Efficiency of the Enzymatic Conversion of Flavone Glycosides Isolated from Carrot Leaves and Anti-Inflammatory Effects of Enzyme-Treated Carrot Leaves

In traditional oriental medicine, carrots (Daucus carota L.) are considered effective medicinal herbs; however, the use of D. carota leaves (DCL) as therapeutic agents has not been explored in depth. Therefore, we aimed to demonstrate the value of DCL, generally treated as waste while developing plants for wide industrial availability. Six flavone glycosides were isolated and identified from DCL, and their constituents were identified and quantitated using an NMR and HPLC/UV method, which was optimized and validated. The structure of chrysoeriol-7-rutinoside from DCL was elucidated for the first time. The method exhibited adequate relative standard deviation (<1.89%) and recovery (94.89–105.97%). The deglycosylation of DCL flavone glycosides by Viscozyme L and Pectinex was assessed. Upon converting the reaction contents to percentages, the luteolin, apigenin, and chrysoeriol groups showed values of 85.8, 33.1, and 88.7%, respectively. The enzyme-treated DCL had a higher inhibitory effect on TNF-α and IL-2 expression than that of the carrot roots or carrot leaves without enzyme treatments. These results highlight the importance of carrot leaves and could be used as baseline standardization data for commercial development.


Introduction
Carrot (Daucus carota), an important vegetable consumed by humans worldwide, is a vital source of nutrients and contains large quantities of carotenoids [1,2], such as provitamin A, β-carotene, and lutein. Carrots have long been used as antifungal, antibacterial, and nephroprotective agents [3,4] owing to their rich nutrient contents, including essential oils. In ancient Rome, carrots were used for treating pain and for detoxification. Furthermore, in traditional oriental medicine, carrots are considered effective remedies for constipation, dysentery, anemia, bladder inflammation, and measles; they also prevent kidney stones [5].
Carrots originated in Afghanistan and were first cultivated in China in the 13th and 14th centuries and in Korea in the 16th and 17th centuries [6]. However, only the roots have been used as therapeutic agents, and the use of carrot leaves has not been explored. In fact, carrots are mainly used as an essential food source rather than a therapeutic agent. The leaves are discarded and sometimes used to feed livestock in the private sector; however, with the mass production of quality feed, carrot leaves are not used for feed.
Recent reports have revealed some chemical components and biological activities that can gradually increase the value of carrot leaves over that of carrot roots. According to previous reports, β-carotene content in carrot leaves is equivalent to or higher than that in carrot roots, even after heat treatment. Furthermore, essential oil content is significantly higher in the leaves than in the roots [6]. The total phenolic content is twice as much in the leaves as in the roots, and the antioxidant activity of water extracts of leaves is also twice as strong as that of the roots [7].
Furthermore, there are reports of an abundant content of flavones, such as luteolin and apigenin, in the leaves, but these have not been compared with the components in carrot roots [8]. In fact, luteolin and apigenin are mostly present in green leafy spices. The most extensively studied effects of flavones are their anti-inflammatory and anticancer activities [9,10]. Moreover, the intracellular penetration and activity of the aglycones of flavone glycosides, in which the sugar is removed, are known to be increased [11,12]. We believe that determining the exact flavone compounds in carrot leaves and roots and verifying their anti-inflammatory properties will aid in identifying novel applications of carrot leaves. The primary focus of this study was to demonstrate the value of the carrot leaves, which are generally treated as waste while developing plants for wide industrial availability.
In this study, six flavone glycosides (1)(2)(3)(4)(5)(6) were isolated and identified from the n-BuOH layer of carrot leaves during standardization of methods for the separation of bioactives. In addition, the flavone glycosides were treated with Viscozyme and Pectinex, and three hydrolyzed flavones were isolated and quantitatively analyzed ( Figure 1). The anti-inflammatory activities of carrot roots (DCR) and leaves (DCL) and enzyme-treated DCL were also investigated.
Molecules 2023, 28, x FOR PEER REVIEW 2 of higher in the leaves than in the roots [6]. The total phenolic content is twice as much in th leaves as in the roots, and the antioxidant activity of water extracts of leaves is also twi as strong as that of the roots [7]. Furthermore, there are reports of an abundant content of flavones, such as luteol and apigenin, in the leaves, but these have not been compared with the components carrot roots [8]. In fact, luteolin and apigenin are mostly present in green leafy spices. Th most extensively studied effects of flavones are their anti-inflammatory and anticanc activities [9,10]. Moreover, the intracellular penetration and activity of the aglycones flavone glycosides, in which the sugar is removed, are known to be increased [11,12]. W believe that determining the exact flavone compounds in carrot leaves and roots an verifying their anti-inflammatory properties will aid in identifying novel applications carrot leaves. The primary focus of this study was to demonstrate the value of the carr leaves, which are generally treated as waste while developing plants for wide industri availability.
In this study, six flavone glycosides (1-6) were isolated and identified from the BuOH layer of carrot leaves during standardization of methods for the separation bioactives. In addition, the flavone glycosides were treated with Viscozyme and Pectine and three hydrolyzed flavones were isolated and quantitatively analyzed ( Figure 1). Th anti-inflammatory activities of carrot roots (DCR) and leaves (DCL) and enzyme-treate DCL were also investigated.

