determination of bioactive compounds in cortex phellodendri

DIETARY SUPPLEMENTS

Determination of Bioactive Compounds in Cortex Phellodendri byHigh-Performance Liquid Chromatography

QI YANG

Changzheng Hospital, Second Military Medical University, Department of Pharmacy, 415 Fengyang Rd, Shanghai 200003,People’s Republic of ChinaFENG ZHANG

Changzheng Hospital, Second Military Medical University, Department of Pharmacy, 415 Fengyang Rd, Shanghai 200003,China and Second Military Medical University, School of Pharmacy, 325 Guohe Rd, Shanghai 200433, People’s Republicof ChinaSHOU-HONG GAO

Changzheng Hospital, Second Military Medical University, Department of Pharmacy, Shanghai 200003, People’s Republic of ChinaLIAN-NA SUN

1

Second Military Medical University, School of Pharmacy, 325 Guohe Rd, Shanghai 200433, People’s Republic of ChinaWAN-SHENG CHEN

1

Changzheng Hospital, Second Military Medical University, Department of Pharmacy, 415 Fengyang Rd, Shanghai 200003,People’s Republic of China

An HPLC method combined with a photodiode array detector was developed for quantitativedetermination of five bioactive compounds thatbelong to two subclasses, including limonin,phellodendrine, jatrorrhizine, palmatine, andberberine in Cortex Phellodendri. The analysis wasperformed on an Agilent Diamonsil C18 column (4.6´ 250 mm, 5 mm) using a gradient of acetonitrile and0.3% aqueous diethylamine phosphate (v/v), a flowrate of 0.8 mL/min, and a detection wavelength of220 nm. The calibration curve was linear over therange of 2.5–100.0 mg/mL for both phellodendrineand jatrorrhizine, 5.0–200.0 mg/mL for palmatine, and 7.5–300.0 mg/mL for both berberine and limonin. The average recoveries ranged from 97.56 to 102.53%with RSD £ 1.00%. Samples from differentgeographical locations were analyzed to evaluatethe applicability of the established method, and theresults indicated that the method was efficient,sensitive, and reliable for determining limonin andfour alkaloids in Cortex Phellodendri.

Cortex Phellodendri (CP, “Huangbo” in Chinese) is thedried bark of two botanical species, Phellodendron amurense Rupr. (PAR) or P. chinense Schneid (PCS)

(both Family Rutaceae). It has been widely used as a drug inTraditional Chinese Medicine for the treatment of diarrhea ordysentery, jaundice, morbid leucorrhea, swelling pains in the

knees and feet, urinary tract infections, and infections of thebody surface (1). Alkaloid components are active ingredientsthat exhibit a wide spectrum of biological and pharmacologicalactivities, including these effects: antimicrobial (2, 3),anti-inflammation (4), inhibition of aldose reductase (5, 6),insecticidal (7, 8), anticancer (9–11), anti-inflammatory (12),and antihypersensitivity (13, 14). Limonin (also namedobaculactone) is another important phytochemical in CP; it isalso abundant in plants such as Rutaceae and Meliaceae.Currently, investigations are being conducted on its therapeuticeffects, such as anti-HIV (15), antioxidant (16),CYP3A4-inhibitory (17), antineoplastic (18, 19), multidrugresistance reversal (20), and neuroprotective activities (21).

In contrast to previously reported methods for analysis ofalkaloids in CP (22–29) or limonin in CP (27, 30), all thesamples were also determined by an HPLC-UV method in our study. Although HPLC provides limited information forelucidation of molecular structure, it is still the most populartechnique for determining active compounds because of itsrapidity, simplicity, and convenience (31). This paper presents an HPLC-UV method capable of simultaneous determinationof five bioactive compounds that belong to two subclasses,including limonin, phellodendrine, jatrorrhizine, palmatine,and berberine (Figure 1) in CP at a single wavelength. Thedifference in bioactive contents of PAR and PCS was alsoobserved when this method was used to evaluate the quality of CP for its safe clinical use.

Experimental

Chemicals and Plant Materials

Acetonitrile was of HPLC grade (Merck, Darmstadt,Germany). Diethylamine phosphate was of analytical grade.HPLC grade water was prepared using a Milli-Q Water

YANG ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 3, 2010 855

Received September 20, 2009. Accepted by AP January 4, 2010.1 Corresponding author’s e-mail: [email protected] and

[email protected]

purification system (Millipore, Billerica, MA). Standards ofphellodendrine, jatrorrhizine, palmatine, berberine, andlimonin were purchased from the National Institute forControl of Biological and Pharmaceutical Products (Beijing,China). CP samples collected from eight different locations inChina (CP1-CP8) and samples purchased in different drugstores (CP9-CP27) were authenticated by Han-Ming Zhang,Department of Pharmacognosy, School of Pharmacy, SecondMilitary Medical University, Shanghai, China. Voucherspecimens were deposited at the Department ofPharmacognosy, School of Pharmacy, Second MilitaryMedical University.

