Screening and Analysis of the Potential Bioactive Components of Poria cocos (Schw.) Wolf by HPLC and HPLC-MSn with the Aid of Chemometrics

The aim of the present study was to establish a new method based on Similarity Analysis (SA), Cluster Analysis (CA) and Principal Component Analysis (PCA) to determine the quality of different samples of Poria cocos (Schw.) Wolf obtained from Yunnan, Hubei, Guizhou, Fujian, Henan, Guangxi, Anhui and Sichuan in China. For this purpose 15 samples from the different habitats were analyzed by HPLC-PAD and HPLC-MSn. Twenty-three compounds were detected by HPLC-MSn, of which twenty compounds were tentatively identified by comparing their retention times and mass spectrometry data with that of reference compounds and reviewing the literature. The characteristic fragmentations were summarized. 3-epi-Dehydrotumulosic acid (F13), 3-oxo-16α,25-dihydroxylanosta-7,9(11),24(31)-trien-21-oic acid (F4), 3-oxo-6,16α-dihydroxylanosta-7,9(11),24(31)-trien-21-oic acid (F7) and dehydropachymic acid (F15) were deemed to be suitable marker compounds to distinguish between samples of different quality according to CA and PCA. This study provides helpful chemical information for further anti-tumor activity and active mechanism research on P. cocos. The results proved that fingerprint combined with a chemometric approach is a simple, rapid and effective method for the quality discrimination of P. cocos.

The therapeutic effects of traditional Chinese medicines (TCMs) are based on the complex interactions of complicated chemical constituents as a whole system, so methods are needed in order to control the quality of the complex system. In this case, HPLC fingerprints of key components provide a new approach for quality control of traditional Chinese medicines. There are many studies about fingerprints analysis combined with chemometrics for the quality control of traditional Chinese medicines and to find the bioactive components [23][24][25][26][27].
Some studies on the fingerprints of Poria cocos (Schw.) Wolf have been reported [28][29][30][31], but in those reports only a few compounds were identified by HPLC-MS n and the characteristic fragmentations were not summarized. No marker compounds were found from cluster analysis (CA) and principal component analysis (PCA).
In the present study, nineteen common peaks and four other peaks which have not been detected using HPLC were identified by high-resolution liquid mass spectrometry. To the best of our knowledge, this is the first time that so many compounds were identified and their characteristic fragmentations summarized. We also found for the first time that 3-epi-dehydrotumulosic acid (F13), 3-oxo-16α, 25-dihydroxylanosta-7,9(11),24(31)-trien-21-oic acid (F4), 3-oxo-6,16α-dihydroxylanosta- 7,9(11),24(31)-trien-21-oic acid (F7) and dehydropachymic acid (F15) might be suitable marker compounds to distinguish between P. cocos samples with different quality according to CA and PCA. This study provides helpful chemical information for further anti-tumor activity and active mechanism research on P. cocos. The method developed in our study also provides a scientific foundation for the origin discrimination and quality control of P. cocos.

Validation of the Method
The relative retention time, relative peak area and similarities were used to evaluate the quality of the fingerprints. Dehydrotumulosic acid (peak 8) which is a large single peak in the middle of the chromatogram, was assigned as the reference peak to calculate relative retention times and relative peak areas.
The precision was determined by replicate injection with the same sample solution six consecutive times. The RSDs of relative retention time and relative peak area of the common peaks were all below 0.87% and 1.47%, respectively; the similarities of different chromatograms were all above 0.995.
The repeatability was evaluated by the analysis of six prepared samples. The RSDs of relative retention time and relative retention time of the common peaks were all below 1.59% and 1.97%, respectively; the similarities of different chromatograms were all above 0.995.
Stability testing was performed with one sample over 24 h. The RSDs of relative retention time and relative retention time of the common peaks were all below 0.96% and 1.98%; the similarities of different chromatograms were all 1.000. All these results indicated that the samples remained stable during the testing period and the conditions for the fingerprint analysis were satisfactory.

