Assay of free ferulic acid and total ferulic acid for quality assessment of Angelica sinensis

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Assay of free ferulic acid and total ferulic acid for quality assessment of Angelica sinensis
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  Journal of Chromatography A, 1068 (2005) 209–219 Assay of free ferulic acid and total ferulic acid for qualityassessment of   Angelica sinensis Guang-Hua Lu, Kelvin Chan ∗ , Kelvin Leung, Chi-Leung Chan,Zhong-Zhen Zhao, Zhi-Hong Jiang  Research and Development Division, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China Received 23 November 2004; received in revised form 22 January 2005; accepted 28 January 2005 Abstract Activity of Chinese Danggui (DG), the processed root of   Angelica sinensis  (Oliv.) Diels, is linked to the ferulic acid content but the stabilityof ferulic acid during extraction for medicinal use is not known. The stabilities of ferulic acid and coniferyl ferulate were evaluated in theextracts of DG using a variety of extraction solvents. These included various combinations and proportions of methanol, water, formic acid,1M aqueous hydrochloric acid and 2% sodium hydrogen carbonate (NaHCO 3 ) in water. Coniferyl ferulate was found liable to hydrolyzeinto ferulic acid in neutral, strongly acidic and basic solvents, where heat and water could facilitate this hydrolysis. However, the hydrolysiswas relatively resisted in weakly organic acid. Based on the stability evaluation, two new terms, namely: free ferulic acid and total ferulicacid, were suggested and defined. Free ferulic acid refers to the natural content of ferulic acid in herbs. Total ferulic acid means the sumof free ferulic acid plus the amount of related hydrolyzed components. Meanwhile, the high-performance liquid chromatographic (HPLC)method was developed to assay free ferulic acid and total ferulic acid in DG using methanol–formic acid (95:5) and methanol–2% NaHCO 3  inwater (95:5) as extraction solvents, respectively. Ten DG samples were investigated on their contents of free and total ferulic acid. The resultsindicated that the amount variety of free ferulic acid was larger than that of their counterparts, and the ratio of total ferulic acid to free ferulicacid was 4.07 ± 2.73 (mean ± SD,  n =10). The chemical assay of DG using total ferulic acid content would be a better choice to assess theherbal quality and was recommended.© 2005 Elsevier B.V. All rights reserved. Keywords:  Ferulic acid; Coniferyl ferulate;  Angelica sinensis ; HPLC; Pharmaceutical analysis 1. Introduction Chinese Danggui (Radix  Angelicae Sinensis , DG) is theprocessed root of   A. sinensis  (Oliv.) Diels, which is oneof the widely used traditional Chinese medicinal (TCM)materials to enrich blood, activate blood circulation, regulatemenstruation, relieve pain and relax bowels, etc. There areover eighty composite formulae of TCM containing DG [1].Furthermore, this herb is commonly used as a female tonic,dietary supplements and one of the cosmetic ingredientssold in China, Europe, USA and/or other countries [2–5].Its medicinal value has been demonstrated by numerous ∗ Correspondingauthor.Tel.:+8685234115303;fax:+8685234115317.  E-mail address:  profchan@hkbu.edu.hk (K. Chan). clinical trials, pre-clinical studies and traditional or modernexperiences [6–11].Ferulic acid was isolated from DG and was also foundin other plants [12–14]. Pharmacological studies showedthat ferulic acid and/or sodium ferulate had been found toinhibit platelet aggregation, increase coronary blood flow,relax or stimulate smooth muscle, possess anti-arrhythmicaffects, anti-oxidate, immunostimulate, anti-inflammatoryeffects, etc. [2,6,15–19]. Some of these bioactivities wererelated to the medicinal functions of DG. Therefore, ferulicacid was widely used as one of the marker compounds toassess the quality of DG and its products [20–26]. However,the reported content of ferulic acid in DG varies withinthe range of 0.211–1.43mg/g, and which were quantifiedby a variety of methods (Table 1) [20–36]. Apart from the variation in natural abundance among the samples, the 0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2005.01.082  210  G.