Apparatus and Chromatographic Conditions

An Agilent 1200 LC system, equipped with aquaternary solvent delivery system and an autosampler andUV detector, was used in this study. A Diamonsil C18

column (4.6 ´ 250 mm, 5 mm, Dikma Technologies,Beijing, China) connected with a Diamonsil SB-C18 guard

column (4 ´ 20 mm, 5 mm; Dikma) was used for all analyses.The mobile phase consisted of acetonitrile (eluent A)and 0.3% aqueous diethylamine phosphate (v/v, eluent B).The column was eluted at 0.8 mL/min under a linear gradientfrom 15% eluent A to 35% for 25 min, to 70% for 5 min, andto 95% for 25 min. The sample injection volume was 10 mL.Compounds were detected at 220 nm with a photodiode arraydetector at 25°C.

Sample Preparation

The dried CP samples (CP1-CP27) were comminuted(60 mesh), and the powders (0.25 g) were suspended in 1%phosphoric acid–methanol (50 mL) in a beaker flask (100 mL)overnight, and weighed again. Then the beaker flask was sealed and extracted by ultrasonication at room temperature for60 min. Methanol was added into the beaker flask to reach theinitial weight after cooling. The supernatant (about 10 mL)was filtered, and the filtrate was filtered through a 0.45 mmmembrane filter; aliquots were submitted to preparative HPLC.

856 YANG ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 3, 2010

Figure 1. Chemical structure of compounds under study.

Table 1. Regression curves, linearity, LOD, and LOQ for five bioactive markers

Compound Regression equationa r Linear range, mg/mL LOD, mg/mL LOQ, mg/mL

1 y = 24.908 x – 13.025 0.9998 2.5–100.0 0.8 2.5

2 y = 34.554 x – 18.191 0.9995 2.5–100.0 0.8 2.5

3 y = 42.546 x – 28.490 0.9999 5.0–200.0 1.7 5.0

4 y = 38.650 x + 36.676 0.9999 7.5–300.0 2.5 7.5

5 y = 3.6711 x + 40.245 0.9994 7.5–300.0 2.5 7.5

a In the regression equation, y = a x + b, x refers to the concentration of the five compounds (mg/mL) and y refers to the peak area (n = 5).

Preparation of Standard Solution

A stock solution (2.0 mg/mL) was prepared in methanol for every standard. Phellodendrine, jatrorrhizine, palmatine,berberine, and limonin standards (100, 100, 200, 300, and300 mL) were separately mixed into a beaker flask (2 mL) anddiluted to the final volume with 15% acetonitrile. A serialdilution of the solution was made with 15% acetonitrile toprepare standard solutions at concentrations of 1/2, 1/4, 1/8,1/10, 1/16, 1/20, and 1/40 for establishment of calibrationcurves.

Linearity

Five consecutive injections of each standard solution wereprepared. Calibration curves were calculated to determine thecorrelation coefficient (r) for each standard component. Themethod was linear over the range of 2.5–100.0 mg/mL for bothlimonin and phellodendrine, 5.0–200.0 mg/mL for jatrorrhizine, and 7.5–300.0 mg/mL for both palmatine and berberine. All thecompounds showed good linearity (r > 0.999) within theconcentration range (Table 1).

LOD and LOQ

The LOD and LOQ were determined based on S/N valuesof approximately 3:1 and 10:1, respectively. The lowestconcentration of the calibration curves was found to be LOQ,and the solution was diluted to three-fold above LOD. Fiveinjections of the LOQ and three injections of the LODsolutions were prepared, and the RSD% for the LOQ solutionwas determined (Table 1).

Precision

A sample (Tonghua, Jilin, CP12) was prepared asdescribed above and subjected to HPLC analysis five times on the same day to evaluate intraday repeatability, and once eachday for 3 consecutive days to assess interday repeatability.The analytical precision from the results was indicated by theRSD; the intraday and interday precisions were almost<2.00% (Table 2). The results also indicated that thesecompounds were stable in methanol solution.

Recovery

The samples (Meihekou, Jilin, CP16), fortified by addingknown quantities of the mixed standard compounds, wereextracted and analyzed three times by our HPLC method. Theratio of detected amount to added amount was used tocalculate the recovery (Table 3). The mean recovery of themethod was 97.56–102.53%, with RSD <1.00%. The resultsof the recovery test showed that the method was accurate.

