further isolation and characterization of the bioactive compounds [45, 46]. Eight compounds were isolated and identified after repeated chromatography of this fraction using normal and reversed phase silica gel and Sephadex LH-20 as adsorbents (Figure 4). These compounds were subsequently characterized as myricitrin (1) [47], afzelin (2) [48], apigenin (3) [49], taraxerol (4) [50], lupeol (5) [51], hispidulin (6) [52], physalin E (7) [53], and physalin D (8) [54], based on the detailed analysis (Table 2) of the NMR spectra (Figures S1–S16) and the published data. This is the first time that compounds 7 and 8 were isolated from the Ruellia genus. Compounds 1–6 are known to be present in R. tuberosa.Figure 4
Chemical structure of isolated compounds. (a) Myricitrin (1): R1=R2=R4=H; R3 = O-Rhamnosyl. Afzelin (2): R1=R2=R4=H; R3=O-Rhamnosyl. Apigenin (3): R1=R2=R3=R4=H. Hispidulin (6): R1=R2=R3=H; R4=OCH3. (b) Taraxerol (4). (c) Lupol (5). (d) Physalin E (7) R1=H, R2=OH Physalin E (8) R1=OH, R2=H.Table 2
1H NMR (500 MHz) and 13C NMR (125 MHz) NMR spectral data of compounds 4, 5, 7, and 8 in CDCl3 and 1-3 and 6 in CD3OD.
Pos.Cpd. 1 (CD3OD)MyricitrinCpd. 2 (CD3OD)AfzelinCpd. 3 (CD3OD)ApigeninCpd.6 (CD3OD)HispidulinδH ppm (J, Hz)δC ppmδH ppm (J, Hz)δC ppmδH ppm (J, Hz)δC ppmδH ppm (J, Hz)δC ppm2159.5159.3166.3165.13136.3136.26.61 (1H, s)103.86.65 (1H, s)103.54179.7179.6183.9183.55163.2163.2163.2153.966.22 (1H,d, 2.0)99.86.22 (1H, d, 2)99.96.23 (1H, d, 2.0 Hz)100.1132.17165.9166.0166.0157.786.39 (1H,d, 2.0)94.76.39 (1H, d, 2)94.86.48 (1H, d, 2.0 Hz)95.06.63 (1H, s)94.69158.5158.6159.4153.910105.9105.9105.3105.71′121.9122.7123.3123.12′6.97 (1H,s)109.67.77 (1H, d, 9.0)131.97.87 (1H, d, 7.0)129.47.97 (1H, d, 8.5)129.23′146.96.95 (1H, d, 8.5)116.56.95 (2H, d, 7.0)117.07.07(1H, d, 8.5)116.84′137.9161.6162.7161.95′146.96.95 (1H, d, 8.5)116.56.95 (2H, d, 7.0)117.07.07(1H, d, 8.5)116.86′6.97 (1H,s)109.67.77 (1H, d, 9.0)131.97.87 (1H, d, 7.0)129.47.97 (1H, d, 8.5)129.2OCH33.80 (3H, s)60.6Rha unit1″5.34 (1H,d, 1.5)103.65.39 (1H, d, 1.5)103.52″4.23 (1H,s)71.94.24 (1H;dd; 3.5; 1.5)72.03″3.81 (1H,dd, 9.3; 3.3)72.13.73 (1H;dd; 9.0; 