-H. Lu et al. / J. Chromatogr. A 1068 (2005) 209–219 Table 1The variation in contents of ferulic acid for  Angelica sinensis  (Oliv.) Diels analyzed by the different methods in literaturesExtraction solvent Extraction method Analytical method Content (mg/g) ReferenceMethanol Reflux TLC 1.08 27Methanol Reflux HPLC 0.418–1.20 28Methanol Sonication HPLC 0.423–1.03 20, 21Methanol Sonication CE 0.211–0.226 22Methanol–formic acid (95:5) Immersion HPLC 0.425 29Methanol–formic acid (95:5) Immersion TLC 0.529 30Methanol–formic acid (95:5) Soxhlet TLC 0.673–1.17 23Methanol–formic acid (95:5) Sonication HPLC 0.233–0.479 31Aqueous methanol Sonication HPLC 0.35–1.43 2470% methanol Reflux HPLC 0.915–1.37 32Ethanol Reflux HPLC 0.468 3370% Ethanol Sonication CE 0.486–1.02 3450% Ethanol Reflux HPLC 0.582–0.606 35, 36Diethyl ether–methanol (20:1) Reflux HPLC 0.271 25Water Reflux CE 0.415 26 nature of extraction solvents and methods were likely to bea critical cause. In the reported literatures, DG sample wascommonly extracted using a variety of solvents, namely:methanol, methanol–formic acid (95:5), ethanol, diethylether–methanol (20:1) and/or water under reflux, sonication,immersion or soxhlet extraction (Table 1) [20–36]. However, coniferyl ferulate, the ester of ferulic acid, was also foundin DG sample [37–39]. Kobayashi et al. reported thatconiferyl ferulate was liable to hydrolyze into ferulic acidand coniferyl alcohol even if the pulverized sample of  Cnidium officinale  Makino was heated in water for 1h(Fig. 1) [40]. According to this reported result, coniferyl ferulate is likely to be hydrolyzed in a variable extent indifferent extraction solvents and therefore resulting in avarietyoflevelofferulicaciddeterminedinherbs.Itisworthnoting that TCM prescription is usually prepared and thendecocted in boiling water. In this regard, coniferyl ferulate inTCM prescription materials are being easily converted intoferulic acid during extraction with boiling water. Therefore,ferulic acid remained as the major chemical constituent inthis aqueous extract. If this is true, ferulic acid should be theprincipal functional compound instead of coniferyl ferulatein DG material according to the TCM practice. Anotherconcern is about the reported levels of ferulic acid in litera-tures which might not represent the natural content in herbsor the actual amount for medicinal functions. Therefore,it is of top importance to examine the stability of ferulicacid in the different extraction conditions and to develop anaccurate method for ferulic acid in DG sample for its qualityassessment and evaluation of the therapeutic effect of DG.This paper focuses mainly on studying the stability andrelationshipofferulicacidwithconiferylferulate,anddevel-oping a quantitative analysis method for the assay of ferulicacid in DG sample. Firstly, ferulic acid and coniferyl ferulatewere identified in the HPLC chromatograms of DG extractsbased on the on-line HPLC-atmospheric pressure chemicalionization (APCI)-MS and UV techniques. Then, the stabil-ities of ferulic acid and coniferyl ferulate were examined inextracts of DG samples by comparing their amounts using avariety of solvent/solvent combination and extraction meth-ods.Thesesolventsincludemethanol,methanol–formicacid,methanol–formic acid–water, methanol–hydrochloric acid(HCl)inwater,methanol–water,waterandmethanol–sodiumhydrogencarbonate(NaHCO 3 )inwater(Table2).Theresultsshowed that coniferyl ferulate was liable to hydrolyze intoferulic acid in neutral, strongly acidic or basic media includ-ing methanol, methanol–water, water, methanol–1M HCl inwater and