Access the full text.
Sign up today, get DeepDyve free for 14 days.
Rehmannia glutinosa (Gaertn.) DC., belonging to the family Scrophulariaceae, is an important medicinal herb culti- vated in East Asia. Traditionally, R. glutinosa is steam processed to increase its efficacy in treating various ailments such as diabetes, hematinic deficiencies and adrenal disorder. However, standardization of processed R. glutinosa 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 R. glutinosa steam processing methods by evaluating some key parameters such as steaming temperature, number of steaming times, steaming duration, and additive supplementation. R. glutinosa samples were steam processed at different temperatures (100 °C, 110 °C, and 120 °C), various steaming times (1 to 5 times), several steaming duration (1 to 4 h), and additives supplementation (rice wine, 5% EtOH, 10% EtOH, 20% EtOH, 30% EtOH, and 40% EtOH). As the result, 2 h, 3 replications, and supplementation with 20% EtOH at 120 °C were iden- tified as the optimal conditions for R. glutinosa steam processing. Optimized processed R. glutinosa (SPRR 20%EtOH) resulted in significantly higher content of 5-HMF (7648.60 ± 150.08 µg/g) and iso-verbacoside (203.80 ± 10.72 µg/g) compared with unprocessed R. glutinosa (UPR). Compared to those of other samples, SPRR 20% EtOH samples had higher total flavonoid (55.36 ± 1.68 mg/g) and phenolic (69.24 ± 4.56 mg/g) contents and stronger DPPH antioxidant activity (56%). Furthermore, SPRR 20% EtOH had excellent anti-inflammatory activity, as evidenced by the suppression of inducible nitric oxide synthase (iNOS) caused by activation of nuclear factor-κB (NF-κB) through p-p65 pathway in LPS-stimulated RAW 264.7 cells. These findings will provide a basis towards industrialization of R. glutinosa processing technology that will be very helpful for oriental medication field. Keywords Rehmannia glutinosa roots steam processing, Anti-oxidant activity, Anti-inflammatory activity, Herbal medicine processing, 5-hydroxymethyl-2-furaldehyde (5-HMF) Yuseong Chung and Endang Rahmat are equally contributed for Co-first authorship *Correspondence: Youngmin Kang firstname.lastname@example.org Herbal Medicine Resources Research Center, Korea Institute of Oriental Medicine (KIOM), 111 Geonjae-ro, Naju-si, Jeollanam-do 58245, Republic of Korea Korean Convergence Medicine Major, University of Science and Technology (UST ), Gajeong-ro, Yuseong-gu, Daejeon, Republic of Korea © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Chung et al. Applied Biological Chemistry (2023) 66:17 Page 2 of 13 market demand that is safe and possess high level of effi - Introduction cacy. Therefore, this study aimed to optimize the steam Rehmannia glutinosa (Gaertn.) DC. (Scrophulariaceae) processing conditions by assessing some key parame- is an herb with oriental medicine applications such as ters such as steaming temperature, number of steaming for treating diabetes, hematinic deficiencies and adre - times, steaming duration, and additives supplementation. nal disorder . It has been cultivated in China, Japan, In addition, we evaluated its marker compounds (catal- and Korea for over 1000 years . Depending on the pol, 5-HMF, verbascoside, and iso-verbascoside) compo- processing method, R. glutinosa roots are classified sition, total phenolic and flavonoid content, anti-oxidant into three types for medicinal purposes, fresh roots and anti-inflammatory activities to verify the efficacy of (Rehmanniae Radix Crudus), dried roots (Rehmanniae processed R. glutinosa roots under optimized steaming Radix), and processed roots (Rehmanniae Radix Pre- conditions. parata) . Fresh and dried roots affect the Yin , decrease heat from the blood, and increase body fluids Materials and methods and salivation, while steamed roots are mainly used to Materials and reagents treat Yang deficiencies, facilitating nourishment and Raw roots of R. glutinosa were collected from Geum- bone marrow improvement [1, 4]. san Farm in Chungcheongnam-do province, Korea. To About 140 compounds have been isolated from prevent decay, the roots were dried in an oven at 60 °C unprocessed R. glutinosa, including monoterpenoids, for 48 h. Before and after processing, the moisture triterpenes, phenyl-ethyl alcohol glycosides, phenolic, content of each sample was measured using a mois- ligans, flavonoids, and polysaccharides [5 –7]. Among ture analyzer balance (Kett Electric Laboratory, Tokyo, these, the majority of the constituents are iridoid gly- Japan). Chromatography grade water, acetonitrile, and cosides (catalpol and harpagide) and phenylethanoid methanol (Merck, Darmstadt Germany) were used for glycosides (verbacoside and iso-verbacoside). Recent high-performance liquid chromatography (HPLC). pharmacological studies have reported that R. glutinosa 1.1-diphenyl-2picrylhydrazyl (DPPH), gallic acid, and possesses anti-oxidant, hypoglycemic, anti-inflamma - all other reagents were purchased from Sigma-Aldrich tory, anti-cancer, and immunoregulatory effects [7 ]. In (St. Louis, MO, USA). Primary antibody of COX-2 and addition, processed R. glutinosa roots protect against iNOS were supplied by Cell Signaling (Denvers, MA, aging, oxidation, and cancer and improve marrow and USA), NF-κB and