3.5)72.14″3.33 (1H,m)73.33.34 (1H;m)73.25″3.54 (1H,m)72.03.34 (1H;m)71.96″0.99 (3H,d, 6.0)17.70.94 (3H;d; 5.5)17.6Pos.Cpd 4 (CDCl3)TaraxerolCpd 5 (CDCl3)LupeolδH ppm (J, Hz)δC ppmδH ppm (J, Hz)δC ppm11.63 (2H,m)37.738.821.61 (2H,m)27.11.62 (2H,m)27.533.19 (1H,dd, 10.5 và 3.5)79.03.19 (1H,t, 10.5)79.0438.738.950.78 (1H,dd, 11.5 và 2.5)55.50.67 (1H,t, 9.8)55.461.62 (1H,m); 1.47 (1H, m)18.81.38 (2H,m)18.472.03 (1H,td, 12.5 and 3.0);1.38 (1H, m)41.334.3839.040.991.40 (1H,m)49.31.25 (1H,m)50.51037.537.2111.63 (1H,m); 1.49 (1H, m)17.521.0121.32 (1H,m); 0.97 (1H, m)37.725.21335.838.114158.142.9155.53 (1H,dd, 3.5 and 8.5)116.827.5161.91 (1H,dd, 14.5 and 3.0)1.58 (1H, m)36.735.61738.043.0180,96 (1H,m)48.71.35 (1H,m)48.4191.38 (1H,m); 1.02 (1H, m)35.12.38 (1H,m)48.02028.8151.0211.62 (1H,m); 1.54 (1H, m)33.729.9221.38 (1H,m); 1.27 (1H, m)33.140.0230.97 (3H,s)28.00.98 (3H,s)28.0240.80 (3H,s)15.40.76 (3H,s)15.4250.92 (3H,s)15.40.83 (3H,s)16.1260.82 (3H,s)29.81.2 (3H,s)16.0271.09 (3H,s)25.90.99 (3H,s)14.6280.91 (3H,s)29.90.79 (3H,s)18.0290.95 (3H,s)33.34.57 (1H, s)4.69 (1H,s)109.3300.91 (3H,s)21.31.68 (3H, s)19.3Pos.Cpd. 7 (CDCl3) PhysalinECpd.8 (CDCl3) PhysalinDδH ppm (J, Hz) 500 MHzδC ppm 125 MHzδH ppm (J, Hz)500 MHzδC ppm125 MHz1207.7206.625.82 (1H,dd, 10.4 and 2.4)128.65.87 (1H,dd, 10.5 and 2.0)127.536.70 (1H,dd, 10.2 and 2.8)144.46.66 (1H,dd, 10.5 and 2.0)143.243.33 (1H,d, 23.6)36.52.06 (1H,m)3.30 (1H, d, 20.0)35.35778.477.2627.63.66 (1H,d, 2.5)73.3774.82.04 (1H,m)2.06 (1H, m)38.383.27 (1H, dd, 10.4 and 8.8)39.82.44 (1H, dd, 12.0 and 3.5)35.3927.63.07 (1H,dd, 10.5 and 8.5)30.91055.654.5111.13 (1H,m, H-11)25.91.86 (1H,t, 14.0)1.17 (1H, m)25.61226.41.53 (1H,dd, 17.05 and 10.5)2.24 (1H, s)26.11380.980.614108.8107.515210.4208.2162.59 (1H,s)56.52.40 (1H,m)55.71780.980.918173.9172.3191.27 (3H,s)14.31.29 (3H,s)13.82082.280.9211.97 (3H,s)22.22.01 (3H,s)21.3224.61 (1H, t, 2.0)78.94.55 (1H,t, 2.0)77.22332.12.11 (1H,m)2.07 (1H, m)32.82431.830.4252.80 (1H,d, 4.0)51.52.48 (1H,d, 4.0)50.826170.2167.3274.43 (1H,dd, 12.8 and 4.4)3.80 (1H, d, 13.6)61.84.50 (1H,dd, 13.5 and 4.5)3.77 (1H, d, 13.5)60.5281.32 (3H,s)26.41.27 (3H, s)26.5