methanol–2% of NaHCO 3  in water resulting ina variety amount of ferulic acid being determined when theDG samples were extracted by different methods. However,arelativelystableamountofferulicacidcouldbeobtainedinherb extracted with methanol–formic acid or methanol–2%NaHCO 3  in water.Based on the observation, two new terms, ‘free ferulicacid’ and ‘total ferulic acid’, were suggested and defined.Free ferulic acid means the freely available ferulic acid andrepresents the natural content of ferulic acid in herb. Totalferulic acid is the sum of free plus the ferulic acid obtainedfrom hydrolysis of conjugated ferulate, which representsthe amount of ferulic acid in medicinal function. Through a Fig. 1. Chemical structure of (a) ferulic acid, (b) coniferyl ferulate and (c) coniferyl alcohol.  G.-H. Lu et al. / J. Chromatogr. A 1068 (2005) 209–219  211Table 2Comparison of ferulic acid and coniferyl ferulate in the sample of   Angelica sinensis  (Oliv.) Diels extracted with different solvents and methodsNumber Solvent Method Temperature Time (min) Ferulic acid a Coniferyl ferulate a Ratio b 1 Methanol Sonication Ambient 100 1097 . 7 691 . 5 1 . 592 Methanol Sonication 50 ◦ C 100 1429 . 2 315 . 1 4 . 543 Methanol Reflux Boiling 60 1445 . 5 52 . 7 27 . 444 Methanol Reflux Boiling 120 1463 . 7 46 . 8 31 . 315 Methanol Reflux Boiling 180 1657 . 1 4 . 7 351 . 086 Methanol–formic acid (99:1) Sonication Ambient 100 656 . 0 1200 . 5 0 . 557 Methanol–formic acid (95:5) Sonication Ambient 100 665 . 2 1205 . 6 0 . 558 Methanol–formic acid (90:10) Sonication Ambient 100 681 . 2 1188 . 3 0 . 579 Methanol–formic acid–water (90:5:5) Sonication Ambient 100 707 . 7 1227 . 1 0 . 5810 Methanol–formic acid (99:1) Sonication 50 ◦ C 100 693 . 3 1163 . 4 0 . 6011 Methanol–formic acid (95:5) Sonication 50 ◦ C 100 720 . 7 1164 . 1 0 . 6212 Methanol–formic acid (90:10) Sonication 50 ◦ C 100 756 . 6 1155 . 3 0 . 6513 Methanol–formic acid–water (90:5:5) Sonication 50 ◦ C 100 751 . 2 1136 . 5 0 . 6614 Methanol–1M HCl (95:5) Sonication Ambient 100 1281 . 4 481 . 8 2 . 6615 Methanol–1M HCl (90:10) Sonication Ambient 100 1396 . 4 510 . 5 2 . 7416 Methanol–1M HCl (95:5) Sonication 50 ◦ C 100 1501 . 7 81 . 8 18 . 3617 Methanol–water (95:5) Sonication Ambient 100 1239 . 5 545 . 5 2 . 2718 Methanol–water (90:10) Sonication Ambient 100 1289 . 3 519 . 6 2 . 4819 Methanol–water (85:15) Sonication Ambient 100 1329 . 9 508 . 1 2 . 6220 Methanol–water (80:20) Sonication Ambient 100 1560 . 3 365 . 9 4 . 2621 Methanol–water (70:30) Sonication Ambient 100 1700 . 8 136 . 8 12 . 4322 Methanol–water (95:5) Sonication 50 ◦ C 100 1634 . 0 120 . 2 13 . 5923 Methanol–water (95:5) Reflux Boiling 60 1676 . 1 22 . 1 75 . 9424 Methanol–water (95:5) Reflux Boiling 120 1698 . 8 2 . 3 745 . 0725 Water Sonication Ambient 100 573 . 2  c N/A26 Water Sonication 50 ◦ C 100 1081 . 8  c N/A27 Water Reflux Boiling 120 1134 . 6  c N/A28 Methanol–2% NaHCO 3  in water (99:1) Sonication Ambient 100 1648 . 3 25 . 0 65 . 9329 Methanol–2% NaHCO 3  in water (97:3) Sonication Ambient 100 1629 . 9  c N/A30 Methanol–2% NaHCO 3  in water (95:5) Sonication Ambient 100 1704 . 8  c N/A31 Methanol–2% NaHCO 3  in water (93:7) Sonication Ambient 100 1673 . 5  c N/A32 Methanol–2% NaHCO 3  in water (90:10) Sonication Ambient 100 1677 . 4  c N/AN/A: not applicable. a Specific peak area. The value is the ratio of peak area to sample weight, mAUs/g. b Ratio of the peak area of ferulic acid to coniferyl ferulate. c Not detected. series of method validation, two new HPLC methods weredeveloped to quantitatively analyze free ferulic acid andtotal ferulic acid in DG samples, respectively. Altogether 10DG samples including four whole roots, two root heads, tworootlets and two slices were investigated on their contents of free ferulic acid and total ferulic acid (Table 3). The resultsindicated that the variety of the content of free ferulic acidin herbs were generally