all other western luminal reagents essence . However, steam processing could alter the were purchased from Santa Cruz. contents of some compounds, thereby affecting phar - macological activity . According to the Korean Pharmacopoeia, processed R. glutinosa roots (Rehmanniae Radix Preparata) are Optimization of processing methods required to have a 5-hydroxymethyl-2-furaldehyde Dried R. glutinosa roots were cut into 20 mm slices, (5-HMF) content of greater than 0.1% to guarantee soaked in water for 16 h, and followed by steam pro- quality [10, 11]. Moreover, the verbascoside content, cessing (Fig. 1). All samples were subjected to steam according to the Chinese Pharmacopoeia, is required to processing (KSP-240 L, Kyungseo E&P, South Korea) be ≥ 0.20% in processed R. glutinosa roots . There - at various temperatures (100 °C, 110 °C, and 120 °C), fore, it’s important to develop R. glutinosa processing various steaming times (1 to 5 times), several steaming condition that meet those criteria to maintain its quality durations (1 to 4 h), and supplementation with vari- and increase the efficacy. However, this could be started ous additives (distilled water, rice wine, and different by choosing high quality R. glutinosa raw material. In the concentration of ethyl alcohol [5%, 10%, 20%, 30%, and previous study, we developed high quality R. glutinosa 40%]). All processed samples were dried in an oven at seedlings trough in vitro culture approach (Korea patent 60 °C for 48 h. For further study, all processed samples no. 10-1881305) and its productivity was verified in the were pulverized with a grinder (250G New Type Pulver- larger outdoor field . Moreover, our team had also izing Machine, Model RT-N04-2 V, Taiwan) and filtered developed a high quality of adventitious roots of R. gluti- through a 60-mesh sieve to make sample powder. A ten- nosa for industrial applications . These materials can fold volume of 70% methanol was added to the sample be used to produce high quality of processed R. glutinosa powder (v/w), and the mixture was subjected to ultra- roots. sonic extraction for 1 h. Finally, three types of samples However, standardization of processed R. glutinosa were obtained for further experiments: unprocessed R. root is highly needed to increase its quality to fulfill global Chung et al. Applied Biological Chemistry (2023) 66:17 Page 3 of 13 Fig. 1 The overall schematic diagram of R. glutinosa processing method by steaming treatment glutinosa (UPR), processed R. glutinosa (PRR), and sup- Preparation and analysis of samples plemented processed R. glutinosa (SPRR). HPLC analysis conditions and parameters for constituent analysis Chromatography was performed following the National Measurement of R. glutinosa moisture content Institute of Food and Drug Safety Evaluation of Ori- The moisture content of all R. glutinosa samples was ental Medicine guideline. HPLC was performed using determined using the Kett FD-720 infrared mois- the Waters Alliance 2695 HPLC system (Milford, MA, ture analyzer (Kett Electric Laboratory, Tokyo, Japan). USA) equipped with a Capcell Pak UG120 C18 analyti- The measurement was performed following previ - cal column (250 × 4.6 mm, 5 µm; Shiseido, Japan) using ously described study . Two grams of all samples water (A) and acetonitrile (B) as the mobile phase. were placed to the weighing dish, then the instrument The gradient elution was as follows: 0–15 min, 95% A; automatically calculated the moisture content of the 15–25 min, 85% A; 25–45 min, 70% A; 45–60 min, 95% R. glutinosa at setting temperature 105 °C. The results A. Absorbance was detected at 205 and 280 nm and the showed as percentages (%). column was kept at 25 °C. The injection volume was 10 µL, and the flow rate was set at 0.8 mL/min. Chung et al. Applied Biological Chemistry (2023) 66:17 Page 4 of 13 Fourier transform near‑infrared spectroscopy (FT‑NIR) root (100 µL) was added to 1 mL of diethylene glycol and analysis 10 µL of sodium hydroxide (NaOH). After incubation at FT-NIR was performed following previously described 37 °C for 1 h, absorbance was measured at 420 nm (Spec- methods . Absorbance spectra for ground R. glu- traMax i3x; Molecular Devices). Rutin was used to gener- tinosa in a glass vial (5 mm internal diameter) were ate standard curves for quantification. collected using the TANGO FT-NIR spectrometer (Billerica, MA, USA) with the InGaAs detector, a Determination of anti‑inflammatory activity broadband light source (50 W), interferometer, and a Cell culture quartz halogen lamp. TANGO FT-NIR was completely Cells were cultured according to previously described controlled using OPUS (version 6.5). The spectra for methods . The RAW 264.7 macrophage cell line was R. glutinosa samples were detected using three posi- obtained from the American Type Culture Collection tions per sample. The spectral conditions for TANGO (Manassas, VA, USA). The cell lines were cultured in FT-NIR were a resolution of 16 cm, mirror speed of Dulbecco’s Modified Eagle Medium (DMEM) supple - 0.9494 cm/s, and spectral range of 800 to 2500 nm. To mented with 1% (v/v) streptomycin/penicillin and 10% avoid surface interaction and the penetration of light fetal bovine serum at 37 °C with a 5% CO atmosphere. into samples, incidence