Introduction

Rehmannia glutinosa (Gaertn.) DC. (Scrophulariaceae) is an herb with oriental medicine applications such as for treating diabetes, hematinic deficiencies and adrenal disorder [1]. It has been cultivated in China, Japan, and Korea for over 1000 years [2]. Depending on the processing method, R. glutinosa roots are classified into three types for medicinal purposes, fresh roots (Rehmanniae Radix Crudus), dried roots (Rehmanniae Radix), and processed roots (Rehmanniae Radix Preparata) [3]. Fresh and dried roots affect the Yin, decrease heat from the blood, and increase body fluids and salivation, while steamed roots are mainly used to treat Yang deficiencies, facilitating nourishment and bone marrow improvement [1, 4].

About 140 compounds have been isolated from unprocessed R. glutinosa, including monoterpenoids, triterpenes, phenyl-ethyl alcohol glycosides, phenolic, ligans, flavonoids, and polysaccharides [5,6,7]. Among these, the majority of the constituents are iridoid glycosides (catalpol and harpagide) and phenylethanoid glycosides (verbacoside and iso-verbacoside). Recent pharmacological studies have reported that R. glutinosa possesses anti-oxidant, hypoglycemic, anti-inflammatory, anti-cancer, and immunoregulatory effects [7]. In addition, processed R. glutinosa roots protect against aging, oxidation, and cancer and improve marrow and essence [8]. However, steam processing could alter the contents of some compounds, thereby affecting pharmacological activity [9].

According to the Korean Pharmacopoeia, processed R. glutinosa roots (Rehmanniae Radix Preparata) are required to have a 5-hydroxymethyl-2-furaldehyde (5-HMF) content of greater than 0.1% to guarantee quality [10, 11]. Moreover, the verbascoside content, according to the Chinese Pharmacopoeia, is required to be ≥ 0.20% in processed R. glutinosa roots [12]. Therefore, it’s important to develop R. glutinosa processing condition that meet those criteria to maintain its quality and increase the efficacy. However, this could be started by choosing high quality R. glutinosa raw material. In the previous study, we developed high quality R. glutinosa seedlings trough in vitro culture approach (Korea patent no. 10-1881305) and its productivity was verified in the larger outdoor field [13]. Moreover, our team had also developed a high quality of adventitious roots of R. glutinosa for industrial applications [14]. These materials can be used to produce high quality of processed R. glutinosa roots.

However, standardization of processed R. glutinosa root is highly needed to increase its quality to fulfill global market demand that is safe and possess high level of efficacy. Therefore, this study aimed to optimize the steam processing conditions by assessing some key parameters such as steaming temperature, number of steaming times, steaming duration, and additives supplementation. In addition, we evaluated its marker compounds (catalpol, 5-HMF, verbascoside, and iso-verbascoside) composition, total phenolic and flavonoid content, anti-oxidant and anti-inflammatory activities to verify the efficacy of processed R. glutinosa roots under optimized steaming conditions.

Materials and methods

Materials and reagents

Raw roots of R. glutinosa were collected from Geumsan Farm in Chungcheongnam-do province, Korea. To prevent decay, the roots were dried in an oven at 60 °C for 48 h. Before and after processing, the moisture content of each sample was measured using a moisture analyzer balance (Kett Electric Laboratory, Tokyo, Japan). Chromatography grade water, acetonitrile, and methanol (Merck, Darmstadt Germany) were used for high-performance liquid chromatography (HPLC). 1.1-diphenyl-2picrylhydrazyl (DPPH), gallic acid, and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibody of COX-2 and iNOS were supplied by Cell Signaling (Denvers, MA, USA), NF-κB and all other western luminal reagents were purchased from Santa Cruz.