larger than that of total ferulic acid,and the average amount of total ferulic acid was more thanfour times of that of free ferulic acid. Total ferulic acidshould be a better chemical marker for assessment of herbalquality. Table 3Contents of free ferulic acid and total ferulic acid in the samples of   Angelica sinensis  (Oliv.) DielsSample (voucher number) Sampling part Source Free ferulic acid a Total ferulic acid a Ratio b 1 (020118-09) Whole root Minxian, Gansu, China 0.124  ±  0.002 1.21  ±  0.007 9.732 (020407-01) Whole root Dangchang, Gansu, China 0.377  ±  0.004 0.899  ±  0.002 2.383 (030328-02) Whole root Pingwu, Sichuan, China 0.358  ±  0.001 1.26  ±  0.036 3.534 (020812-01) Whole root Diqing, Yunnan, China 0.172  ±  0.004 0.956  ±  0.009 5.565 (020407-16) Root head Minxian, Gansu, China 0.184  ±  0.004 0.588  ±  0.007 3.206 (020118-11) Root head Weiyuan, Gansu, China 0.100  ±  0.001 0.767  ±  0.010 7.677 (020407-17) Rootlet Minxian, Gansu, China 0.354  ±  0.001 1.09  ±  0.007 3.088 (020407-03) Rootlet Dangchang, Gansu, China 0.384  ±  0.009 0.942  ±  0.008 2.459 (020407-18) Root slice Minxian, Gansu, China 0.529  ±  0.006 0.811  ±  0.003 1.5310 (020407-08) Root slice Weiyuan, Gansu, China 0.296  ±  0.004 0.477  ±  0.001 1.61 a The value is mean ± SD ( n =4), mg/g. The value is expressed in three significant figures. b The value is the ratio of the content of total ferulic acid to that of free ferulic acid in herbs.  212  G.-H. Lu et al. / J. Chromatogr. A 1068 (2005) 209–219 2. Experimental 2.1. Instrumentation An Agilent/HP 1100 series HPLC-DAD system con-sisting of a vacuum degasser, binary pump, autosampler,thermostated column compartment and diode array de-tection (DAD) (Agilent, Palo Alto, CA, USA) was usedfor acquiring chromatogram, UV spectra and 3D-plotsof retention time–absorbance–wavelength. An AppliedBiosystems/PE-SCIEX API 365 LC–MS–MS system withatmospheric pressure chemical ionization source (AppliedBiosystems, Foster City, CA, USA) was used for mass spec-trometric measurements. A Branson 5210E-MTH ultrasonicprocessor (Branso ultrasonics corporation, CT, USA) wasused for sample extraction. An Alltima C 18  column (5  m,250mm × 4.6mm) with a suitable guard column (C 18 ,5  m, 7.5mm × 4.6mm) was used for chromatographicanalysis. The mobile phase consisted of 1.0% acetic acidin water ( A ) and acetonitrile ( B ) using a gradient programof 19%  B  in 0–18min, 19–100%  B  in 18–60min and100%  B  in 60–75min. The flow rate was 1.0mL/min andcolumn temperature was maintained at 30 ◦ C. DAD detectorwas set at 320nm for acquiring the chromatogram. UVspectra were acquired from 200 to 400nm. The APCI–MSspectra were acquired in both the positive and negativeion mode. 2.2. Solvents and chemicals Analytical grade of methanol (Labscan, Bangkok, Thai-land), formic acid, sodium hydrogen carbonate (Unichem,Warsaw, Poland) and hydrochloric acid (Farco, Beijing,China) were used for preparation of standard and/or samplesolutions. HPLC grade acetonitrile (Labscan, Bangkok,Thailand) and analytical grade of glacial acetic acid(Unichem, Warsaw, Poland) was used for preparation of mobile phase. Deionized water was obtained from a Milli-Qwater system (Millipore, Bedford, MA, USA). Ferulicacid was obtained from the Institute for the Control of Pharmaceutical and Biological Products of the People’sRepublic of China (Beijing, China). 2.3. Plant materials Herb samples of   A. sinensis  (Oliv.) Diels were collectedfrom various cultivation areas in China. Sample vouchernumber, source and sampling part were summarized inTable 3. Voucher specimens were preserved at the Schoolof Chinese Medicine, Hong Kong Baptist University (HongKong, China). Samples 1 and 6 were collected in November2001anddriedinshade.Sample1washarvestedinMinxian,Gansu, China whilst sample 6 was purchased in one of themarkets in Weiyuan, Gansu, China. Samples 2, 5, 7–10were collected and supplied by Gansu Shengtai TraditionalChinese Medicine Development Limited (Gansu, China)in April 2002. Sample 4 was a commercial product fromDiqing Pharmaceutical Company (Yunnan, China), and wasprovided by Professor Hao Zhang (West China School of Pharmacy, Sichuan University, Chengdu, China) in June2002. Sample 3 was harvested in November 2002 and driedin the shade by Professor Liang Li (Institute of MianyangTraditional Chinese Medicine, Sichuan, China). 