angles were set to 75°. Cells were seeded at 3 × 10 cells/mL in 96-well plates. Cells were then treated with R. glutinosa at various con- Determination of anti‑oxidant activity centrations and further cultured with or without LPS DPPH assay (1 μg/mL) for 24 h at 37 °C. A DPPH assay was performed as described previously . Briefly, 1 mL of sample extract (at various concen - trations) was mixed with 1 mL of 20 mM DPPH etha- Cell viability assay nol solution, followed by incubation at 37 °C for 30 min. RAW 264.7 macrophages cell lines was cultured in Dul- The absorbance of the mixture was measured at 517 nm becoo’s Modified Eagle Medium (DMEM) (Gibco, Inv - (SpectraMax i3x; Molecular Devices, Wokingham, UK) itrogen) added with 1% (v/v) penicillin and 10% fetal and absolute ethanol was used as a positive control. The bovine serum (FBS), followed by incubation at 37 °C with scavenging capacity of DPPH radicals was calculated a 5% CO atmosphere. The RAW 264.7 macrophages using the following formula: were treated with all R. glutinosa extracts at concentra- tion of 200 μg/ml followed by culture with or without DPPH radical scavenging activity (%) LPS (1 μg/mL) for 24 h at 37 °C. The determination of the = 1− A -A /A × 100. sample empty control cell lines proliferation on the substrates was done using cell counting kit assay (CCK-8, Dojindo, Kumamoto, Japan). The 96-well plate was used to seed the samples Determination of total polyphenols (2 × 10 cells/mL density per well). ELISA plate reader Total polyphenols were evaluated using Folin-Ciocateu was used to measure the absorbance at 490 nm. reagent according to previously described methods , with modifications. Pulverized sample extraction was performed by using 1 g and 70% methanol with centrifu- Measurement of NO levels gation for 10 min at 8000 × g at 4 °C. The supernatant was NO levels were measured according to previously harvested for analyses. A 500 µL aliquot of each sample described methods , with modifications. RAW 264.7 was mixed with 500 µL of the Folin-Ciocalteu reagent. cells (2 × 10 cells/mL) were plated in 6-well plates. After After 3 min, 500 µL of 10% sodium carbonate (N a CO ) 2 3 cell adhesion, various R. glutinosa extracts in PBS were were added and incubated in dark conditions at 25 °C for treated with or without LPS (6 × 10 U/mL). The super - 1 h. Absorbance was measured at 725 nm using the Spec- natant was harvested to measure NO after treatment traMax i3x (Molecular Devices). Gallic acid was used to for 24 h. Nitrite accumulation in the LPS-induced RAW generate a standard curve for quantification. 264.7 macrophages was measured as an indicator of NO by the Griess reaction method. Determination of total flavonoids Total flavonoids were evaluated by an aluminum chlo - ride colorimetric assay as described previously , with Western blot analysis modifications. The 70% methanolic extract of steamed The nuclear and cytosolic fractions of the RAW264.7 cells were obtained following a previously reported method Chung et al. Applied Biological Chemistry (2023) 66:17 Page 5 of 13 Statistical data analysis . Raw264.7 macrophages (2 × 10 cells/mL) were All experimental data were evaluated by analysis of vari- pretreated with R. glutinosa extracts before the addi- ance (ANOVA) with Dunnett’s post hoc tests or Duncan’s tion of LPS (1 µg/mL) for stimulation in 24-well plates. multiple range tests using Prism (Graph Pad v 5. 03). After 16 h, proteins were extracted by RIPA buffer. The Differences were considered statistically significant at proteins were separated by 10% SDS-PAGE and trans- p ≤ 0.05. ferred to nitrocellulose membranes. The membranes were blocked with 5% (w/v) skim milk, and the blots were Results and discussion incubated for overnight at 4 °C with primary antibodies Eec ff t of steam processing on R. glutinosa moisture (1:1000) for iNOS, COX-2, phospho-NF-κB, and β-actin. content Thereafter, secondary antibodies (1:10,000) were incu - R. glutinosa is a perishable material due to high mois- bated with the membrane for 2 h. Bands were evaluated ture and sugar contents . To maintain its quality, we using western blotting luminal reagent and images were therefore investigated the differences in the moisture obtained using a ChemiDoc MP Imaging System (Bio- content between unprocessed R. glutinosa (fresh and Rad, Hercules, CA, USA). 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 Fig. 2 The changes in biomass root morphology and water content of R. glutinosa samples. a Changes in root morphology appearance of R. glutinosa samples. b The water content of R. glutinosa samples. c. Water content loss of R. glutinosa samples during processing. *UPR Unprocessed R. glutinosa, PRR Processed R. glutinosa. Values are presented as means ± standard deviation. Same letters are not significantly different by Tukey’s test and p = 0.05 Chung et al. Applied Biological Chemistry (2023) 66:17 Page 6 of 13 Fig. 3 Eec ff t of different steaming temperature (100 ℃, 110 ℃, and 120 ℃) on marker compounds content of R. glutinosa samples. a Catalpol content of R. glutinosa samples. b 5-HMF content of R. glutinosa samples. c Verbacoside content of R. glutinosa samples. d Iso-verbacoside content of R. glutinosa samples. *UPR Unprocessed R. glutinosa, PRR