### 3.6. Effects of All Isolated Compounds (1–8) on NO Production in LPS-Induced RAW 264.7 Cells

The eight isolated compounds were further evaluated for their anti-inflammatory activity via NO production in the LPS-induced RAW 264.7 cells. Three compounds6–8 exhibited a strong inhibitory effect on NO generation with the percentage inhibition ranging from 35.67% to 59.20% at a concentration of 40 μg/mL (Figures 2(b) and 2(c)), compared with that of the LPS control cells (p < 0.05). Compound 3 exhibited moderate anti-inflammatory activity at all the evaluated concentrations (p < 0.05). Meanwhile, the four remaining compounds (1–3, 5) exhibited weak activity for the inhibition of NO production.


### 3.7. Effects of Isolated Compounds on Cytokines IL6 and IL10 Production in RAW 264.7 Cells

Compounds6–8 exhibited good NO inhibition activity, which required further investigation. The protein expression of IL-6 and IL-10 increased significantly after stimulation with LPS (p < 0.05). Compared with the LPS-treated control cells, the positive control Dex (1 μM) significantly suppressed the IL6 protein expression by a factor of 4.73 (p < 0.05), while physalin D (8) was the most effective compound, suppressing IL-6 secretion by a factor of 14 at a concentration of 20 μg/mL (p < 0.05). In addition, the level of expression of IL6 decreased by a factor of 3 and 8 (concentration dependent), by compounds 6 and 7, compared with the LPS control (Figure 3(c)). These compounds did not affect the secretion of IL10, and the level of the anti-inflammatory cytokine IL10 was maintained and independent of its concentration (Figure 3(d)).


## 3.1. Antioxidant Activities

The antioxidant capacities of three fractions (n-hexane, ethyl acetate, and methanol) and the crude ethanol extract are listed in Table 1. Aside from the crude ethanol extract, the ethyl acetate fraction exhibited stronger antioxidant activity than the other fractions, exhibiting an IC50 value of 12.29 and 14.70 μg/mL in the DPPH and ABTS experiments, respectively. The antioxidant activity conforms with the high concentration of the total flavonoids (TFs) and polyphenols (TPs) of the ethyl acetate fraction (56.07 mg QE/100 g and 259.12 mg GAE/100 g). Free radicals and oxidants are known to enact a significant dual role as both toxic and beneficial reagents. The imbalance between free radicals and antioxidants (in favor of radicals) in living humans is called oxidative stress, and it causes degenerative diseases such as atherosclerosis, cancer, arthritis, diabetes, and aging [22–24]. Excessive generation of free radicals can trigger inflammation in type 2 diabetes mellitus [25]. In addition, the oxidative stress and inflammation can cause development of diabetic complications [26]. Therefore, antioxidants and anti-inflammatory compounds from plants may play a major role in preventing and treating these complications [27]. Many studies have showed that flavonoids and phenolics appeared to be the good antioxidants [28–31]. In addition to the antioxidant effects, they also exhibited anti-inflammatory activity [32, 33]. The results of this study indicate that the crude ethanol extract and ethyl acetate fraction are potential sources of antioxidant compounds and Ruellia tuberosa may help prevent type 2 diabetes mellitus.Table 1
The antioxidant activity, total polyphenols (TPs), and total flavonoids (TFs) content of crude ethanol extract and its derived fractions.
SampleIC50 (μg/mL)Content (mg/100 g dry sample)DPPHABTS•+PolyphenolsFlavonoidsCrude ethanol extract20.65 ± 3.12∗13.93 ± 2.18∗308.21 ± 4.72c97.8 ± 10.25an-Hexane fraction96.82 ± 5.65∗71.82 ± 5.39∗21.45 ± 2.05a15.75 ± 1.91cEthyl acetate fraction12.69 ± 2.35∗14.70 ± 2.25∗259.12 ± 1.42b56.07 ± 2.23bMethanol fraction62.93 ± 4.17∗49.34 ± 3.62∗158.41 ± 9.47d71.82 ± 3.76cVitamin C (positive control)3.66 ± 0.28—Trolox (positive control)—2.90 ± 0.03Values represent the mean of three replicates (n = 3) ± SD. Mean values followed by the different superscripts (a–d) in a column are significantly different (p < 0.05).∗p < 0.05 vs. positive control.


## 3.2. Effect of RTFs on Cell Viability

To examine the effect ofRuellia tuberosa fractions (RTFs) on inflammation in LPS-induced RAW 264.7 cells, ELISA and nitric oxide (NO) quantitation were performed with and without these fractions. The cytotoxic effect of the fractions and dexamethasone (Dex) on the cells were examined using the Cell Counting Kit-8 (CCK-8) assay. The cells were treated with various RTFs at different concentrations (5–40 µg/mL) or Dex (100 nmol/L) for the indicated times. The response of cell viability was represented by the cell viability index, in which the number of RAW264.7 cells cultured at 0 h was used as a reference point. Interestingly, the number of cells in all treatments indicated the proliferation of RAW264.7 cells after 18 h of exposure. Collectively, these results support the safety of all the fractions at the concentrations examined in RAW264.7 cells. Thus, the concentrations of RTFs in the 5–40 µg/mL range and Dex (100 nmol/L) were used for subsequent inflammatory assays (Figure 1).Figure 1
Effect of (a) extract, (b) compounds 1–4, and (c) compounds 5–8 on RAW 264.7 macrophage cell viability. Cells were incubated for 24 h with the indicated concentrations (5, 10, 20, 30, and 40μg/mL). Data are expressed as mean ± S.D. of three independent experiments. RTH: n-hexane fraction; RTEA: ethyl acetate fraction. RTM: methanol fraction; RTE: crude ethanol extract.
(a)(b)(c)