2.4. Preparation of standard solution for calibrationand linearity studies For assay of free ferulic acid, the stock solution of ferulic acid was prepared at a concentration of 100mg/L inmethanol–formic acid (95:5). Calibration standard solutionswere prepared in the concentration range of 1–15mg/L withmethanol–formic acid (95:5). For assay of total ferulic acid,thestocksolutionofferulicacidwaspreparedataconcentra-tion of 500mg/L in methanol–2% NaHCO 3  in water (95:5).Calibration standard solutions were prepared in the concen-tration range of 5–45mg/L with methanol–2% NaHCO 3  inwater (95:5). An aliquot of 10  L solution for each cali-bration standard solution was injected for HPLC analysis.The calibration curve was constructed by plotting the peak areas of the analyte against the concentration of ferulicacid. 2.5. Sample preparation Representative samples were cut into smaller pieces andfurther grounded into powder, and passed through a 20-mesh(0.9mm) sieve. The grounded powders were stored at about4 ◦ C before use.For assay of free ferulic acid, 0.5g of sample powder wasaccurately weighed and transferred into a 60mL amber vial.Twenty-five milliliters of methanol–formic acid (95:5) wasaddedandsonicatedfor100min.Theextractwasnormalizedto25mLbyaddingadditionalextractionsolvent.Theextractwas filtered through a 0.2  m membrane filter. An aliquotof 10  L solution was injected for HPLC analysis. Sampleduplicates were prepared.For assay of total ferulic acid, the sample preparation wasmade under similar treatment as the assay of free ferulic acidexcept replacing the extraction solvent with methanol–2%NaHCO 3  in water (95:5). 3. Results and discussion 3.1. Identification of ferulic acid and coniferyl ferulate Ferulic acid was identified in the HPLC chromatogramof DG extracts by spiking authentic standard and comparedtheir UV and APCI–MS spectra. Owing to the unavailabil-ity of authentic compound, the identification of coniferylferulate in the chromatogram was based on the on-lineHPLC–APCI–MS and UV spectra [41].  G.-H. Lu et al. / J. Chromatogr. A 1068 (2005) 209–219  213 3.2. Stability of ferulic acid and coniferyl ferulate Bothferulicacidanditsester,coniferylferulate,arecoex-istinginDG[37–39].Kobayashietal.reportedthatconiferylferulatewasreadilyhydrolyzedintoferulicacidandconiferylalcohol that would be easily decomposed [40]. In literature,a variety of extraction solvents and conditions had been em-ployed in the determination of ferulic acid in DG samples.Thecontentofferulicacidwasfoundtobewithintherangeof 0.211–1.43mg/g (Table 1) [20–36]. It was suggested that the significant variation in contents of ferulic acid was probablyattributedinacertainextenttothedifferenceinsampleprepa-ration and extraction procedures. In this study, the stabilitiesandlevelsofferulicacidandconiferylferulateinthedifferentextraction conditions were examined. Aqueous methanolicsolvents in different pH values had been employed for ex-traction by sonication or reflux (Table 2). Although coniferylferulate standard was not available, specific peak area (peak area/sampleweight,mAUs/g)wasusedfortheassaypurposein this study.For the evaluation of optimal extraction time, a compar-ative study of different sonication time, namely, 20, 40, 60,80, 100, 120 and 150min, was conducted by sonication atambient temperature using the ultrasonic processor. Resultsshowedthatafterextractionfor40min,theamountsofferulicacid were not obviously changed further in