Processed R. glutinosa. Values are presented as means ± standard deviation. Same letters are not significantly different by Tukey’s test and p = 0.05 an additional 29% from dried UPR to PRR. These results showed that 120 °C resulted in significantly higher indicated that most of the total water (i.e., 80%) is lost 5-HMF (229.52 ± 74.85 µg/g) and iso-verbascoside during all stage of processing (Fig. 2c). Previous analy- (575.05 ± 74.90 µg/g) contents compared to 100 °C sis have revealed that during long-term storage, samples and 110 °C (Fig. 3). Moreover, steaming tempera- with a moisture content of less than 12% show minimal ture of 120 °C also dramatically reduced the level of quality changes, including changes in appearance and catalpol (4836.43 ± 2295.44 µg/g) and verbacoside texture . However, traditional practice to dry R. gluti- (160.77 ± 20.49 µg/g) compared with 100 °C and 110 °C nosa samples is not really suitable to maintain its quality (Fig. 3). Therefore, 120 °C is the optimum temperature as they applied sun drying that can easily get contami- for steam processing of R. glutinosa roots. nated, thereby reducing quality . Therefore, to main - UPR and PRR samples were investigated for the pres- tain high quality, it is essential to apply modern methods ence of catalpol, 5-HMF, verbascoside, and iso-verbas- for drying . coside influenced by number of steaming times and steaming duration (Fig. 4). R. glutinosa samples were Eec ff t of steam processing on marker compounds content subjected to steaming for 1 h, 2 h, and 4 h with five rep - Steaming temperature, number of steaming times, lications. The results showed that samples processed by steaming duration, and additives were selected as steaming for 1 h with five replications, 2 h with three the main factors for processed R. glutinosa stand- replications, and 4 h with two replications met the mini- ardization. An analysis of steaming temperature mal Korean Pharmacopoeia criteria for 5-HMF content Chung et al. Applied Biological Chemistry (2023) 66:17 Page 7 of 13 Fig. 4 Eec ff t of different steaming times (five times) and steaming duration (1 h, 2 h, and 4 h) on marker compounds content of R. glutinosa samples. a Catalpol content of R. glutinosa samples. b 5-HMF content of R. glutinosa samples. c Verbacoside content of R. glutinosa samples. d Iso-verbacoside content of R. glutinosa samples. *UPR Unprocessed R. glutinosa, PRR Processed R. glutinosa. Values are presented as means ± standard deviation. Same letters are not significantly different by Tukey’s test and p = 0.05 (above 0.1%) . Furthermore, we decided to choose EtOH is the best additive supplement for processed R. steaming for 2 h with three replications as the most glutinosa. In addition, our optimized processing method optimal condition as it’s produced highest content of (SPRR 20% EtOH) had been evaluated by Korea Minis- verbacoside (48.98 ± 12.40 µg/g) and iso-verbacoside try of Food and Drug Safety resulted in high quality of (52.09 ± 29.38 µg/g) (Fig. 4c, d). Although steaming for processed R. glutinosa roots with 0.5% 5-HMF content 4 h with two replications yielded the highest content (Additional file 1: Fig. S1). of 5-HMF among all samples but surplus 5-HMF could induce cell toxicity . In addition, steaming for 4 h FT‑NIR analysis with two replications showed lower iso-verbacoside con- FT-NIR spectroscopy, a non-destructive chemical evalu- tent compared to other samples. ation technique, is used industrially for the rapid charac- Figure 5 shows the effect of additive supplementation terization and identification of the chemical composition on marker compound content of R. glutinosa root sam- of materials . The FT-NIR spectrum provides infor - ples. The results revealed that 5-HMF, verbascoside, and mation on the major chemical bonds constituting the iso-verbascoside contents were higher in SPRR samples chemical composition of plant samples . The result compared with UPR and PRR (Fig. 5). However, all SPRR showed that UPR consist of eight spectrum peaks while samples met the criteria established by the Korean and PRR and SPRR both have seven peaks (Fig. 6a). The spec - Chinese Pharmacopoeia (verbascoside content > 0.02% trum peaks of all samples were observed between 9000 −1 and 5-HMF content > 0.1%). Moreover, among SPRR and 4000 cm of wavenumbers range. UPR showed dif- samples, supplementation with 20% EtOH resulted in ferent pattern of spectra compared with processed R. the highest verbascoside (119.88 ± 5.88 µg/g) and iso- glutinosa samples (PRR and SPRR). This possibly hap - verbascoside contents (203.80 ± 10.72 µg/g). Hence, 20% pened due to the change of chemical composition during Chung et al. Applied Biological Chemistry (2023) 66:17 Page 8 of 13 Fig. 5 Eec ff t of different additive supplements (rice wine, 5% EtOH, 10% EtOH, 20% EtOH, 30% EtOH, and 40% EtOH) on marker compounds content of R. glutinosa samples. a Catalpol content of R. glutinosa samples. b 5-HMF content of R. glutinosa samples. c Verbacoside content of R. glutinosa samples. d Iso-verbacoside 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 Fig. 6 The fourier transform near-infrared (FT-NIR) spectroscopy of characterization all R. glutinosa samples. a FT-NIR spectra of analysed R. glutinosa samples. b Dendogram of analysed FT-NIR showing the heterogeneity of all R. glutinosa samples. *UPR Unprocessed R. glutinosa, PRR, Processed R. glutinosa, SPR, Supplemented Processed R. glutinosa, RW Rice wine Chung et al. Applied Biological Chemistry (2023) 66:17 Page 9 of 13 Fig. 