Optimization of processing methods

Dried R. glutinosa roots were cut into 20 mm slices, soaked in water for 16 h, and followed by steam processing (Fig. 1). All samples were subjected to steam processing (KSP-240 L, Kyungseo E&P, South Korea) at various temperatures (100 °C, 110 °C, and 120 °C), various steaming times (1 to 5 times), several steaming durations (1 to 4 h), and supplementation with various additives (distilled water, rice wine, and different concentration of ethyl alcohol [5%, 10%, 20%, 30%, and 40%]). All processed samples were dried in an oven at 60 °C for 48 h. For further study, all processed samples were pulverized with a grinder (250G New Type Pulverizing Machine, Model RT-N04-2 V, Taiwan) and filtered through a 60-mesh sieve to make sample powder. A tenfold volume of 70% methanol was added to the sample powder (v/w), and the mixture was subjected to ultrasonic extraction for 1 h. Finally, three types of samples were obtained for further experiments: unprocessed R. glutinosa (UPR), processed R. glutinosa (PRR), and supplemented processed R. glutinosa (SPRR).

Fig. 1
figure 1

The overall schematic diagram of R. glutinosa processing method by steaming treatment

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Measurement of R. glutinosa moisture content

The moisture content of all R. glutinosa samples was determined using the Kett FD-720 infrared moisture analyzer (Kett Electric Laboratory, Tokyo, Japan). The measurement was performed following previously described study [15]. Two grams of all samples were placed to the weighing dish, then the instrument automatically calculated the moisture content of the R. glutinosa at setting temperature 105 °C. The results showed as percentages (%).

Preparation and analysis of samples

HPLC analysis conditions and parameters for constituent analysis

Chromatography was performed following the National Institute of Food and Drug Safety Evaluation of Oriental Medicine guideline. HPLC was performed using the Waters Alliance 2695 HPLC system (Milford, MA, USA) equipped with a Capcell Pak UG120 C18 analytical column (250 × 4.6 mm, 5 µm; Shiseido, Japan) using water (A) and acetonitrile (B) as the mobile phase. The gradient elution was as follows: 0–15 min, 95% A; 15–25 min, 85% A; 25–45 min, 70% A; 45–60 min, 95% A. Absorbance was detected at 205 and 280 nm and the column was kept at 25 °C. The injection volume was 10 µL, and the flow rate was set at 0.8 mL/min.

Fourier transform near-infrared spectroscopy (FT-NIR) analysis

FT-NIR was performed following previously described methods [16]. Absorbance spectra for ground R. glutinosa in a glass vial (5 mm internal diameter) were collected using the TANGO FT-NIR spectrometer (Billerica, MA, USA) with the InGaAs detector, a broadband light source (50 W), interferometer, and a quartz halogen lamp. TANGO FT-NIR was completely controlled using OPUS (version 6.5). The spectra for R. glutinosa samples were detected using three positions per sample. The spectral conditions for TANGO FT-NIR were a resolution of 16 cm, mirror speed of 0.9494 cm/s, and spectral range of 800 to 2500 nm. To avoid surface interaction and the penetration of light into samples, incidence angles were set to 75°.

Determination of anti-oxidant activity

DPPH assay

A DPPH assay was performed as described previously [17]. Briefly, 1 mL of sample extract (at various concentrations) was mixed with 1 mL of 20 mM DPPH ethanol solution, followed by incubation at 37 °C for 30 min. The absorbance of the mixture was measured at 517 nm (SpectraMax i3x; Molecular Devices, Wokingham, UK) and absolute ethanol was used as a positive control. The scavenging capacity of DPPH radicals was calculated using the following formula:

$${\text{DPPH radical scavenging activity }}\left( \% \right)\, = \,\left[ {{1 - }\left( {{\text{A}}_{{{\text{sample}}}} {\text{ - A}}_{{{\text{empty}}}} } \right)/{\text{A}}_{{{\text{control}}}} } \right]\, \times \,{1}00.$$

Determination of total polyphenols

Total polyphenols were evaluated using Folin-Ciocateu reagent according to previously described methods [18], with modifications. Pulverized sample extraction was performed by using 1 g and 70% methanol with centrifugation for 10 min at 8000 × g at 4 °C. The supernatant was harvested for analyses. A 500 µL aliquot of each sample was mixed with 500 µL of the Folin-Ciocalteu reagent. After 3 min, 500 µL of 10% sodium carbonate (Na2CO3) were added and incubated in dark conditions at 25 °C for 1 h. Absorbance was measured at 725 nm using the SpectraMax i3x (Molecular Devices). Gallic acid was used to generate a standard curve for quantification.