Similarity Analysis (SA)
The chromatographic profile must be representative of all the samples and have the features of integrity and fuzziness. By analyzing the mutual pattern of chromatograms, the identification and authentication of the samples can be conducted well even if the amounts of some chemical constituents are different from the others.
Fifteen batches of samples from different habitats were determined and the chromatograms analyzed by SES to generate a common pattern R (Figure 1). SES for Chromatographic Fingerprint was performed to calculate the similarities of different chromatograms compared to the common pattern. The results are shown in Table 1. NO. S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 R S1  Figure 1. Overlaid HPLC chromatograms of samples from No. S1-S15. The common pattern (marked R) was obtained by using Similarity Evaluation System (SES) for Chromatographic Fingerprint of TCM.
The conclusion can be drawn from the results that the similarities of different chromatograms compared to the common pattern are all above 0.900 except for samples S4 (0.880), S6 (0.885), S7 (0.872), S11 (0.860) and S13 (0.875), which indicates that the chemical constituents of different samples are not influenced highly by sources. The common pattern is a very positive identification for the samples of P. cocos.

Identification of the Compounds Present
HPLC-ESI-MS n method was employed to identify the components in P. cocos (Figure 2-4) Molecular weight and fragmentation information ( Table 2) were obtained. The possible structures of these 19 common peaks and four other peaks a1, a2, a3 and a4 were deduced as it shown in Figure 5. Under the optimized MS conditions, positive mode and negative mode were used to identify the peaks.   Overlaid HPLC chromatograms of samples from No. S1-S15. The common pattern (marked R) was obtained by using Similarity Evaluation System (SES) for Chromatographic Fingerprint of TCM.
The conclusion can be drawn from the results that the similarities of different chromatograms compared to the common pattern are all above 0.900 except for samples S4 (0.880), S6 (0.885), S7 (0.872), S11 (0.860) and S13 (0.875), which indicates that the chemical constituents of different samples are not influenced highly by sources. The common pattern is a very positive identification for the samples of P. cocos.

Identification of the Compounds Present
HPLC-ESI-MS n method was employed to identify the components in P. cocos (Figures 2-4) Molecular weight and fragmentation information ( Table 2) were obtained. The possible structures of these 19 common peaks and four other peaks a1, a2, a3 and a4 were deduced as it shown in Figure 5. Under the optimized MS conditions, positive mode and negative mode were used to identify the peaks. Overlaid HPLC chromatograms of samples from No. S1-S15. The common pattern (marked R) was obtained by using Similarity Evaluation System (SES) for Chromatographic Fingerprint of TCM.
The conclusion can be drawn from the results that the similarities of different chromatograms compared to the common pattern are all above 0.900 except for samples S4 (0.880), S6 (0.885), S7 (0.872), S11 (0.860) and S13 (0.875), which indicates that the chemical constituents of different samples are not influenced highly by sources. The common pattern is a very positive identification for the samples of P. cocos.

Identification of the Compounds Present
HPLC-ESI-MS n method was employed to identify the components in P. cocos (Figure 2-4) Molecular weight and fragmentation information ( Table 2) were obtained. The possible structures of these 19 common peaks and four other peaks a1, a2, a3 and a4 were deduced as it shown in Figure 5. Under the optimized MS conditions, positive mode and negative mode were used to identify the peaks.   The conclusion can be drawn from the results that the similarities of different chromatograms compared to the common pattern are all above 0.900 except for samples S4 (0.880), S6 (0.885), S7 (0.872), S11 (0.860) and S13 (0.875), which indicates that the chemical constituents of different samples are not influenced highly by sources. The common pattern is a very positive identification for the samples of P. cocos.

Cluster Analysis (CA)
Cluster Analysis is a multivariate analysis technique that is used to sort samples into groups. It is widely applied for fingerprint analysis, because it is a nonparametric data interpretation method and simple to use. CA provides a visual representation of complex data. Average linkage between groups was applied, and Pearson correlation was selected as a measurement. The method can classify different herbs by measuring the peak areas from their corresponding HPLC fingerprints. The common characteristic peaks, which were calculated by the Similarity Evaluation System, were selected for the CA. Cluster analysis of P. cocos samples was performed based on the relative peak areas of all 19 common peaks.
The results of CA are shown in Figure 6, where the quality characteristics are revealed more clearly. The cluster analysis results show that the samples could be divided into three quality clusters. Among them, Cluster I includes the samples S6, S8, S15, S1 and S9, Cluster III includes S2, S5 and the others are in Cluster II. All the compounds in Cluster III had much higher concentrations than the other two clusters.