Stability

Stability was tested using standard and sample solutionsstored at room temperature and analyzed at 0, 2, 4, 8, 16, 24,36, and 48 h. It was shown that the analytes were rather stable(RSD < 2.8%).

Results and Discussion

Optimization of Extraction Method

To obtain optimal extraction efficiency, different extraction techniques and solvents were tested to determine thecombination that gives the highest recovery of the


Table 2. Intraday and interday precision of developed assay for five bioactive markers, sample from Tonghua, Jilin(CP12)

Compound Concentration, mg/mL

Intraday (n = 5) Interday (n = 5)

Detected, mg/mL RSD, % Detected, mg/mL RSD, %

1 50.00 51.11 ± 0.30 0.58 50.20 ± 0.20 0.40

25.00 24.75 ± 0.05 0.18 24.78 ± 0.05 0.18

5.00 5.02 ± 0.02 0.30 5.06 ± 0.03 0.51

2 50.00 58.15 ± 0.24 0.41 58.56 ± 0.57 0.97

25.00 27.63 ± 0.18 0.66 27.30 ± 0.10 0.37

5.00 4.85 ± 0.04 0.89 5.00 ± 0.03 0.57

3 100.00 101.07 ± 0.25 0.24 100.24 ± 0.06 0.06

50.00 49.62 ± 0.03 0.06 49.66 ± 0.03 0.07

10.00 9.97 ± 0.01 0.10 10.07 ± 0.01 0.11

4 150.00 150.76 ± 0.40 0.27 150.20 ± 0.11 0.07

75.00 73.34 ± 0.07 0.10 74.65 ± 0.07 0.09

15.00 14.00 ± 0.03 0.20 15.36 ± 0.03 0.22

5 150.00 149.81 ± 1.05 0.70 150.15 ± 3.71 2.47

75.00 80.22 ± 0.65 0.81 76.08 ± 0.30 0.40

15.00 19.56 ± 0.13 0.66 15.30 ± 0.74 4.86

compounds. First, methanol was chosen as the extractionsolvent because the compounds are well-soluble in it. Figure 2 clearly shows that the highest extraction yield was obtainedusing ultrasonication for 60 min after staying overnight (M4).Then, different solvents were tested by M4, and the resultsshowed that acid methanol (S1, S2, and S3) yielded morecompounds than pure methanol (S4). All compounds wereobtained in S1, S2, and S3 as well, but methanol–water(0.10% phosphoric acid; S3) was the preferred solventbecause it was consistent with the mobile phase.

Optimization of HPLC Conditions

To obtain chromatograms with a good separation outcome,different mobile phase compositions were tested. It was foundthat the peaks were broad and overlapped whenmethanol–water was used. However, when acetonitrile wasapplied instead of methanol, the situation became better,which was consistent with the previous report (32).

HPLC-UV is currently the main choice for the analysis ofalkaloids. However, with traditional silica packings,secondary interactions between analyte and residual silanolsmay induce peak tailing, which can affect the resolution,sensitivity, and reproducibility (33). To avoid thisshortcoming, several stationary phases with speciallydesigned groups have been developed to reduce theaccessibility to free silanols (34), as well as mobile phaseswith electrolyte buffers, ion-pairing reagents, or aminemodifiers to raise ionic strength (32, 35). But the use of a highconcentration buffer or ion-pairing solution will lead toinstrument erosion and a longer time for column balancing,which may affect the quality of HPLC chromatograms (35).Hence, addition of diethylamine phosphate in mobile phasewas chosen as the amine modifier to improve the peak shapeor baseline separation, as it was previously used (24). It wascoordinated with the extraction solvent, methanol withphosphoric acid, which was used in the sample preparation.

Chemical structures similar to those of palmatine andberberine (Figure 1) often lead to coelution, which requiresthe optimization of parameters, i.e., flow rate, temperature,gradient program, and pH of the mobile phase, whichappeared to be the crucial factor. Finally, 0.3% aqueousdiethylamine phosphate was chosen after differentconcentrations were tested. The gradient program should be


Table 3. Analytical results of recoveries (n = 3) of sample from Meihekou, Jilin (CP16)

Compound Original, mg/mL Added, mg Detected, mg/mL Recovery, % Average, % RSD, %

1 30.48 250 35.34 97.15 97.56 0.83

250 35.33 97.05

250 35.40 98.49

2 8.46 1600 45.99 102.66 102.53 0.21

1600 45.87 102.28

1600 45.98 102.64

3 55.07 1000 76.51 97.53 97.62 0.53

1000 76.64 98.18

1000 76.43 97.15

4 146.50 2100 191.13 101.97 102.18 0.20

2100 191.30 102.37

2100 191.22 102.18

5 144.45 2800 201.53 101.47 101.09 0.88

2800 200.74 100.06

2800 201.66 101.72

Figure 2. Compound levels in mg/g (dry weight)obtained with different extraction methods and solvents from CP12 (n = 3).

long enough to avoid incomplete separation among fivestandards. The baseline separation of all compounds wasobtained under the established conditions (Figure 3). The little peak in the front of peak 5 was not impurity, but caused by thegradient elution, which was aimed at time reduction as well asgood separation, so it made no difference in the calculations.