Structure Elucidation of the Isolated Compounds
Six flavone glucosides (1-6) were isolated from DCR during the standardization methods for the separation of bioactives by repeated separation via semi-preparativ high-performance liquid chromatography (HPLC) using an ODS gel column. Th structures of all the isolates were identified and determined based on their spectroscop data, such as their 1 H and 13

Structure Elucidation of the Isolated Compounds
Six flavone glucosides (1-6) were isolated from DCR during the standardization of methods for the separation of bioactives by repeated separation via semi-preparative highperformance liquid chromatography (HPLC) using an ODS gel column. The structures of all the isolates were identified and determined based on their spectroscopic data, such as their 1 H and 13 C nuclear magnetic resonance (NMR), including the 2D NMR ( 1 H-1 H correlated spectroscopy [COSY], heteronuclear multiple quantum coherence [HMQC], heteronuclear multiple bond correlation [HMBC]) and electrospray ionization mass spectrometry (ESI/MS) spectra, and compared with previously reported data.
For compound 2, which was collected as a brown powder, the ESI/MS ion peaks were observed at m/z 449.38 [M + H] + and 447.80 [M − H] + , implying the loss of a rhamnose moiety compared with that in compound 1, which has a luteolin aglycone moiety. In the 13 C NMR spectrum, 21 resonances were observed. As in the NMR spectrum of 1, the resonances of a glucose moiety at δ C 77.7 (C-5 ), 76.9 (C-3 ), 73.7 (C-2 ), and 70.1 (C-4 ) and CH 2 carbon at 61.2 (C-6 ) had one anomeric proton at δ C 100.5 (C-1 ). This anomeric carbon was also assigned by correlation with a proton in the HMBC spectrum as H-1 (δ H 5.04)/C-8 (δ C 100.5). Thus, compound 2 was determined as luteolin-7-O-glucoside (cynaroside) after comparing it with that in previous reports [14,15]. All the other NMR data, besides those for the flavone B-ring, revealed the same structure as of compound 1. Based on the spectroscopic data and literature, compound 3 was determined as apigenin-7-rutinoside [16].
Compounds 5 and 6 were isolated as brown powders, and their molecular weights were determined as 432 and 462, respectively, using ESI/MS. Both compounds had one glycoside, 7-O-glucoside, which was verified, similar to compound 2. The aglycones of compounds 5 and 6 were apigenin [18] and chrysoeriol [19], similar to those of compounds 3 and 4, respectively. Thus, compounds 5 and 6 were apigenin-7-O-glucoside [20] and chrysoeriol-7-O-glucoside, respectively. Some of the isolated and identified flavone glycosides, 1-6, have been reported in carrots. In a previous study in which several flavonoids, including flavones from DCL, were separated, as in this study, five compounds, except for compound 4, were isolated, and compound 2 was shown to be the main flavone [21][22][23]. Furthermore, in other studies, compounds 1 and 2 were identified in the seeds and green leaves of carrots. Compounds 1 and 2 have been isolated from the methanol extracts of seeds at increased amounts, compared with the amounts isolated from DCL (30 mg of 1 and 15 mg of 2 from the seeds, and 10 mg of 1 and 7 mg of 2 from the DCL). However, the isolation and identification of compound 4 from DCL and the entire carrots have not yet been described. The structure of compound 4, chrysoeriol-7-rutinoside, from the DCL was elucidated for the first time. Compounds 2 and 5 were detected in DCL via LC/MS analysis in recent studies [24,25].
In other studies, all the compounds isolated in this study, 1-6, were analyzed using ultrahigh-performance liquid chromatography/ESI/MS [26,27]; however, there are no verified reports on the flavone content in DCL. Therefore, a standardized, reproducible analysis for quality control was necessary to enable the industrial utilization of DCL. We developed a reliable analysis employing the HPLC/UV system and using the flavones isolated in this study as standards.

