Quantitation and Radical Scavenging Activity Evaluation of Iridoids and Phenylethanoids from the Roots of Phlomis umbrosa (Turcz.) using DPPH Free Radical and DPPH-HPLC Methods, and their Cytotoxicity원문보기
Le, Duc Dat
(Drug Research and Development Center, College of Pharmacy, Catholic University of Daegu)
,
Nguyen, Duc Hung
(Drug Research and Development Center, College of Pharmacy, Catholic University of Daegu)
,
Zhao, Bing Tian
(Drug Research and Development Center, College of Pharmacy, Catholic University of Daegu)
,
Min, Byung Sun
(Drug Research and Development Center, College of Pharmacy, Catholic University of Daegu)
,
Song, Si Whan
(ChemOn Inc., Gyeonggi Bio-Research Center)
,
Woo, Mi Hee
(Drug Research and Development Center, College of Pharmacy, Catholic University of Daegu)
The roots of Phlomis umbrosa (Turcz.) (Phlomidis Radix) have been traditionally used to treat cold, reduce swelling and staunch bleeding. Four iridoids (1 - 3 and 5) and six phenylethanoid derivatives (4, and 6 - 10) were isolated from the roots of P. umbrosa. A simple, sensitive, and reliable analy...
The roots of Phlomis umbrosa (Turcz.) (Phlomidis Radix) have been traditionally used to treat cold, reduce swelling and staunch bleeding. Four iridoids (1 - 3 and 5) and six phenylethanoid derivatives (4, and 6 - 10) were isolated from the roots of P. umbrosa. A simple, sensitive, and reliable analytical HPLC/PDA method was developed, validated, and applied to determine 10 marker compounds in Phlomidis Radix. Furthermore, the isolates were evaluated for cytotoxic and anti-oxidant activities as well as DPPH-HPLC method. Among them, compounds 4 and 6 - 9 displayed potent anti-oxidant capacities using DPPH assay with $IC_{50}$ values of $27.7{\pm}2.4$, $10.2{\pm}1.1$, $18.0{\pm}0.8$, $19.1{\pm}0.3$, and $19.9{\pm}0.6{\mu}M$, and compounds 6, 8, and 9 displayed significant cytotoxic activity against HL-60 with $IC_{50}$ values of $35.4{\pm}3.1$, $18.6{\pm}2.0$, and $42.9{\pm}3.0{\mu}M$, respectively.
The roots of Phlomis umbrosa (Turcz.) (Phlomidis Radix) have been traditionally used to treat cold, reduce swelling and staunch bleeding. Four iridoids (1 - 3 and 5) and six phenylethanoid derivatives (4, and 6 - 10) were isolated from the roots of P. umbrosa. A simple, sensitive, and reliable analytical HPLC/PDA method was developed, validated, and applied to determine 10 marker compounds in Phlomidis Radix. Furthermore, the isolates were evaluated for cytotoxic and anti-oxidant activities as well as DPPH-HPLC method. Among them, compounds 4 and 6 - 9 displayed potent anti-oxidant capacities using DPPH assay with $IC_{50}$ values of $27.7{\pm}2.4$, $10.2{\pm}1.1$, $18.0{\pm}0.8$, $19.1{\pm}0.3$, and $19.9{\pm}0.6{\mu}M$, and compounds 6, 8, and 9 displayed significant cytotoxic activity against HL-60 with $IC_{50}$ values of $35.4{\pm}3.1$, $18.6{\pm}2.0$, and $42.9{\pm}3.0{\mu}M$, respectively.
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문제 정의
There are several studies using HPLC development method to find bioactive constituents from medicinal plants. The aims of this study were focused on the isolation and the development of new HPLC analytical method for the major compounds from the roots of P. umbrosa. Additionally, their anti-oxidant activity was evaluated based on above developed method.
가설 설정
b Adriamycin was used as the positive control.
제안 방법
A gradient RP-C18 HPLC system was performed for the simultaneous quantitative determination of 10 compounds (1 – 10).
Determination of DPPH-HPLC assay − The antioxidant activities of marker compounds in methanol extract were carried out using a DPPH-HPLC assay9 with a slight modification.