## 3.3. Effect of RTFs on NO Production in LPS-Induced RAW 264.7 Cells

To assess the inflammatory ability of all fractions, LPS-induced inflation in macrophages was used as anin vitro model in this study. Treatment with LPS (1 µg/mL) alone induced a nonsignificant increase in the viability of RAW264.7 cells compared with noninduced RAW264.7 cells. Thus, LPS (1 µg/mL) was used to activate RAW264.7 cells in this study. The RAW 264.7 cells were treated with various concentrations (5–40 μg/mL) of RTFs, and inflammation was stimulated by LPS. The data indicated that NO generation in LPS-induced cells was significantly inhibited in a dose-dependent manner by the ethanol extract and the different fractions compared with LPS control cells (p < 0.05) (Figure 2). At a concentration of 40 µg/mL, the ethyl acetate and methanol fractions decreased the NO production by 45.63% and 48.76% (p < 0.05), respectively, compared with that of the LPS-treated cells. The positive control, Dex (100 nmol/L), decreased NO production by 51.83% (p < 0.05). The results indicated that the two potentially effective extracts were the ethyl acetate and methanol fractions.Figure 2
Effects of (a) extract and (b) compounds1–4 and (c) 5–8 on the production of NO in LPS-induced RAW264.7 macrophages. Cells were pretreated for 2 h with the indicated concentrations (5, 10, 20, 30, and 40 μg/mL) and then stimulated with LPS (1 μg/mL) for another 24 h. Data are expressed as mean ± S.D. of three independent experiments. ∗p <  0.05 versus the LPS group; #p <  0.05 versus the positive group. RTH: (n)-hexane fraction; RTEA: ethyl acetate fraction. RTM: methanol fraction; RTE: crude ethanol extract.
(a)(b)(c)


## 3.4. Effects of RTFs on Cytokines IL-6 and IL-10 Production in RAW 264.7 Cells

After 18 h of incubating with LPS (1μg/mL), RAW264.7 cells were cultured with various concentrations (5, 10, 20, 30, and 40 μg/mL) of RTFs for 45 min following the determination by the ELISA assay of proinflammatory cytokines (IL-6) and anti-inflammatory cytokines (IL-10) in macrophage cells according to the manufacturer’s protocol. Both IL-6 and lL-10 were strongly expressed in LPS-activated RAW264.7 cells (Figures 3(a) and 3(b)). The expression levels of IL-6 and IL-10 in the native control RAW264.7 cells were 0.03 ± 0.0027 ng/mL and 0.078 ± 0.0048 ng/ml, respectively, which increased significantly to 1.20 ± 0.0148 ng/mL (p < 0.05) and 0.97 ± 0.0186 ng/ml (p < 0.05) in the LPS-induced RAW264.7 cells, respectively. Interestingly, coculture with RTFs suppressed the expression of IL-6 but did not increase IL-10 at all the tested concentrations. Many proinflammatory cytokines play an important role in the inflammatory response and have been shown to promote the risk of type 2 diabetes mellitus [34, 35]. Moreover, high content of anti-inflammatory and proinflammatory cytokines such as IL-6 and IL-10 is found in the plasma of type 2 diabetic patients [36, 37]. The glucose homeostasis is regulated by IL6 cytokine [38]. The studies showed that patients with type 2 diabetes had a higher level of IL-6 compared with nondiabetic persons [39–41]. Meanwhile, IL-10 cytokine possesses both immunosuppressive and antiangiogenic function [42]. The level of IL-10 in people with impaired glucose tolerance or type 2 diabetes was lower than that in healthy control [43]. The present results show that RTFs possess a good anti-inflammatory activity, so they might efficiently prevent or slow down type 2 diabetes.Figure 3
Effects on the secretion of IL-6 and IL-10 of the (a, b) extract and (c, d) compounds 6–8 in LPS-induced RAW 264.7 macrophages. Cells were pretreated for 2 h with indicated concentrations (5, 10, 20, 30, and 40 μg/mL) and then stimulated with LPS (1 μg/mL) for another 24 h. Data are expressed as mean ± S.D. of three independent experiments. ∗p < 0.05 versus the LPS group; #p < 0.05 versus the positive group. RTH: (n)-hexane fraction; RTEA: ethyl acetate fraction; RTM: methanol fraction; RTE: crude ethanol extract.
(a)(b)(c)(d)The level expression of IL-6 was reduced to 5.99 and 5.72 times (p < 0.05) after a 45 min incubation with the methanol and ethyl acetate fractions at 30 µg/mL compared with the LPS-treated control cells, respectively. The inhibition of the IL-6 production by these two fractions (at a concentration of 30 µg/mL) was equivalent to that of the positive control (p < 0.05). The hexane fraction and ethanol extract reduced the release of IL-6 by three to four times compared with the LPS-activated cells. For IL-10, the RTFs significantly maintained the same level of IL-10 in the LPS-induced cells, although the concentration of RTFs increased from 5 µg/mL to 40 µg/mL.Collectively, the obtained data indicated that all the extracts and fractions can suppress the inflammation-associated expression of IL-6 in the LPS-stimulated RAW 264.7 cells. However, the tendency of IL-6 was upregulated with an increase in the RTF concentration (>5µg/mL), whereas the IL-10 expression was independent of the RTF concentration. Specifically, the strongest inflammatory inhibition was observed in the methanol and ethyl acetate fractions. This suggests that there may have been active compounds present in the those fractions that exhibited counteracting effects [44–46].