Principal Components Analysis (PCA)
To evaluate the variations in quality of the 15 samples, PCA was carried out with the relative amounts of each identified component. The contents of 19 fingerprint peaks were applied to evaluate the sample variations. Figure 7 shows the score plots obtained by PCA. The first six principal components accounted for 89.329% of the total variance. Examination of the score plots indicates that the main components responsible for the separation were 3-epi-dehydrotumulosic acid (F13), 6α-hydroxyldehydropachymic acid (F6), 24(31)-trien-21-oic acid (F4), 24(31)-trien-21-oic acid (F7), 3-oxo-6,16α-dihydroxylanosta-7, 9 (F15), 29-hydroxydehydrotumulosic acid (F1), dehydropachymic acid (F12), as shown in Table 3. These components were deemed to be the marker compounds of sample variation. This result is in accord with the one obtained from the cluster analysis (CA). The combination of PCA and CA was thus a useful tool for quality control and evaluation of P. cocos.

Principal Components Analysis (PCA)
To evaluate the variations in quality of the 15 samples, PCA was carried out with the relative amounts of each identified component. The contents of 19 fingerprint peaks were applied to evaluate the sample variations. Figure 7 shows the score plots obtained by PCA. The first six principal components accounted for 89.329% of the total variance. Examination of the score plots indicates that the main components responsible for the separation were 3-epi-dehydrotumulosic acid (F13), 6α-hydroxyldehydropachymic acid (F6), 24(31)-trien-21-oic acid (F4), 24(31)-trien-21-oic acid (F7), 3-oxo-6,16α-dihydroxylanosta-7, 9 (F15), 29-hydroxydehydrotumulosic acid (F1), dehydropachymic acid (F12), as shown in Table 3. These components were deemed to be the marker compounds of sample variation. This result is in accord with the one obtained from the cluster analysis (CA). The combination of PCA and CA was thus a useful tool for quality control and evaluation of P. cocos.

Samples and Reagents
Fifteen P. cocos samples were purchased from different regions of China and were authenticated by Professor Chun-Sheng Liu (School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China). The samples were harvested between July and September. The samples were processed as follows: the sediment was removed after them digging up, and the material was piled to "sweat", spread out until the surface was dry, then "sweated" again. This was repeated several times until the surface of the samples was wrinkled and the water in the sample was almost dissipated. Samples were then dried in the shade, peeled and cut into cubes. The surface of the blocks is white or faint red in color. Each sample (three replicates) was placed in a dark and dry environment. The regions where the 15 samples were obtained are shown in Table 4. Pachymic acid (Batch number: 130306, purity ≥ 98%) and dehydroeburicoic acid (Batch number: 131027, purity ≥ 98%) were obtained from Chengdu MUST BioTechnology Co., Ltd. (Chengdu, China); HPLC grade acetonitrile and acetic acid were obtained from Fisher (Waltham, MA, USA); distilled water was bought from Watsons (Beijing, China) and was filtered through a 0.45 µm membrane (Dikma, Beijing, China) prior to use. All other reagents were of analytical grade. Chuxiong, Yunnan S10 Xinxiang, Henan S3 Dali, Yunnan S11 Yulin, Guangxi S4 Lijiang, Yunnan S12 Jinzhai, Anhui S5 Luotian, Hubei S13 Chengdu, Sichuan S6 Shennongjia, Hubei S14 Suining, Sichuan S7 Yundu, Guizhou S15 Yuexi, Anhui S8 Fujian

Preparation of Reference Substance
Stock solutions of individual reference substance were prepared by dissolving each compound in 50% methanol at a concentration of 212 µg·mL −1 for pachymic acid and 22.9 µg·mL −1 for dehydroeburicoic acid. Both solutions were stored at approximately 4 °C.