HPLC instrument and chromatographic conditions −The quantitative analyses were conducted on an HPLC chromatography (Waters, Houston, TX, USA) equipped with an autosampler, degasser, quaternary solvent pump,and photodiode array detector (PDA).
The estimate for the LOQ was calculated using S/N ratio of 10. Intra-day (n = 5) and inter-day (n = 5) precisions and accuracies were evaluated by analyzing sets of five independent samples at the low, mid, and high concentration levels. The precision was expressed as RSD% and the accuracy was expressed as bias.
Method validation − The validation parameters of the developed HPLC-PDA method for the roots of P.umbrosa were linearity, limit of detection (LOD) and limit of quantitation (LOQ), accuracy, precision, stability, and robustness.
Precision and accuracy − The accuracy of the developed HPLC method was determined by analyzing the known amounts of analytes spiked into methanol extract solution of the roots of P. umbrosa.
umbrosa were grinded for 5 min and were then sieved through a 250 μmsieve to ensure required sample homogeneity. The mixtures of solvent systems were used as following: 95%, 75%, 50%, 25% ethanol and 100%, 75%, 50%, 25% methanol for extraction solvents. The P.
The quantitative analyses were conducted on an HPLC chromatography (Waters,Houston, TX, USA) and a Kinetex C18 column (4.6 × 250 mm, 5 μm particle size; Phenomenex, Torrance, CA,USA).
In comparison between ultra-sonication and reflux method using 100% methanol extraction solvent, the sample assay results were superior after extraction with sonication than with reflux. The time needed for complete extraction was determined with five lengths of time (30, 45, 60, 75, and 90 min) in 100% methanol via sonication at room temperature. The extraction time for 60 min yielded similar sample assay results as 75 min.
umbrosa through storing extract solution in the dark at 4 ℃ and room temperature(25 ℃). The two samples were analyzed in triplicate at 0,1, 3, 7, 15, and 30 days separation.
The concentration range is generally chosen as per International Conference on Harmonization guidelines. Triplicate analysis for each analyte was carried out. The linearity regression parameters of the calibration curves with correlation coefficients ranging between 0.
대상 데이터
Four comparing samples (PU2 − PU5) were harvested in August 2016, at different geographic regionsin Korea (PU2: Uiseong-gun, Gyeongsangbuk-do; PU3:Andong-si, Gyeongsangbuk-do; PU4: Yeongcheon-si,Gyeongsangbuk-do; PU5: Taebaek-si, Gangwon-do).
Samples 1 − 5 were marked as PU1 −PU5. Sample PU1 (Yeongwol-gun, Gangwon-do) was collected in March 2016, and used for isolation and validation. Four comparing samples (PU2 − PU5) were harvested in August 2016, at different geographic regionsin Korea (PU2: Uiseong-gun, Gyeongsangbuk-do; PU3:Andong-si, Gyeongsangbuk-do; PU4: Yeongcheon-si,Gyeongsangbuk-do; PU5: Taebaek-si, Gangwon-do).
The voucher specimens (PU1 − PU5) of the plants were deposited in Herbarium at College of Pharmacy, Catholic University of Daegu, Korea.
이론/모형
The cytotoxic activity of the isolates (1 − 10) was evaluated against HL-60 as well as MCF-7 and Hela cell lines. The inhibitory process was assessed by MTT assay. According to the results, compounds (6, 8, and 9) significantly showed cytotoxic effects against HL-60 cell lines with IC50 values of 35.
성능/효과
The LOD of these isolated constituents weredetermined to be 0.013 to 0.430 μg/mL and the LOQ was 0.042 to 1.435 µg/mL indicating that the developed method for the roots of P. umbrosa exhibited good sensitivity.
The results were shown in Table 2. The method precision was measured by six successive injections, and the precisions were less than 3.28% in intra-day and 4.83% in inter-day.
The method validation indicated that the regression equations of the marker compounds were linear and this method was precise, accurate, and reliable for quantitation of the 10 marker compounds (1 − 10).
The results exhibited that all the marker compounds (1 − 10) in the D. asperoides samples disappeared in the chromatogram(Fig. 2C) except for compound 4 (0.919 ± 0.002%).
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