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 steam processing . For instance, high temperature of SPRR samples, supplementation with 20% EtOH (SPRR steam changed some active compound composition such 20%EtOH) showed highest antioxidant activity. This is as decreased content of catalpol and verbacoside and correlated with its high content of verbacoside and iso- increased content of 5-HMF from unprocessed to pro- verbacoside compared to other SPRR samples (Fig. 5c, cessed R. glutinosa (Fig. 5). Meanwhile, PRR and SPRR d). Several previous reports showed that verbacoside have similar pattern of FT-NIR spectra indicating the and iso-verbacoside are good antioxidant agents . homogeneity of certain functional classes of chemicals within these materials. Additionally, Ward’s algorithm was applied to clus- Eec ff t of steam processing on total flavonoids and phenolic ter all spectra produced from FT-NIR analysis (Fig. 6b). contents According to Ward’s algorithm grouping, processed R. The total phenolic and flavonoid contents of all R. glu - glutinosa samples (PRR and SPRR) display more homo- tinosa samples are presented in Fig. 7b, c, respectively. geneity among themselves than unprocessed R. glutinosa The results indicated that steaming process clearly (UPR). Moreover, all SPRR samples indicated more simi- influences phenolic and flavonoid contents. Compared lar heterogeneity compared to the rest of the samples. with unprocessed sample (UPR), all processed sam- The similar heterogeneity recorded in the dendrogram of ples (PRR and SPRR) showed higher total phenolic and the respective grouped samples of R. glutinosa roots was flavonoids contents. Many reports have said that heat caused by higher similarity between the distinct near-IR treatment could increase the total phenolic and flavo - spectra of these samples . This suggests a close chem - noids content in the plants . Among all SPRR sam- ical phylogenetic relationship between the samples. ples, supplementation with 20% EtOH significantly increased the total phenolic and flavonoids content of Eec ff t of steam processing on antioxidant activity 69.24 ± 4.56 mg/g and 55.36 ± 1.68 mg/g , respectively. The antioxidant activity level all R. glutinosa samples This result is in line with the level of antioxidant activ - were evaluated by a DPPH assay. As shown in Fig. 7a, ity where optimum result obtained by supplementa- all of processed R. glutinosa (PRR and SPRR) have sig- tion with 20% EtOH. Therefore, the increased in total nificantly higher antioxidant activity when compared phenolic and flavonoids content could also level up with that in unprocessed sample (UPR) (18%). Previ- the antioxidant activity. Flavonoids and phenolic com- ous studies have shown that heat treatment causes pounds are widely used in Oriental medicine and food chemical changes, such as changes in Maillard reac- products due to their capability in promoting antioxi- tion products, including 5-HMF production. In this dant activity . regard, an increase in 5-HMF may have contributed to the increase in anti-oxidant capacity detected by the DPPH scavenging assay . In addition, among Chung et al. Applied Biological Chemistry (2023) 66:17 Page 10 of 13 Fig. 8 Eec ff t 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 Eec ff t of steam processing on anti‑inflammatory activity NO suppression ability. Several previous studies have We tested the effects of R. glutinosa extracts on cell also reported anti-inflammatory potential of 5-HMF, viability to detect cytotoxicity in RAW264.7 cells by the verbascoside and iso-verbascoside . CCK assay. As shown in Fig. 8a, all R. glutinosa samples To verify the efficacy of optimized processed R. gluti- at 200 µg/mL did not cause cell death. This is consistent nosa (SPRR 20% EtOH) on anti-inflammatory activity, with previous research showing that unprocessed and RAW 264.7 pro-inflammatory factors (iNOS, NF- κB) processed R. glutinosa do not exert cytotoxicity . and cytokine (COX-2) release was evaluated by western Therefore, all R. glutinosa samples at concentration blot . The increase in iNOS protein expression levels of 200 µg/mL is considered safe for further study and in LPS-stimulated macrophages is related to a substantial applications. increase in NO production, and iNOS acts as an impor- NO is a free radical synthesized in tissues and cells. tant regulator of inflammation and immune defense Low NO levels contribute to homeostasis, while high . The result showed that processed R. glutinosa sup - NO levels are associated with inflammation and vari - plemented with 20% EtOH (SPRR 20% EtOH) strongly ous diseases. Thus, NO levels are an indicator of inhibited iNOS expression to levels in untreated sam- anti-inflammatory activity . NO production was ples (Fig. 9a). The suppressive effect of SPRR 20% EtOH distinctly diminished after LPS-induced macrophages samples was concentration-dependent, in which a high were treated with all R. glutinosa extracts. Moreover, concentration (i.e., 200 µg/mL) resulted in a significantly processed R. glutinosa showed higher inhibition activ- higher inhibition value than that of a low concentra- ity on NO production (81%) compared with unpro- tion (100 µg/mL). However, levels of pro-inflammatory cessed R. glutinosa (86%) (Fig. 8). This is consistent with COX-2 were not affected by SPRR 20% EtOH samples their effect on antioxidant activity where processed R. (Fig. 9b). This indicates that the ability of processed R. glutinosa showing stronger effect than unprocessed R. glutinosa samples to induce anti-inflammatory only glutinosa. This could be related with the increased level through the inhibtion of iNOS expression. of some active compounds (5-HMF, iso-verbascoside, The activation of NF-κB has a critical role in the induc - flavonoid, and phenolics) in processed R. glutinosa. tion of the inflammatory factors COX-2 and iNOS . However, there were no significant differences in NO The nuclear transcription factor NF-κB is a key media - inhibition effect among processed R. glutinosa samples. tor of inflammation and plays an important role in Due to its high 5-HMF, verbacoside, and iso-verbaco- cytokine expression . Various factors acting upstream side contents, SPRR 20% EtOH was chosen for further of NF-κB activation, including NH2-terminal kinase western blot analysis to uncover its molecular mecha- (JNK), extracellular signal-regulated protein kinase nism of action for anti-inflammatory activity based on (ERK), mitogen-activated protein kinase (MAPK), and Chung et al. Applied Biological Chemistry (2023) 66:17 Page 11 of 13 Fig. 9 Inhibition effect of processed R.glutinosa sample extracts on the expression of iNOS, COX-2, and NFκB in LPS-stimulated macrophages. a Eec ff t of processed R.glutinosa extracts on iNOS expression. b Eec ff t of processed R.glutinosa extracts on COX-2 expression. c Eec ff t 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 Abbreviations p-65 phosphorylation (p65) [40, 42]. The NF-κB levels UPR Unprocessed R. glutinosa through p-p65 pathway in LPS-stimulated RAW264.7 PRR Processed R. glutinosa macrophages and SPRR 20% EtOH samples are sum- SPRR Supplemented processed R. glutinosa 5-HMF 5-Hydroxymethyl-2-furaldehyde marized in Fig. 9c. Processed R. glutinosa supplemented NO Nitric oxide with 20% EtOH significantly inhibited p-p65 expression iNOS Inducible nitric oxide synthase in a concentration-dependent manner (100 and 200 µg/ NF-κB Nuclear fac tor-kappa B LPS Lipopolysaccharide mL). These results indicated that processed R. glutinosa COX-2 Cyclooxygenase-2 supplemented with 20% EtOH exerts anti-inflammatory HPLC High-performance liquid chromatography effects by the inhibition of iNOS via the NF-κB activation. DPPH 1.1-Diphenyl-2picrylhydrazyl FT-NIR F ourier transform near-infrared spectroscopy Moreover, these results support those of previous stud- ies demonstrating that polysaccharide derivatives such as Supplementary Information 5-HMF isolated from steam processed R. glutinosa effec - The online version contains supplementary material available at https:// doi. tively suppress the expression of pro-inflammatory fac - org/ 10. 1186/ s13765- 023- 00773-7. tors . Moreover, the increase in iso-verbascoside after steam processing could contribute to the anti-inflamma - Additional file 1: Figure S1. Certificate of analysis of optimized pro - tory effect . cessed R. glutinosa (SPRR 20% EtOH) accredited by Korea ministry of Food and Drug Safety. Chung et al. Applied Biological Chemistry (2023) 66:17 Page 12 of 13 Acknowledgements 10. Youn UJ, Gu B-S, Kim KH, Ha C, Jung IC (2018) Variation of main compo- We thank Korea Institute of Oriental Medicine (KIOM) for the research fund nents according to the number of steaming and drying of Rehmanniae with Grant Number: KSN2013320 and KSN2021320. radix preparata. J Pharmacopuncture 21:112. https:// doi. org/ 10. 3831/ KPI. 2018. 21. 014 Author contributions 11. Chung Y, Kang YM (2021) New insight of perspective requirements in YC and ER are co-first authors. YC: Conceptualization, methodology, data standardization and modernization for prepared Rehmannia Root. Korean collection and curation, writing original draft. ER: Data supervision, review, and Herb Med Inf 9:9–28. https:// doi. org/ 10. 2267/ KHMI-9- 1-2 manuscript editing. HHN: Western blotting supervision. AL: HPLC supervision. 12. Wei GD, Liu H, We XS, Guo YX (2013) Discussion on the quality marker of JHP: Anti-inflammatory study supervision. BCM: Project administration. YK: Rehmanniae radix praeparata in the Chinese pharmacopoeia. Zhong Yao Supervision, project administration, review and editing. All authors read and Cai 36:853–856 approved the final manuscript. 13. Kang YM, Kim C, Moon BC (2016) Optimization of in vitro cultures for production of seedling and rootstock of Rehmannia glutinosa (Gaertn.) Funding DC. JALS 50:81–93. https:// doi. org/ 10. 1439/ jals. 2016. 50.5. 81 This work was supported by the Development of Sustainable Application for 14. Rahmat E, Okello D, Kim H, Lee J, Chung Y, Komakech R, Kang Y (2021) Standard Herbal Resources (KSN2013320 & KSN2021320), Development of Scale-up production of Rehmannia glutinosa adventitious root biomass in Foundational Techniques for the Domestic Production of Herbal Medicines bioreactors and improvement of its acteoside content by elicitation. Ind (K18405), and Korea Institute of Oriental Medicine through the Ministry of Crops Prod 172:114059. https:// doi. org/ 10. 