these extracts.Considering the possible difference in the nature of herbalsamples, a duration of 100min was chosen as the optimalextraction time for sonication extraction whilst 60min wasselected as the optimal extraction time under refluxed condi-tion to ensure the quantitative extraction of ferulic acid.The stabilities and amounts of ferulic acid and coniferylferulate with different extraction conditions are described asfollows: 3.2.1. Sample extraction with methanol Methanolwasthemostcommonlyusedextractionsolventintheassayofferulicacidinherbsinliteratures.DGsampleswere usually extracted with methanol by sonication for20–30min or under reflux for 3h (Table 1) [20–22,27–28]. In this study, the amounts of ferulic acid and coniferylferulate were compared in the methanolic extracts of DGby sonication for 100min at ambient temperature, 50 ◦ Cand reflux for 60, 120 and 180min, respectively (Table 2,numbers 1–5). In Fig. 2, the remarkable differences inlevels were observed in the chromatographic patterns. Theresults showed that their extracts were obviously differentin relative levels. The differences among the two extremitiesin specific peak areas were 1.5 times for ferulic acid and147.1 times for coniferyl ferulate. The ratios of peak area of ferulic acid to coniferyl ferulate were also ranging from 1.59to 351.1. It indicated that coniferyl ferulate was unstablein methanol at elevated temperatures. In general, the higherthe extraction temperature was, the more coniferyl ferulatewere hydrolyzed and hence the larger peak area of ferulicacid was being observed. This chemical transformation of  Fig. 2. Chromatograms of the root of   Angelica sinensis  (Oliv.) Diels ex-tracted with methanol by sonication for 100min at (1) ambient temperatureand (2) 50 ◦ C, reflux for (3) 60min, (4) 120min and (5) 180min, respec-tively. (a) Ferulic acid; (b) coniferyl ferulate (analytical column: AlltimaC 18 ,5  m,250mm × 4.6mm;guardcolumn:C 18 ,5  m,7.5mm × 4.6mm;injected sample volume: 10  L; mobile phase: 1.0% acetic acid in water( A ) and acetonitrile ( B ) using a gradient program of 19%  B  in 0–18min,19–100%  B  in 18–60min and 100%  B  in 60–75min; flow rate: 1.0mL/min;temperature: 30 ◦ C; measured at UV 320nm.). coniferyl ferulate to ferulic acid could be visualized in the3D-plots of retention time–absorbance–wavelength (Fig. 3).The stabilities of ferulic acid and coniferyl ferulate inmethanolic extract during storage were further evaluated.DG sample was extracted with methanol by sonication for100min at ambient temperature. The amounts of ferulic acidand coniferyl ferulate were determined in the sample overthe storage period of 0.7–127h after sonication. By compar-ing peak areas of ferulic acid and coniferyl ferulate, the peak area of ferulic acid was increased by 60.5% at 127h withthe RSD of 19.0% ( n =11) while that of coniferyl ferulatewas decreased by 58.1% with the RSD of 26.1% ( n =11)(Fig. 4A). It indicated that coniferyl ferulate was relativelyunstable and readily decomposed into ferulic acid whilst theamount of ferulic acid was almost quantitatively increased inthe methanolic extracts during storage. 3.2.2. Sample extraction with methanol–formic acid  Methanol–formic acid (95:5) was another widely usedsolvent for the assay of feruilic acid in DG sample in somerecent publications (Table 1) [23,29–31]. The amounts of ferulic acid and coniferyl ferulate in DG extracts wereevaluated with methanol–formic acid (99:1, 95:5 and 90:10)and methanol–formic acid–water (90:5:5) by sonicationfor 100min at ambient temperature and 50 ◦ C, respectively(Table 2, numbers 6–13). The results showed that thechromatographic pattern of DG samples were similar(Fig. 5). The RSD of the specific peak area were 5.3% ( n =8)for ferulic acid and 2.6% ( n =8) for coniferyl ferulate inthese extracts. The ratio of the peak area of ferulic acid to
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