A comparative study of the antioxidant and intestinal protective effects of extracts from different parts of Java tea (Orthosiphon stamineus)

Abstract The aim of this study was to compare the free radical scavenging ability and intestinal epithelial cell protective effects of Java tea (Orthosiphon stamineus) root extracts (ORE), stem extracts (OSE), and leaf extracts (OLE) to determine the potential of Java tea by‐products. The Java tea extracts were prepared using a standard water–ethanol method. The antioxidant activity and intestinal protective effects were tested by H2O2‐induced cell model and high‐fat diet‐induced mice model, respectively. The results showed that the total phenolic acid and flavonoid content and relative content were different in the ORE, OSE, and OLE. ORE had the highest total polyphenol and flavonoid content, the highest free radical scavenging rate, and the highest intracellular free radical scavenging rate. However, the yeast content in the ORE was lower than that in the OSE and OLE. All the Java tea extracts protected mouse intestine from high‐fat diet‐induced oxidative injury. This study indicates the potential of Java tea extracts as food or feed additives to protect the intestine from oxidative stress.


| INTRODUCTION
The intestinal epithelium not only has the functions of nutrition digestion and absorption, but also is a barrier against antigens and pathogens (Suzuki, 2013). The intestine is exposed to a complex microenvironment that includes chyme, enterobacteria, various digestive juices, and immune factors. Imbalances in this microenvironment contribute to oxidative stress in the intestinal epithelium (Miranda-Bautista, Bañares, & Vaquero, 2017).
However, the published reports evaluating O. stamineus extracts do not describe their effects in different organs. Only O. stamineus leaves and stems are routinely sold in Chinese markets, with the roots being discarded as a by-product. This processing method is not only inconvenient for the consumer, but also a waste of O. stamineus resources.
In fact, a previous study showed the stem and root of O. stamineus also possessed high antioxidant activities (Xue et al., 2016) and could be used as food or feed additives.
The aim of this study was to compare the main phenolic compounds with antioxidant activity from O. stamineus extracts, to determine the potential of these extracts as antioxidant additives and their protective effects on intestinal cells. Medical University), and a voucher specimen was retained in our laboratory for future reference. The roots, stems, and leaves were separated and then dried in a drying oven. The extracts were prepared using a water-ethanol method (Yam et al., 2007). Briefly, a 20 g dry powder of O. stamineus root and stem of leaves were subjected to an ultrasonic extractor at 50°C for 15 min in extracted with 1 L of 50% ethanol. The resulting Orthosiphon stamineus extracts were filtered and concentrated by applying vacuum rotary evaporation method.

| Materials and plant extracts
The concentrated liquid extract was freeze-dried, and the powder stored at −20°C until use.

| Animals and model treatment
Fifty male C57BL/6 mice weighing 18-20 g were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. After being acclimated for 1 week, the mice were randomly divided into normal control (NC), high-fat control (FC), root extract (R), stem extract (S), and leaf extract (L) groups, with 10 mice in each group. The mice were housed in standard cages under controlled temperature conditions (22 ± 2 °C) with a 12-h light/dark cycle. The NC group received only a normal diet (D12450B, Research Diet Inc.) containing 4.3% fat, and other groups received a high-fat diet (D12492; Research Diet Inc., New Brunswick, USA) containing 35% fat. The mice in the R, S, and T groups were orally administered O. stamineus root extracts (ORE), stem extracts (OSE), and leaf extracts (OLE) at a dose of 100 mg/kg body weight, while the mice in the NC and FC groups were orally administered saline. The oral administration lasted for 8 weeks. At the end of the study, blood samples were collected by eyeball removal. Jejunum is the longest segment in small intestine. In this study, jejunum samples of mice were washed immediately with ice-cold PBS and stored at −80 °C prior to analysis. These experiments were carried out in accordance with local guidelines for the care of laboratory animals and were approved by the institution's ethics committee for research using laboratory animals.

| Total phenolics and flavonoids analysis of O. stamineus extract
The total polyphenol content in the extracts was determined by the Folin-Ciocalteu method using gallic acid as the standard. Total flavonoids in the extract were determined using the method by Rana et al. (2015), with quercetin as the standard (Taga, Miller, & Pratt, 1984).

| HPLC-MS analysis
HPLC-MS analyses were performed using an Acquity UPLC BEH-C18 column (100 × 2.1 mm, 1.7 μm) at 45 °C with a mobile phase at a flow rate of 0.4 ml/min. The mobile phase consisted of 0.1% formic acid in water (phase A) and acetonitrile (phase B). The mobile phase was con-
The DPPH radical scavenging activities of ORE, OSE, and OLE were determined according to Wu, Jiang, Jing, Zheng, and Yan (2017).

| H 2 O 2 challenge assay with IPEC-J2 cell model
The H 2 O 2 -induced IPEC-J2 cell oxidative stress model was included according to a previous study . In this study, IPEC-J2 cells were divided into five groups. The PBS group was the control group. In the test groups, 50 μg/ml of the ORE, OSE, or OLE was added to the final concentration for 24 hr before analysis, and then, 1 mmol/L H 2 O 2 was added for 1 hr before testing. In the H 2 O 2 group, 1 mmol/L H 2 O 2 was added to the final concentration for 1 hr before the test. An intracellular total ROS assay and cell viability assay were performed.
The cell viability assay was performed using the cell counting kit method as described above. The inhibition ratio was calculated as:

Cell viability in relation to the control group =
where A test is the absorbance of the ORE, OSE, or OLE group, and A control is the absorbance of the control group.