Application of the Method

Different CP samples of the two species were assessed byour method (Table 4). We found that the contents of thecompounds varied greatly in the different samples. Thecontents of berberine, which was considered the dominantalkaloid in CP, varied from 6.77 to 49.87 mg/g in PCS,and 3.72 to 14.65 mg/g in PAR, which further supports thedivision of CP into PAR and PCS based on their composition

differences. The composition variation between the twospecies may be mainly due to climatic and geographicalcharacteristics. In addition, the differences observed insamples of the same species could be attributed to harvestingtime, drying process, and storage conditions. However, thecontent of jatrorrhizine ranged from 0.23 to 0.93 mg/g in allsamples and was relatively stable. On the other hand, PAR had a lower content of phellodendrine than PCS. In contrast, thecontents of palmatine and limonin were significantly higherthan those found in PCS. The content of limonin ranged from10.31 to 15.48 mg/g in PAR samples and was relatively stable.

Berberine was found to be the major alkaloid in CP, whichexplains why the standard derived from its contentsdiffers between PAR and PCS, and based on which, CP hasbeen clarified in two entries in the latest edition of Chinese


Figure 3. HPLC chromatograms of (A) standard mixture, (B) CP4, and (C) CP9. The marker compounds:(1) phellodendrine; (2) jatrorrhizine; (3) palmatine; (4) berberine; and (5) limonin.

Pharmacopoeia (1). Some other herbs, such as CoptidisRhizome, Radix Berberidis, and Folium Mahonicum, are alsorich in berberine, which indicates that the evaluation of CP byberberine only lacks specificity (24). Furthermore, mostbioactivities of CP were associated with the bioactivecompounds; therefore, the quantification of the compoundsshould be considered in the quality of CP. Toensure the safety, quality, and efficacy of the relatedpreparations, it is necessary to establish a simple, specific, and multiple-component analytical technique for determination of these compounds in CP.

Conclusions

We successfully developed a single HPLC-UV assay forthe simultaneous analysis of five bioactive constituents that

belong to two subclasses of CP. The method was successfullyused to determine these constituents in two species of CP, andmight be an effective, exclusive means of evaluation for QC of CP. The results of the present study contribute to theknowledge of the major composition of CP, and allow for acomparison of samples of two species from different sources.

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Table 4. Quantities of five bioactive markers in CP in different regions aggregated (mg/g crude drug; n = 3)

Sample No.a Origin Species

Content, mg/gb

1 2 3 4 5

CP1 Henan P. Chinense Schneid. 4.11 0.56 0.37 38.55 2.37

CP2 Sichuan1 P. Chinense Schneid. 1.43 0.32 2.97 6.77 10.74

CP3 Sichuan2 P. Chinense Schneid. 2.16 0.61 3.78 9.03 14.18

CP4 Jilin1 P. amurense Rupr. 1.88 0.41 4.10 4.70 13.45

CP5 Jilin2 P. amurense Rupr. 2.30 0.62 3.84 9.23 12.40

CP6 Heilongjiang P. amurense Rupr. 2.12 0.57 3.97 8.53 12.91

CP7 Liaoning1 P. amurense Rupr. 1.79 0.44 2.98 6.31 10.31

CP8 Liaoning2 P. amurense Rupr. 2.54 0.62 4.53 10.05 12.70

CP9 Chengdu, Sichuan P. Chinense Schneid. 5.26 0.40 0.26 42.75 3.39

CP10 Yaan, Sichuan P. Chinense Schneid. 2.94 0.27 0.61 18.72 5.58

CP11 Xinjiang P. Chinense Schneid. 5.64 0.26 0.12 49.87 1.73

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CP24 Ginan, Shandong P. amurense Rupr. 2.77 0.93 4.84 10.92 14.28

CP25 Shenzhen, Guangdong P. amurense Rupr. 1.84 0.50 3.69 7.13 11.91

CP26 Changchun2, Jilin P. amurense Rupr. 1.06 0.23 2.81 6.72 11.17

CP27 Nanchang, Jiangxi P. amurense Rupr. 2.16 0.76 5.17 12.33 15.41

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determination of bioactive compounds in cortex phellodendri

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