Optimization of Analytical Conditions and Quantitation
To develop an accurate and reliable HPLC method for the flavones, 1-6, and their hydrolyzed aglycones, 7-9, identified in DCL, the sample extraction solvents, HPLC column resins, wavelengths, solvent gradient system, and column oven temperatures were optimized. MeOH, ethanol (EtOH), H 2 O, and their mixtures are commonly used to extract vegetable flavonoids and polar glycosides. Therefore, we examined the yield of compounds from DCL under several solvent conditions. The mixture of 70% EtOH in H 2 O was found to be the most appropriate solvent for extraction. The other solvents, such as 100% MeOH or EtOH, resulted in a poor yield of glycosides, and the flavones were poorly soluble in H 2 O. Furthermore, the aqueous MeOH solvent resulted in a lower yield than that obtained using the EtOH and H 2 O mixture, which was not attractive from the industrial perspective. Extraction in 70% EtOH, performed twice with ultra-sonication for 5 min, was found to be optimal. With regard to the optimal HPLC conditions, a YMC-Triart C18 column (5 µm, 4.6 mm × 250 mm) was used based on a typical ODS resin, which increased the resolution of the polar components. An important factor in quantitating flavonoids is UV absorbance; the maximum absorbance is generally observed at 300-350 nm. Therefore, the maximum absorbance of the nine standard compounds was investigated, and 346 nm was selected as the wavelength representative of all the standard compounds. A solvent gradient system comprising ACN (A) and H 2 O with 0.1% phosphoric acid as a buffer (B) was the most suitable for separating the standard compounds in the leaf extracts without impurities. The We employed the optimized conditions described above to compare flavone glycosides 1-6 and flavones 7-9 and assess the activities of DCL. Based on the analysis of five replicate samples of DCL and DCR, we found that the DCL contained all the standard compounds except 8, and that the glycosides were the major components; the content of the flavone glycosides was as follows: 2.7378 ± 0.0295 mg/g of 1, 6.6595 ± 0.0105 mg/g of 2, 1.4797 ± 0.0150 mg/g of 3, 2.7711 ± 0.0148 mg/g of 4, 1.0465 ± 0.0110 mg/g of 5, and 0.8607 ± 0.0128 mg/g of 6. The content of flavones was as follows: 0.0593 ± 0.0004 of 7 and 0.2947 ± 0.0019 mg/g of 8. The content of compounds 4 and 6 in the DCR was quantified as 0.0682 ± 0.0009 of 4 and 1.0062 ± 0.0082 mg/g of 6; however, the other compounds were not detected (Table 1). DCR, along with its rich and diverse nutrients, contains carbohydrates as approximately 10.6% of the main energy source and 2.71-4.53% of its constituent free-sugars [28]. Thus, these glycosides were predictable; moreover, the results were different from our expectations. This fact is also evidenced by previous studies, in which the glycosides identified by us were reported from DCL, whereas only luteolin and apigenin were identified from DCR [29]. Only two chrysoeriols (4 and 6) were obtained from the BuOH extract layer of the DCR extract using the same extraction and fractionation process as for DCL. The structure of the compounds was verified using spectrometric data and by comparison with the standard compounds. as for DCL. The structure of the compounds was verified using spectrometric data and by comparison with the standard compounds.   0.2947 ± 0.0019 N.D.
a Not detected, b Standard error (mg/g).