1016/j. indcr op. 2021. 114059 Science and ICT, Republic of Korea. 15. Cho JS, Choi JY, Moon KD (2020) Hyperspectral imaging technology for monitoring of moisture contents of dried persimmons during drying Availability of data and materials process. Food Sci Biotechnol 29:1407–1412. https:// doi. org/ 10. 1007/ The data will be made available by the corresponding author upon reasonable s10068- 020- 00791-x request. 16. Kim YJ, Ma KH, Han JW, Lee SH, Chang JK, Han SH (2020) Quality charac- teristics of Rehmannia glutinosa dried at different drying temperature. Korean J Food Preserv 27:17–24. https:// doi. org/ 10. 1100/ kjfp. 2020. 27.1. 17 Declarations 17. Jing N, Wang M, Gao M, Zhong Z, Ma Y, Wei A (2021) Color sensory characteristics, nutritional components and antioxidant capacity of Ethics approval and consent to participate Zanthoxylum bungeanum maxim as affected by different drying methods. Not applicable. Ind Crops and Prod 160:113167. https:// doi. org/ 10. 1016/j. indcr op. 2020. Competing interests 18. Mu Y, Gao W, Lv S, Li F, Lu Y, Zhao C (2021) The antioxidant capacity and The authors declare that they have no known competing financial interests antioxidant system of jerusalem artichoke (Helianthus tuberosus L.) tubers or personal relationships that could have appeared to influence the work in relation to inulin during storage at different low temperatures. Ind reported in this paper. Crops Prod 161:113411. https:// doi. org/ 10. 1016/j. indcr op. 2020. 113229 19. Baek JY, Lim SY (2016) Flavonoid and phenol contents and antioxidant effect of wine by-product extracts. J Life Sci 26:948–954. https:// doi. org/ Received: 9 December 2022 Accepted: 13 February 2023 10. 5352/ JLS. 2016. 26.8. 948 20. Vimala B, Hussain H, Nazaimoon WMW (2012) Eec ff ts of edible bird’s nest on tumour necrosis factor-alpha secretion, nitric oxide production and cell viability of lipopolysaccharide-stimulated RAW 264.7 macrophages. Food Agr Immunol 23:303–314. https:// doi. org/ 10. 1080/ 09540 105. 2011. References 1. Zhang RX, Li MX, Jia ZP (2008) Rehmannia glutinosa: review of botany, 21. Mostafa ES, Nawwar MA, Mostafa DA, Ragab MF, Swilam N (2020) Karafsin, chemistry and pharmacology. J Ethnopharmacol 117:199–214 a unique mono-acylated flavonoid apiofurnoside from the leaves of 2. Li XJ, Jiang C, Xu N, Li JX, Meng FY, Zhai HQ (2018) Sorting and identifica- apium graveolens var secalinum alef: in vitro and in vivo anti-inflamma- tion of Rehmannia glutinosa germplasm resources based on EST-SSR, tory assessment. Ind Crops Prod 158:112901. https:// doi. org/ 10. 1016/j. scanning electron microscopy micromorphology, and quantitative indcr op. 2020. 112901 taxonomy. Ind Crops Prod 123:303–314. https:// doi. org/ 10. 1016/j. indcr op. 22. Nam HH, Nan L, Choo BK (2018) Dichloromethane extracts of geranium 2018. 06. 088 koreanum kom. alleviates esophagus damage in acute reflux esophagitis- 3. Hong SP, Kim YC, Kim KH, Park JH, Park MK (1993) Characteristic compo- induced rats by anti-inflammatory activities. Inter J Mol Sci 19:3622. nent of Rehmanniae radix preparata compared to Rehmanniae radix and https:// doi. org/ 10. 3390/ ijms1 91136 22 Rehmanniae radix Crudus. Anal Sci Technol 6:401–404 23. Zhang G, Ji J, Liu Q, Zhang L, Gong G, Jin Z, Ren S (1993) Collected edition 4. Han K, Bose S, Kim YM, Chin YW, Kim BS, Wang JH, Lee JH, Kim H (2015) of identification and assessment of common TCD. Heilongjiang Science Rehmannia glutinosa reduced waist circumferences of Korean obese and Technique Press, Harbin, pp 301–302 women possibly through modulation of gut microbiota. Food Funct 24. Kim YJ, Ma KH, Han JW, Lee SH, Chang JK, Han SH (2020) Quality charac- 6:2684–2692. https:// doi. org/ 10. 1039/ C5FO0 0232J teristics of Rehmannia glutinosa dried at different drying temperature. 5. Fu G, Du X (2015) Research advance on chemical constituents and Korean J Food Preserv 27:17–24. https:// doi. org/ 10. 1100/ kjfp. 2020. 27.1. 17 pharmacological activities of Rehmannia glutinosa. China Med Pharm 25. Lee HJ, Oh SH, Choi KH (2008) Studies on the general composition, 15:39–41 rheometric and microbiological change of pacific saury, coloabis saira 6. Li H, Meng X (2015) Research progress on chemical constituents and kwamaegi on the storage temperatures and durations. Korean J Food pharmacological activities of Rehmannia glutinosa. Drug Eval Res Nutr 21:165–175 38:218–228 26. Rhim JW, Xi Y, Jeong WC, Ham KS, Chung HS, Kim ES (2009) Eec ff t of 7. Liu C, Ma R, Wang L, Zhu R, Liu H, Guo Y, Zhao B, Zhao S, Tang J, Li Y drying method on antioxidant activity of Jiwhang (Rehmannia glutinosa). (2017) Rehmanniae radix in osteoporosis: a review of traditional CHINESE Food Sci Biotechnol 18:1464–1469 medicinal uses, phytochemistry, pharmacokinetics and pharmacology. J 27. Lee CK, Seo JM (2004) Changes of the constituents in the Rehmanniae Ethnopharmacol 198:351–362. https:// doi. org/ 10. 1016/j. jep. 2017. 01. 021 radix preparata during processing. J Korean Soc Food Sci Nutr 33:1748– 8. Luo C, Qin S, Liang P, Ling W, Zhao C (2020) Advances in modern research 1752. https:// doi. org/ 10. 