Determination of total flavonoids

Total flavonoids were evaluated by an aluminum chloride colorimetric assay as described previously [19], with modifications. The 70% methanolic extract of steamed root (100 µL) was added to 1 mL of diethylene glycol and 10 µL of sodium hydroxide (NaOH). After incubation at 37 °C for 1 h, absorbance was measured at 420 nm (SpectraMax i3x; Molecular Devices). Rutin was used to generate standard curves for quantification.

Determination of anti-inflammatory activity

Cell culture

Cells were cultured according to previously described methods [20]. The RAW 264.7 macrophage cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). The cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 1% (v/v) streptomycin/penicillin and 10% fetal bovine serum at 37 °C with a 5% CO2 atmosphere. Cells were seeded at 3 × 105 cells/mL in 96-well plates. Cells were then treated with R. glutinosa at various concentrations and further cultured with or without LPS (1 μg/mL) for 24 h at 37 °C.

Cell viability assay

RAW 264.7 macrophages cell lines was cultured in Dulbecoo’s Modified Eagle Medium (DMEM) (Gibco, Invitrogen) added with 1% (v/v) penicillin and 10% fetal bovine serum (FBS), followed by incubation at 37 °C with a 5% CO2 atmosphere. The RAW 264.7 macrophages were treated with all R. glutinosa extracts at concentration of 200 μg/ml followed by culture with or without LPS (1 μg/mL) for 24 h at 37 °C. The determination of the cell lines proliferation on the substrates was done using cell counting kit assay (CCK-8, Do**do, Kumamoto, Japan). The 96-well plate was used to seed the samples (2 × 105 cells/mL density per well). ELISA plate reader was used to measure the absorbance at 490 nm.

Measurement of NO levels

NO levels were measured according to previously described methods [21], with modifications. RAW 264.7 cells (2 × 105 cells/mL) were plated in 6-well plates. After cell adhesion, various R. glutinosa extracts in PBS were treated with or without LPS (6 × 103 U/mL). The supernatant was harvested to measure NO after treatment for 24 h. Nitrite accumulation in the LPS-induced RAW 264.7 macrophages was measured as an indicator of NO by the Griess reaction method.

Western blot analysis

The nuclear and cytosolic fractions of the RAW264.7 cells were obtained following a previously reported method [22]. Raw264.7 macrophages (2 × 106 cells/mL) were pretreated with R. glutinosa extracts before the addition of LPS (1 µg/mL) for stimulation in 24-well plates. After 16 h, proteins were extracted by RIPA buffer. The proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% (w/v) skim milk, and the blots were incubated for overnight at 4 °C with primary antibodies (1:1000) for iNOS, COX-2, phospho-NF-κB, and β-actin. Thereafter, secondary antibodies (1:10,000) were incubated with the membrane for 2 h. Bands were evaluated using western blotting luminal reagent and images were obtained using a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).

Statistical data analysis

All experimental data were evaluated by analysis of variance (ANOVA) with Dunnett’s post hoc tests or Duncan’s multiple range tests using Prism (Graph Pad v 5. 03). Differences were considered statistically significant at p ≤ 0.05.