Samples and Reagents
Fifteen P. cocos samples were purchased from different regions of China and were authenticated by Professor Chun-Sheng Liu (School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China). The samples were harvested between July and September. The samples were processed as follows: the sediment was removed after them digging up, and the material was piled to "sweat", spread out until the surface was dry, then "sweated" again. This was repeated several times until the surface of the samples was wrinkled and the water in the sample was almost dissipated. Samples were then dried in the shade, peeled and cut into cubes. The surface of the blocks is white or faint red in color. Each sample (three replicates) was placed in a dark and dry environment. The regions where the 15 samples were obtained are shown in Table 4. Pachymic acid (Batch number: 130306, purity ě 98%) and dehydroeburicoic acid (Batch number: 131027, purity ě 98%) were obtained from Chengdu MUST BioTechnology Co., Ltd. (Chengdu, China); HPLC grade acetonitrile and acetic acid were obtained from Fisher (Waltham, MA, USA); distilled water was bought from Watsons (Beijing, China) and was filtered through a 0.45 µm membrane (Dikma, Beijing, China) prior to use. All other reagents were of analytical grade. Chuxiong, Yunnan S10 Xinxiang, Henan S3 Dali, Yunnan S11 Yulin, Guangxi S4 Lijiang, Yunnan S12 Jinzhai, Anhui S5 Luotian, Hubei S13 Chengdu, Sichuan S6 Shennongjia, Hubei S14 Suining, Sichuan S7 Yundu, Guizhou S15 Yuexi, Anhui S8 Fujian

Preparation of Reference Substance
Stock solutions of individual reference substance were prepared by dissolving each compound in 50% methanol at a concentration of 212 µg¨mL´1 for pachymic acid and 22.9 µg¨mL´1 for dehydroeburicoic acid. Both solutions were stored at approximately 4˝C.

Preparation of Sample Solution
Dried powder of P. cocos from different regions (1 g) was accurately weighed out and transferred into a 100 mL conical flask. Methanol (10 mL) was added to the flask and the flask with the methanol and powder was accurately weighed and placed in an ultrasonic extraction device and extracted for 30 min. The flask was weighed again and methanol was added to make up the weight. The solution was filtered through a 0.45 µm membrane filter for fingerprint analysis.
The LCMS-IT-TOF instrument (Shimadzu, Kyoto, Japan) was equipped with an ESI source used in positive and negative ionization mode. The interface and MS parameters were as follows: nebulizer pressure, 100 kPa; dry gas, N 2 (1.5 L/min); drying gas temperature, 200˝C; spray capillary voltage, 4000 V; scan range, m/z 100-1500.

Conclusions
The therapeutic effects of traditional Chinese medicines (TCM) are based on the complex interactions of complicated chemical constituents as a whole system. HPLC and HPLC-MS n fingerprint analysis combined with chemometrics were employed to study the complex P. cocos system. Triterpenoid acids were the most important chemical components in the samples, which had a variety of potential biological activities, according to previous extensive phytochemical and pharmacological studies. The qualitative analysis and quantification of triterpenoid acids can better reflect the therapeutic effects and quality of P. cocos. The chromatographic method is predominantly to control the quality and stability of the complex system. This study provided a systematic method for the quality control of P. cocos by HPLC fingerprinting and the HPLC-MS n evaluation system based on Similarity Analysis (SA), Cluster Analysis (CA) and Principal Component Analysis (PCA). As a result, a common mutual pattern was established by determining and comparing the fingerprints of 15 samples of P. cocos from different regions. Twenty-three compounds were detected by HPLC-MS n , of which twenty were tentatively identified by comparing their retention times, and mass spectrometry data with that of reference compounds and literature data. The characteristic fragmentations were summarized. 3-epi-Dehydrotumulosic acid (F13), 3-oxo-16α,25-dihydroxy-lanosta-7,9(11),24(31)-trien-21-oic acid (F4), 3-oxo-6,16α-dihydroxylanosta-7,9(11),24(31)-trien-21-oic acid (F7) and dehydropachymic acid (F15) were deemed to be the markers to distinguish between P. cocos samples of different quality. The proposed method can be used to improve the quality control of P. cocos, thus ensuring the effectiveness of Poria herbs. There are still three peaks-F9, F11 and a3-which were not identified by HPLC-MS n , of which F9 and F11 were used as marker compounds to distinguish the P. cocos of different quality. These two components require further study.