3746/ jkfn. 2004. 33. 10. 1748 on pharmacological effects of radix Rehmanniae. AIP Conf Proc 2252:1–8. 28. Ke Y, Yin Z, Chen N, Chen P, Liu J, Ou S, Li G (2022) Formation and identi- https:// doi. org/ 10. 1063/5. 00203 48 fication of a 5-(hydroxymethyl)-2-furfural-zingerone condensate and its 9. Albach DC, Li H-Q, Zhao N, Jensen SR (2007) Molecular systematics and cytotoxicity in caco-2 cells. Front Nutr 9:893991. https:// doi. org/ 10. 3389/ phytochemistry of Rehmannia (Scrophulariaceae). Biochem Syst Ecol fnut. 2022. 893991 35:293–300. https:// doi. org/ 10. 1016/j. bse. 2006. 11. 003 Chung et al. Applied Biological Chemistry (2023) 66:17 Page 13 of 13 29. Komakech R, Kim YG, Kim WJ, Omujal F, Yang S, Moon BC, Okello D, Rahmat E, Nambatya Kyeyune G, Matsabisa MG (2020) A micropropaga- tion protocol for the endangered medicinal tree prunus africana (Hook f.) kalkman: genetic fidelity and physiological parameter assessment. Front Plant Sci 11:1871. https:// doi. org/ 10. 3389/ fpls. 2020. 548003 30. Wang CY, Tang L, Li L, Zhou Q, Li YJ, Li J, Wang YZ (2020) Geographic authentication of eucommia ulmoides leaves using multivariate analysis and preliminary study on the compositional response to environment. Front Plant Sci 11:79. https:// doi. org/ 10. 3389/ fpls. 2020. 00079 31. Kwon Y, Yu S, Choi GS, Kim JH, Baik M, Su ST, Kim W (2019) Puffing of Rehmannia glutinosa enhances anti-oxidant capacity and down-regulates IL-6 production in RAW 264.7 cells. Food Sci Biotechnol 28:1235–1240. https:// doi. org/ 10. 1007/ s10068- 019- 00566-z 32. Srivastava S, Mishra G, Mishra HN (2018) Identification and differentia- tion of insect infested rice grains varieties with FTNIR spectroscopy and hierarchical cluster analysis. Food Chem 268:402–410. https:// doi. org/ 10. 1016/j. foodc hem. 2018. 06. 095 33. Sanchez PM, Villarreal ML, Herrera-Ruiz M, Zamilpa A, Jiménez-Ferrer E, Trejo-Tapia G (2013) In vivo anti-inflammatory and anti-ulcerogenic activities of extracts from wild growing and in vitro plants of castilleja tenuiflora benth. (Orobanchaceae). J Ethnopharmacol 150:1032–1037. https:// doi. org/ 10. 1016/j. jep. 2013. 10. 002 34. Jiratanan T, Liu RH (2004) Antioxidant activity of processed table beets (beta vulgaris var, conditiva) and green beans (Phaseolus vulgaris L.). J Agric Food Chem 52:2659–2670. https:// doi. org/ 10. 1021/ jf034 861d 35. Nabavi S, Ebrahimzadeh M, Nabavi S, Hamidinia A, Bekhradnia A (2008) Determination of antioxidant activity, phenol and flavonoids content of parrotia persica mey. Pharmacol Online 2:560–567 36. Seo HS (2008) The experimental study on anti-inflammation and anti-oxidation of indigo naturalis and Rehmanniae radix. J Korean Med Ophthalmol Otolaryngol Dermatol 21:104–110 37. Tung Y T, Chua MT, Wang SY, Chang ST (2008) Anti-inflammation activities of essential oil and its constituents from indigenous cinnamon (Cin‑ namomum osmophloeum) twigs. Biores Technol 99:3908–3913. https:// doi. org/ 10. 1016/j. biort ech. 2007. 07. 050 38. Guo J, Chen J, Lu X, Guo Z, Huang Z, Zeng S, Zhang Y, Zheng B (2018) Proteomic analysis reveals inflammation modulation of κ/ι-carrageenan hexaoses in lipopolysaccharide-induced RAW264. 7 macrophages. J Agric Food Chem 66:4758–4767. https:// doi. org/ 10. 1021/ acs. jafc. 8b011 44 39. Nam HH, Yang S, Kim HS, Kim MJ, Kim JS, Lee JH (2021) Role of semisul- cospira gottschei extract as medicinal food on reflux esophagitis in rats. Food Sci Nutr 9:3114–3122. https:// doi. org/ 10. 1002/ fsn3. 2270 40. Huang GJ, Huang SS, Deng JS (2012) Anti-inflammatory activities of inotilone from phellinus linteus through the inhibition of MMP-9, NF-κB, and MAPK activation in vitro and in vivo. PloS ONE 7:e35922. https:// doi. org/ 10. 1371/ journ al. pone. 00359 22 41. Jing W, Chunhua M, Shumin W (2015) Eec ff ts of acteoside on lipopoly- saccharide-induced inflammation in acute lung injury via regulation of NF-κB pathway in vivo and in vitro. Toxicol Appl Pharmacol 285:128–135. https:// doi. org/ 10. 1016/j. taap. 2015. 04. 004 42. Liu W, Huang S, Li Y, Li Y, Li D, Wu P, Wang Q, Zheng X, Zhang K (2018) Glycyrrhizic acid from licorice down-regulates inflammatory responses via blocking MAPK and PI3K/Akt-dependent NF-κB signalling pathways in TPA-induced skin inflammation. Med Chem Comm 9:1502–1510. https:// doi. org/ 10. 1039/ C8MD0 0288F 43. Lu MK, Chang CC, Chao CH, Hsu YC (2022) Structural changes, and anti- inflammatory, anti-cancer potential of polysaccharides from multiple processing of Rehmannia glutinosa. Int J Biol Macromol 206:621–632. https:// doi. org/ 10. 1016/j. ijbio mac. 2022. 02. 112 44. Alipieva K, Korkina L, Orhan IE, Georgiev MI (2014) Verbascoside—a review of its occurrence, (bio) synthesis and pharmacological signifi- cance. Biotechnol Adv 32:1065–1076. https:// doi. org/ 10. 1016/j. biote chadv. 2014. 07. 001 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
Applied Biological Chemistry – Springer Journals
Published: Mar 1, 2023
Keywords: Rehmannia glutinosa roots steam processing; Anti-oxidant activity; Anti-inflammatory activity; Herbal medicine processing; 5-hydroxymethyl-2-furaldehyde (5-HMF)
Access the full text.
Sign up today, get DeepDyve free for 14 days.