Results and discussion

Effect of steam processing on R. glutinosa moisture content

R. glutinosa is a perishable material due to high moisture and sugar contents [23]. To maintain its quality, we therefore investigated the differences in the moisture content between unprocessed R. glutinosa (fresh and dried UPR) and processed R. glutinosa (PRR) (Fig. 2). The average water contents excluding other constituents were 77% for fresh UPR, 12% for dried UPR, and 0.1% for PRR (Fig. 2b). Total moisture was reduced by about 75% during processing from fresh to dried UPR and by an additional 29% from dried UPR to PRR. These results indicated that most of the total water (i.e., 80%) is lost during all stage of processing (Fig. 2c). Previous analysis have revealed that during long-term storage, samples with a moisture content of less than 12% show minimal quality changes, including changes in appearance and texture [24]. However, traditional practice to dry R. glutinosa samples is not really suitable to maintain its quality as they applied sun drying that can easily get contaminated, thereby reducing quality [25]. Therefore, to maintain high quality, it is essential to apply modern methods for drying [32]. This suggests a close chemical phylogenetic relationship between the samples.

Effect of steam processing on antioxidant activity

The antioxidant activity level all R. glutinosa samples were evaluated by a DPPH assay. As shown in Fig. 7a, all of processed R. glutinosa (PRR and SPRR) have significantly higher antioxidant activity when compared with that in unprocessed sample (UPR) (18%). Previous studies have shown that heat treatment causes chemical changes, such as changes in Maillard reaction products, including 5-HMF production. In this regard, an increase in 5-HMF may have contributed to the increase in anti-oxidant capacity detected by the DPPH scavenging assay [31]. In addition, among SPRR samples, supplementation with 20% EtOH (SPRR 20%EtOH) showed highest antioxidant activity. This is correlated with its high content of verbacoside and iso-verbacoside compared to other SPRR samples (Fig. 5c, d). Several previous reports showed that verbacoside and iso-verbacoside are good antioxidant agents [33].

Fig. 7
figure 7

Percentage of DPPH antioxidant activity, total phenolic, and total flavonoid content of all R. glutinosa sample extracts. a Antioxidant activity of R. glutinosa samples. b Total phenolics content of of R. glutinosa samples. c Total flavonoids content of R. glutinosa samples. *UPR Unprocessed R. glutinosa, PRR Processed R. glutinosa, SPRR Supplemented Processed R. glutinosa, RW Rice wine. Values are presented as means ± standard deviation. Same letters are not significantly different by Tukey’s test and p = 0.05

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Effect of steam processing on total flavonoids and phenolic contents

The total phenolic and flavonoid contents of all R. glutinosa samples are presented in Fig. 7b, c, respectively. The results indicated that steaming process clearly influences phenolic and flavonoid contents. Compared with unprocessed sample (UPR), all processed samples (PRR and SPRR) showed higher total phenolic and flavonoids contents. Many reports have said that heat treatment could increase the total phenolic and flavonoids content in the plants [34]. Among all SPRR samples, supplementation with 20% EtOH significantly increased the total phenolic and flavonoids content of 69.24 ± 4.56 mg/g and 55.36 ± 1.68 mg/g, respectively. This result is in line with the level of antioxidant activity where optimum result obtained by supplementation with 20% EtOH. Therefore, the increased in total phenolic and flavonoids content could also level up the antioxidant activity. Flavonoids and phenolic compounds are widely used in Oriental medicine and food products due to their capability in promoting antioxidant activity [35].

Effect of steam processing on anti-inflammatory activity

We tested the effects of R. glutinosa extracts on cell viability to detect cytotoxicity in RAW264.7 cells by the CCK assay. As shown in Fig. 8a, all R. glutinosa samples at 200 µg/mL did not cause cell death. This is consistent with previous research showing that unprocessed and processed R. glutinosa do not exert cytotoxicity [36]. Therefore, all R. glutinosa samples at concentration of 200 µg/mL is considered safe for further study and applications.