## 3.5. Isolation of Potentially Anti-Inflammatory Compounds

The anti-inflammatory results indicated that ethyl acetate and methanol were the potential fractions that would contain the anti-inflammatory compounds. These fractions exhibited the highest activity in the downregulation of the proinflammatory cytokine IL-6 secretion and NO production. Moreover, the ethyl acetate fraction contained the highest concentration of phenolic compounds and exhibited the strongest radical scavenging activities in the DPPH and ABTS assays compared with the other fractions. Therefore, the ethyl acetate fraction was chosen for further isolation and characterization of the bioactive compounds [45, 46]. Eight compounds were isolated and identified after repeated chromatography of this fraction using normal and reversed phase silica gel and Sephadex LH-20 as adsorbents (Figure 4). These compounds were subsequently characterized as myricitrin (1) [47], afzelin (2) [48], apigenin (3) [49], taraxerol (4) [50], lupeol (5) [51], hispidulin (6) [52], physalin E (7) [53], and physalin D (8) [54], based on the detailed analysis (Table 2) of the NMR spectra (Figures S1–S16) and the published data. This is the first time that compounds 7 and 8 were isolated from the Ruellia genus. Compounds 1–6 are known to be present in R. tuberosa.Figure 4
Chemical structure of isolated compounds. (a) Myricitrin (1): R1=R2=R4=H; R3 = O-Rhamnosyl. Afzelin (2): R1=R2=R4=H; R3=O-Rhamnosyl. Apigenin (3): R1=R2=R3=R4=H. Hispidulin (6): R1=R2=R3=H; R4=OCH3. (b) Taraxerol (4). (c) Lupol (5). (d) Physalin E (7) R1=H, R2=OH Physalin E (8) R1=OH, R2=H.Table 2
1H NMR (500 MHz) and 13C NMR (125 MHz) NMR spectral data of compounds 4, 5, 7, and 8 in CDCl3 and 1-3 and 6 in CD3OD.
Pos.Cpd. 1 (CD3OD)MyricitrinCpd. 2 (CD3OD)AfzelinCpd. 3 (CD3OD)ApigeninCpd.6 (CD3OD)HispidulinδH ppm (J, Hz)δC ppmδH ppm (J, Hz)δC ppmδH ppm (J, Hz)δC ppmδH ppm (J, Hz)δC ppm2159.5159.3166.3165.13136.3136.26.61 (1H, s)103.86.65 (1H, s)103.54179.7179.6183.9183.55163.2163.2163.2153.966.22 (1H,d, 2.0)99.86.22 (1H, d, 2)99.96.23 (1H, d, 2.0 Hz)100.1132.17165.9166.0166.0157.786.39 (1H,d, 2.0)94.76.39 (1H, d, 2)94.86.48 (1H, d, 2.0 Hz)95.06.63 (1H, s)94.69158.5158.6159.4153.910105.9105.9105.3105.71′121.9122.7123.3123.12′6.97 (1H,s)109.67.77 (1H, d, 9.0)131.97.87 (1H, d, 7.0)129.47.97 (1H, d, 8.5)129.23′146.96.95 (1H, d, 8.5)116.56.95 (2H, d, 7.0)117.07.07(1H, d, 8.5)116.84′137.9161.6162.7161.95′146.96.95 (1H, d, 8.5)116.56.95 (2H, d, 7.0)117.07.07(1H, d, 8.5)116.86′6.97 (1H,s)109.67.77 (1H, d, 9.0)131.97.87 (1H, d, 7.0)129.47.97 (1H, d, 8.5)129.2OCH33.80 (3H, s)60.6Rha unit1″5.34 (1H,d, 1.5)103.65.39 (1H, d, 1.5)103.52″4.23 (1H,s)71.94.24 (1H;dd; 3.5; 1.5)72.03″3.81 (1H,dd, 9.3; 3.3)72.13.73 (1H;dd; 9.0; 3.5)72.14″3.33 (1H,m)73.33.34 (1H;m)73.25″3.54 (1H,m)72.03.34 (1H;m)71.96″0.99 (3H,d, 6.0)17.70.94 (3H;d; 5.5)17.6Pos.Cpd 4 (CDCl3)TaraxerolCpd 5 (CDCl3)LupeolδH ppm (J, Hz)δC ppmδH ppm (J, Hz)δC ppm11.63 (2H,m)37.738.821.61 (2H,m)27.11.62 (2H,m)27.533.19 (1H,dd, 10.5 và 3.5)79.03.19 (1H,t, 10.5)79.0438.738.950.78 (1H,dd, 11.5 và 2.5)55.50.67 (1H,t, 9.8)55.461.62 (1H,m); 1.47 (1H, m)18.81.38 (2H,m)18.472.03 (1H,td, 12.5 and 3.0);1.38 (1H, m)41.334.3839.040.991.40 (1H,m)49.31.25 (1H,m)50.51037.537.2111.63 (1H,m); 1.49 (1H, m)17.521.0121.32 (1H,m); 0.97 (1H, m)37.725.21335.838.114158.142.9155.53 (1H,dd, 3.5 and 8.5)116.827.5161.91 (1H,dd, 14.5 and 3.0)1.58 (1H, m)36.735.61738.043.0180,96 (1H,m)48.71.35 (1H,m)48.4191.38 (1H,m); 1.02 (1H, m)35.12.38 (1H,m)48.02028.8151.0211.62 (1H,m); 1.54 (1H, m)33.729.9221.38 (1H,m); 1.27 (1H, m)33.140.0230.97 (3H,s)28.00.98 (3H,s)28.0240.80 (3H,s)15.40.76 (3H,s)15.4250.92 (3H,s)15.40.83 (3H,s)16.1260.82 (3H,s)29.81.2 (3H,s)16.0271.09 (3H,s)25.90.99 (3H,s)14.6280.91 (3H,s)29.90.79 (3H,s)18.0290.95 (3H,s)33.34.57 (1H, s)4.69 (1H,s)109.3300.91 (3H,s)21.31.68 (3H, s)19.3Pos.Cpd. 7 (CDCl3) PhysalinECpd.8 (CDCl3) PhysalinDδH ppm (J, Hz) 500 MHzδC ppm 125 MHzδH ppm (J, Hz)500 MHzδC ppm125 MHz1207.7206.625.82 (1H,dd, 10.4 and 2.4)128.65.87 (1H,dd, 10.5 and 2.0)127.536.70 (1H,dd, 10.2 and 2.8)144.46.66 (1H,dd, 10.5 and 2.0)143.243.33 (1H,d, 23.6)36.52.06 (1H,m)3.30 (1H, d, 20.0)35.35778.477.2627.63.66 (1H,d, 2.5)73.3774.82.04 (1H,m)2.06 (1H, m)38.383.27 (1H, dd, 10.4 and 8.8)39.82.44 (1H, dd, 12.0 and 3.5)35.3927.63.07 (1H,dd, 10.5 and 8.5)30.91055.654.5111.13 (1H,m, H-11)25.91.86 (1H,t, 14.0)1.17 (1H, m)25.61226.41.53 (1H,dd, 17.05 and 10.5)2.24 (1H, s)26.11380.980.614108.8107.515210.4208.2162.59 (1H,s)56.52.40 (1H,m)55.71780.980.918173.9172.3191.27 (3H,s)14.31.29 (3H,s)13.82082.280.9211.97 (3H,s)22.22.01 (3H,s)21.3224.61 (1H, t, 2.0)78.94.55 (1H,t, 2.0)77.22332.12.11 (1H,m)2.07 (1H, m)32.82431.830.4252.80 (1H,d, 4.0)51.52.48 (1H,d, 4.0)50.826170.2167.3274.43 (1H,dd, 12.8 and 4.4)3.80 (1H, d, 13.6)61.84.50 (1H,dd, 13.5 and 4.5)3.77 (1H, d, 13.5)60.5281.32 (3H,s)26.41.27 (3H, s)26.5