Deglycosylation of Flavone Glycosides by Enzyme Treatment
In nature, most flavonoids exist as nonabsorbable and biologically inactive glycosides. The presence of a glycoside moiety in a flavonoid increases its molecular weight, rendering it unusable by the human body, and reduces its activity [30]. The bioavailability of glucosides is increased by hydrolysis of the glucose moiety using enzymatic conversion [31]. The effect of the enzymatic complexes, Viscozyme L containing cellulase (Viscozyme) and Pectinase (Pectinex), on flavone glycosides from DCL has been examined.
There are reports confirming that the antioxidant activities of flavone glycosides from DCL increase, along with the total flavonoid and phenol content, when treated with enzymes [32]. Therefore, in this study, the deglycosylation of flavone glycosides from DCL by treatment with Viscozyme L and Pectinex was assessed, and the hydrolyzed compounds were analyzed using the HPLC method.
After treatment of the DCL sample with the enzymes at a concentration of 0.1% to verify the efficiency of the enzyme reaction over time, the decomposition and generation of the compounds were monitored for up to 24 h. All the flavone glycosides (1-6) in DCL were gradually hydrolyzed upon treatment with Viscozyme L (Table 4). Their content was determined to be 0.03 ± 0.00 for 1, 0.22 ± 0.00 for 2, 0.02 ± 0.00 for 3, 0.59 ± 0.10 for 4, 0.06 ± 0.03 for 5, and 0.07 ± 0.04 mg/mL for 6. Using Pectinex, the final content was 0.21 ± 0.01 for 1, 2.02 ± 0.12 for 2, 0.10 ± 0.01 for 3, 0.94 ± 0.05 for 4, 0.40 ± 0.02 for 5, and 0.26 ± 0.01 mg/mL for 6. In contrast, the deglycosylated flavones were gradually produced until 24 h had passed, and their final content was 6.34 ± 0.09 for 7, 0.40 ± 0.05 for 8, and 1.72 ± 0.03 mg/g for 9 using Viscozyme L and 5.43 ± 0.09 for 7, 0.35 ± 0.01 for 8, and 1.45 ± 0.09 mg/g for 9 using Pectinex. Although there were no significant differences in these enzymes, as illustrated in the conversion rate graph over response time (Figure 3), considering the data for the converted content comprehensively, Viscozyme L was more efficient than Pectinex at the same concentration. Based on the law of mass conservation, to verify the loss of compounds in the samples during enzyme treatment, the molecular weight of each flavone glycoside was estimated as a percentage of the molecular weight when the sugar was degraded. Furthermore, by substituting this in the flavone glycoside before and after the enzyme reaction, the content of relatively pure flavone was estimated, and the total content of each flavone was compared. Thus, the three groups of the flavone glycoside aglycones and content of the converted flavones, including luteolin, apigenin, and chrysoeriol, were compared. The total content of the luteolin group, including compounds 1, 2, and 7, was 6.41 mg/mL at 0.5 h and 5.50 mg/mL at 24 h after treatment with Viscozyme L. The total content of the apigenin group, including compounds 3, 5, and 8, was 1.36 mg/mL at 0.5 h and 0.45 mg/mL at 24 h after treatment with Viscozyme L. In the chrysoeriol group, including compounds 4, 6, and 7, the total content was 2.31 mg/mL at 0.5 h and 2.05 mg/mL at 24 h after the Viscozyme L treatment. Upon converting the reaction contents to % values, the luteolin, apigenin, and chrysoeriol groups showed values of 85.8, 33.1, and 88.7%, respectively. These values validated the conversion yield of the flavone glycosides in the sample, allowing a more realistic correlation with the results of sample activation following the enzyme reaction (Figures 2 and 3).
The treatment of 1 g DCL extract in 70% ethanol with Viscozyme L and Pectinex resulted in 15.91 mg/g and 14.83 mg/g of total flavonoids, respectively. There was no significant difference in the conversion efficiency between these enzymes, but Viscozyme L showed a higher conversion rate to flavonoids than Pectinex. A similar trend was observed for the enzymatic conversion of flavone glycosides isolated from DCL.
following the enzyme reaction (Figures 2 and 3).
The treatment of 1 g DCL extract in 70% ethanol with Viscozyme L and Pec resulted in 15.91 mg/g and 14.83 mg/g of total flavonoids, respectively. There w significant difference in the conversion efficiency between these enzymes, but Visco L showed a higher conversion rate to flavonoids than Pectinex. A similar trend observed for the enzymatic conversion of flavone glycosides isolated from DCL.