Fig. 8
figure 8

Effect of R.glutinosa samples extracts (200 μg/ml) on cell viability and nitric oxide (NO) production in LPS-stimulated macrophages cell lines. a Cell viability of LPS-stimulated macrophages treated with R. glutinosa extracts. b Inhibition of NO production by all R. glutinosa sample extracts. *UPR Unprocessed R. glutinosa, PRR Processed R. glutinosa, SPRR Supplemented Processed R. glutinosa, RW Rice wine. Values are presented as means ± standard deviation. Same letters are not significantly different by Tukey’s test and p = 0.05

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NO is a free radical synthesized in tissues and cells. Low NO levels contribute to homeostasis, while high NO levels are associated with inflammation and various diseases. Thus, NO levels are an indicator of anti-inflammatory activity [37]. NO production was distinctly diminished after LPS-induced macrophages were treated with all R. glutinosa extracts. Moreover, processed R. glutinosa showed higher inhibition activity on NO production (81%) compared with unprocessed R. glutinosa (86%) (Fig. 8). This is consistent with their effect on antioxidant activity where processed R. glutinosa showing stronger effect than unprocessed R. glutinosa. This could be related with the increased level of some active compounds (5-HMF, iso-verbascoside, flavonoid, and phenolics) in processed R. glutinosa. However, there were no significant differences in NO inhibition effect among processed R. glutinosa samples. Due to its high 5-HMF, verbacoside, and iso-verbacoside contents, SPRR 20% EtOH was chosen for further western blot analysis to uncover its molecular mechanism of action for anti-inflammatory activity based on NO suppression ability. Several previous studies have also reported anti-inflammatory potential of 5-HMF, verbascoside and iso-verbascoside [33].

To verify the efficacy of optimized processed R. glutinosa (SPRR 20% EtOH) on anti-inflammatory activity, RAW 264.7 pro-inflammatory factors (iNOS, NF- κB) and cytokine (COX-2) release was evaluated by western blot [38]. The increase in iNOS protein expression levels in LPS-stimulated macrophages is related to a substantial increase in NO production, and iNOS acts as an important regulator of inflammation and immune defense [39]. The result showed that processed R. glutinosa supplemented with 20% EtOH (SPRR 20% EtOH) strongly inhibited iNOS expression to levels in untreated samples (Fig. 9a). The suppressive effect of SPRR 20% EtOH samples was concentration-dependent, in which a high concentration (i.e., 200 µg/mL) resulted in a significantly higher inhibition value than that of a low concentration (100 µg/mL). However, levels of pro-inflammatory COX-2 were not affected by SPRR 20% EtOH samples (Fig. 9b). This indicates that the ability of processed R. glutinosa samples to induce anti-inflammatory only through the inhibtion of iNOS expression.

Fig. 9
figure 9

Inhibition effect of processed R.glutinosa sample extracts on the expression of iNOS, COX-2, and NFκB in LPS-stimulated macrophages. a Effect of processed R.glutinosa extracts on iNOS expression. b Effect of processed R.glutinosa extracts on COX-2 expression. c Effect of processed R.glutinosa extracts on NF-κB expression. *SPRR Supplemented Processed R. glutinosa; RW. Values are presented as means ± standard deviation. Same letters are not significantly different by Tukey’s test and p = 0.05

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The activation of NF-κB has a critical role in the induction of the inflammatory factors COX-2 and iNOS [40]. The nuclear transcription factor NF-κB is a key mediator of inflammation and plays an important role in cytokine expression [41]. Various factors acting upstream of NF-κB activation, including NH2-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), mitogen-activated protein kinase (MAPK), and p-65 phosphorylation (p65) [40, 42]. The NF-κB levels through p-p65 pathway in LPS-stimulated RAW264.7 macrophages and SPRR 20% EtOH samples are summarized in Fig. 9c. Processed R. glutinosa supplemented with 20% EtOH significantly inhibited p-p65 expression in a concentration-dependent manner (100 and 200 µg/mL). These results indicated that processed R. glutinosa supplemented with 20% EtOH exerts anti-inflammatory effects by the inhibition of iNOS via the NF-κB activation. Moreover, these results support those of previous studies demonstrating that polysaccharide derivatives such as 5-HMF isolated from steam processed R. glutinosa effectively suppress the expression of pro-inflammatory factors [43]. Moreover, the increase in iso-verbascoside after steam processing could contribute to the anti-inflammatory effect [44].