## 3.6. Effects of All Isolated Compounds (1–8) on NO Production in LPS-Induced RAW 264.7 Cells

The eight isolated compounds were further evaluated for their anti-inflammatory activity via NO production in the LPS-induced RAW 264.7 cells. Three compounds6–8 exhibited a strong inhibitory effect on NO generation with the percentage inhibition ranging from 35.67% to 59.20% at a concentration of 40 μg/mL (Figures 2(b) and 2(c)), compared with that of the LPS control cells (p < 0.05). Compound 3 exhibited moderate anti-inflammatory activity at all the evaluated concentrations (p < 0.05). Meanwhile, the four remaining compounds (1–3, 5) exhibited weak activity for the inhibition of NO production.


## 3.7. Effects of Isolated Compounds on Cytokines IL6 and IL10 Production in RAW 264.7 Cells

Compounds6–8 exhibited good NO inhibition activity, which required further investigation. The protein expression of IL-6 and IL-10 increased significantly after stimulation with LPS (p < 0.05). Compared with the LPS-treated control cells, the positive control Dex (1 μM) significantly suppressed the IL6 protein expression by a factor of 4.73 (p < 0.05), while physalin D (8) was the most effective compound, suppressing IL-6 secretion by a factor of 14 at a concentration of 20 μg/mL (p < 0.05). In addition, the level of expression of IL6 decreased by a factor of 3 and 8 (concentration dependent), by compounds 6 and 7, compared with the LPS control (Figure 3(c)). These compounds did not affect the secretion of IL10, and the level of the anti-inflammatory cytokine IL10 was maintained and independent of its concentration (Figure 3(d)).


## 4. Conclusions

The present study was designed to evaluate the total flavonoids (TFs) and total polyphenols (TPs) combined within vitro antioxidant and anti-inflammatory activities of the crude extract and fractions from aerial part of Ruellia tuberosa L. The ethyl acetate fraction (RTE) exhibited the most effective antioxidant activity against DPPH and ABTS radicals and also contained a high quantity of TFs and TPs. In the anti-inflammatory activity assay, the methanol and ethyl acetate fractions demonstrated the highest activity in the downregulation of the proinflammatory cytokine, IL6 secretion, and NO production at a concentration of 30 µg/mL in LPS-induced RAW 264.7 cells. Eight compounds were isolated and characterized from the RTE, of which three compounds 6–8 exhibited a strong inhibitory effect on NO generation. Physalin D (8) was the most effective compound when it inhibited IL-6 secretion by a factor of 14 at a concentration of 20 μg/mL. Compounds 6 and 7 decreased the level of protein IL6 from 3 to 8 times compared with the LPS control group in a dose-dependent manner. Based on these data, physalin D (8), physalin E (7), and hispidulin (6) are potentially bioactive compounds for the treatment of inflammation symptoms in type 2 diabetes mellitus from herbal medicines based on Ruellia tuberosa L.



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*Source: 4644641-2022-01-25.xml*