Anti-Inflammatory Activities of the Samples
We determined the effects of DCR, DCL, and enzyme-treated DCL on the expression of TNF-α and IL-2 in stimulated human T lymphocyte cells. TNF-α and IL-2 are crucial to several immune-mediated inflammatory diseases [33][34][35][36]. As illustrated in Figure 4, cells treated with phorbol 12-myristate 13-acetate (PMA)/phytohemagglutinin-L (PHA) expressed higher levels of TNF-α and IL-2 mRNAs than non-treated cells. The treatment with DCR and DCL extracts considerably decreased the expression of TNF-α and IL-2 under PMA and PHA stimulation. In addition, DCL extract exhibited a stronger inhibitory activity than DCR. When treated with Viscozyme and Pectinex, the DCL had a higher mRNA inhibitory effect than that of the DCR and untreated DCL. Furthermore, Jurkat cells stimulated with PMA/PHA were used to verify the effects of DCR and DCL on TNF-α and IL-2 expression. Similar to the mRNA expression results, the DCL displayed more potent inhibitory effects than the DCR. The inhibitory effect of enzyme-treated DCL on the expression of TNF-α and IL-2 was higher than that of DCR. These results indicated that TNF-α was differentially regulated at the transcriptional level, and the release of cytokines was associated with the enzyme treatment. The enzyme treatment of DCL resulted in a stronger inhibitory effect than that in DCR or DCL without enzyme treatment.  . (A,B) The mRNA levels of TNF-α and IL-2 were assayed using RT-PCR. Jurkat cells were stimulated using PMA (100 ng/mL)/PHA (10 ng/mL) and treated with various extracts, as indicated for 3 h. The mRNA levels of TNF-α and IL-2 were determined using qRT-PCR and normalized to those of GAPDH. (C,D) TNF-α and IL-2 levels were measured using an enzyme-linked immunosorbent assay (ELISA). Cells were stimulated with PMA (100 ng/mL)/PHA (10 ng/mL) and cotreated with various extracts, as indicated for 24 h. After simulation, the supernatants were collected for ELISA. The values are presented as the mean ± SD for three independent experiments. ### p < 0.0001 vs. control and **** p < 0.0001, *** p < 0.001, ** p < 0.005, * p < 0.05 vs. PMA/PHA.  . (A,B) The mRNA levels of TNF-α and IL-2 were assayed using RT-PCR. Jurkat cells were stimulated using PMA (100 ng/mL)/PHA (10 ng/mL) and treated with various extracts, as indicated for 3 h. The mRNA levels of TNF-α and IL-2 were determined using qRT-PCR and normalized to those of GAPDH. (C,D) TNF-α and IL-2 levels were measured using an enzyme-linked immunosorbent assay (ELISA). Cells were stimulated with PMA (100 ng/mL)/PHA (10 ng/mL) and cotreated with various extracts, as indicated for 24 h. After simulation, the supernatants were collected for ELISA. The values are presented as the mean ± SD for three independent experiments. ### p < 0.0001 vs. control and **** p < 0.0001, *** p < 0.001, ** p < 0.005, * p < 0.05 vs. PMA/PHA.

Sample Preparation
Carrot seeds were purchased from a local market in Daejeon, Korea, in March 2018 and were cultivated in the Korea Institute of Oriental Medicine (KIOM) for 3 months after being sown in July. Samples used in the separation and analysis were harvested in October 2018. To secure the samples, an expert committee from the KIOM was consulted. The validated samples of the DCR and DCL were separated and deposited as a standard plant in KIOM (KIOM DCL 181010 and KIOM DCR 181011). Each sample was cleaned, and the DCR were chopped into small pieces. Thereafter, the DCR and DCL were dried at 45 • C for 3 days in a drying oven.