| Serum diamine oxidase (DAO) content
Serum was separated by centrifugation at 3,500 g for 15 min at 4°C. Serum concentrations of DAO were measured using a quantitative sandwich enzyme immunoassay technique according to the manufacturer's instructions (Cusabio Biotech Co., Wuhan, China).

| Antioxidant analysis of jejunal homogenates
Jejunal homogenates (10% w/v) were prepared in cold PBS using homogenizer in ice and centrifugation at 4,000 g for 20 min at 4°C. The supernatants were diluted to the optimal content for detecting redox status. The protein content of homogenates was measured using the Coomassie Brilliant G-250 method. The superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) contents of jejunal homogenates were measured by colorimetry at absorbances of 550, 412, and 532 nm, respectively, according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). All absorbances were measured by a microplate reader (Tecan Inc., Mannedorf, Switzerland).

| Statistical analysis
All reaction mixtures were prepared in triplicate, and at least three independent assays were performed for each sample. All data are expressed as mean ± SEM. Data were subjected to one-way ANOVA followed by Duncan's multiple range tests using SPSS version 17.0 software. A p-value <.05 was considered to be statistically significant.

Compound
Root ( Mean values within a column with different superscript letters were significantly different (p < .05).

1
One unit of SOD activity was defined as the amount required to inhibit the reduction in nitro blue tetrazolium by 50% of maximum inhibition in 1 mg tissue protein.

2
One unit of GSH-Px activity was defined as a decrease of μmol/L of GSH per 5 min for 1 mg protein at 37°C after subtraction of the nonenzymatic reaction.

| Antioxidant and cell protective effects of O. stamineus on IPEC-J2 cells
As shown in Figure 2, the extracts of roots, stems, and leaves scavenged intracellular reactive oxygen species (ROS) and significantly increased cell viability under oxidative stress (p < .05). At a concentration of 50 μg/ml, the ORE had the highest intracellular ROS scavenging rate, but the OLE had the greatest cell viability increase (not significantly higher than that of the ORE, p > .05).

| Serum DAO content
As shown in Figure 3, DAO concentrations were increased in high-fat diet mice compared with the control mice (p < .05). O. stamineus root extract-fed mice had DAO concentrations that were significantly lower than those of the high-fat diet mice (p < .05) but still higher than those of control mice (p < .1). No significant differences in DAO concentration were found between ORE-, OSE-, and OLE-treated mice (p > .05).  (Ameer et al., 2012;Sumaryono, Proksch, Wray, Witte, & Hartmann, 1991). Our results show that both total polyphenol and total flavonoids were highest in the root extracts. However, the total polyphenol and total flavonoids yields from O. stamineus leaves are higher than those from the roots.

| Antioxidant effect of O. stamineus on intestinal epithelia
The present study shows rosmarinic acid to be the most abundant phenolic acid of O. stamineus in both leaves and roots. This finding is consistent with the reports by Akowuah, Zhari, Norhayati, and Sadikun (2004) and Lee, Peng, Chang, Huang, and Chyau (2013). The study by Lee et al. (2013) showed rosmarinic acid to be the major contributor to the antioxidant activities of O. stamineus. Interestingly, the present study showed that ursolic acid was also present at very high levels in O. stamineus and that the ursolic acid content of roots and stems was much higher than that of leaves (Table 1). Ursolic acid is a well-known anticancer agent (Chen et al., 2015), while rosmarinic acid shows cellular protective effects (Nabavi et al., 2015). These results suggest that ORE, OSE, and OLE may display different bioactivities on cellular proliferation ( Figure 2); however, the mechanism by which O. stamineus extracts regulate cellular processes needs further study.  26.55, and 11.34 μg/ml, respectively ( Figure 1). The cell model studies also yielded similar results: Root extracts showed the highest intracellular ROS scavenging rate, whereas stem extracts showed the lowest intracellular ROS scavenging rate. The in vivo study confirmed these results, with the R group of mice having the lowest jejunal MDA content, and the S group the highest. MDA is one of the key toxic products of lipid peroxidation, a process that disrupts membrane structure and slows cellular metabolism (Moon & Shibamoto, 2009). The data in Table 2 show the high-fat diet induced a high-lipid peroxidation rate in mice and that O. stamineus extracts reduced this effect. However, the mechanism by which this occurs does not appear to relate to the levels of antioxidative enzymes such as SOD or GSH-Px (Table 2). The findings of a study by Choi et al. (2013) may partly account for this: O. stamineus extracts increased leptin expression in mice, and leptin decreased tissue MDA levels (Hacioglu, Algin, Pasaoglu, Pasaoglu, & Kanbak, 2005). This is an interesting topic, and more data are still needed to confirm this hypothesis. Our results show that O. stamineus extracts can protect intestine from oxidative stress and that not only the leaf but also the stem and root have good oxygen radical and nitrogen radical scavenging activity.
IPEC-J2 is a nontumorigenic epithelial cell line and is a suitable oxidative stress model . Figure 2 shows  showed the highest intracellular free radical scavenging rate, but the yeast content in the ORE was lower than that in the OSE and OLE. Therefore, the establishment of a highly effective extraction method for O. stamineus roots is necessary. These results indicate that Java tea by-products have potential as a food or feed additive for protecting the intestine from oxidative stress. If we could separate the leaves and stems of O. stamineus and process the leaves to drink while processing the stems and roots as food or feed additives, we could not only offer a better drink for human consumption, but also produce a large amount of raw material for animal feed or natural food additives.