Separation and Isolation Procedures
The dried DCL (1 kg) were extracted thrice with 5 L of 70% EtOH at 80 • C for 24 h. The extracted solutions were evaporated under vacuum, and 207.25 g of the 70% EtOH extract was obtained. Furthermore, 185 g of the total extract was suspended in H 2 O (1.5 L) and sequentially partitioned into HEX (3 L × 4), EtOAc, (3 L × 4), and BuOH (3 L × 4). All four separated layers were concentrated using vacuum evaporation. The BuOH extract (18.1 g) was selected based on HPLC chromatogram patterns and results of the biological activity for separation and isolation of the compounds. Column chromatography was conducted using a semi-preparative HPLC with a YMC Triat C18 column (5 µm, 150 mm × 21.20 mm i.d.) at 254 and 320 nm, a flow rate of 15 mL/min, and a gradient system with H 2 O:acetonitrile (ACN) (90:10-75:35 v/v) to separate and identify the active components. After separating the BuOH extract, six fractions (fr. B1-B6) were obtained. The highest content was obtained from fraction B6 (7.2 g), and six main peaks were observed in the chromatogram. Thus, for the next separation step, fraction B6 (1.0 g) was fractionated using semi-preparative HPLC equipped with a YMC Triat C18 column
Dried DCL and DCR were powdered, and 1 g of each was extracted twice with 10 mL of 70% EtOH for 5 min with sonication to prepare the HPLC samples. The extracted sample solutions were filtered using a disposable syringe filter (0.22 µm, 25 mm, CA syringe fitter) from Futecs Co., LTD (Daejeon, Korea). For preparing enzyme-treated DCL samples, 1 g of dried powder was extracted with 3 mL of H 2 O containing 0.1% (v/w) of Viscozyme L or Pectinex, in a water bath set at 50 • C, in five successive treatments: 30 min, 1 h, 3 h, 8 h, and 24 h. EtOH (7 mL extra-pure reagent) was added and the mixture was vortexed for 1 min to deactivate the enzymatic reaction at each time point. All the deactivated samples were sonicated for 5 min and passed through the same disposable syringe filter (0.22 µm, 25 mm, CA syringe fitter) before injecting into the HPLC system.

Validation of the Developed Analytical Method
The HPLC analysis was validated in terms of linearity, accuracy, precision, LOD, and LOQ following the guidelines of the International Conference on Harmonization (ICH). The linearity was established by evaluating r 2 (correlation coefficient) values for the calibration curves generated using 10 serial concentrations. The precision of the analysis was examined by intermediate evaluation using measurements of the intra-and inter-day variability. The intra-day variability was determined by analyzing the sample solution during one of the study days (24 h). In contrast, the inter-day variability was assessed over four days by injecting the sample solutions five times daily. Relative standard deviation (RSD) values were estimated for the retention time and peak area in five experiments. RSD was a measure of precision. Recovery tests were performed to evaluate accuracy in the sample solution spiked with each standard compound. Recovery rates were determined by estimating the mean recovery (%) of the standards from the spiked extract solutions vs. the non-spiked extract sample. LOD and LOQ were determined using the signal-to-noise ratio (S/N), in which S/N ratios of 3 for LOD and 10 for LOQ were used.

Cell Culture and Reagents
Human T lymphocyte cells, Jurkat cells, were obtained from American Type Culture Collection (ATCC). The cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin and maintained in a humidified atmosphere containing 5% CO 2 at 37 • C. PMA and PHA were purchased from Sigma-Aldrich.
3.8. Quantitation of TNF-α and IL-2 in Cell Culture Supernatants Using ELISA TNF-α and IL-2 levels in the cell supernatants were measured using a Human TNFα and IL-2 ELISA kit (Abcam, Cambridge, England), according to the manufacturer's instructions. The OD value was measured at 540 nm using a multi-mode microplate reader (Berthold Technology, Calmbacher, Germany).

Statistical Analysis
Data are presented as means ± SD. Paired Student's t-test was used to compare the groups and ANOVA with Tukey's test was used for multiple comparison using the PRISM software (v6.0; GraphPad Software, San Diego, CA, USA). p-values < 0.05 were considered significant.

Conclusions
In this study, six flavone glycosides were isolated from 70% EtOH DCL extract, and their structures were identified via NMR and MS spectroscopy. Among the isolated compounds 1-6, the structure of compound 4, chrysoeriol-7-rutinoside, from the DCL has been elucidated for the first time. Structural elucidations, DCL quality control, and the reproducibility and accuracy of the HPLC/UV analytical method for the six isolated flavone glycosides along with their three aglycones were fully validated for the first time. From the validation study, all standard compounds in the DCL samples were estimated with accuracy and precision, as confirmed by the recoveries of 94.89-105.97% and RSDs < 1.89.
The effects of the extracts of DCR, DCL, and enzyme-treated DCL extract on the expression of TNF-α and IL-2 in human T lymphocyte cells were investigated using PMA/PHA stimulation. These extracts considerably inhibited the expression of TNF-α and IL-2. The inhibition of TNF-α (p < 0.001) and IL-2 (p < 0.0001) expression by enzyme-treated DCL extract was higher than the inhibition by DCR extract and untreated DCL extract.
The new discovery pertaining to the compounds present in DCL may provide valuable data for standardization of raw materials, which is essential for the development of functional foods and drugs and may also provide valuable information for its general use as food.