Abstract: Comparative studies of different Fritillaria Chinese medicinal materials were carried out using X-ray powder diffraction. Due to existence of plenty of starches in Fritillaria, which is a very good semicrystalline macromolecule, the X-ray diffraction pattern of Fritillaria powders mainly showed the crystal properties of starch. However, the crystal properties of starch contained in Fritillaria were highly different due to the differences of species. These differences in crystal properties of starch resulted in the differences in the diffraction pattern. In addition, the differences in other chemical constituents contained in Fritillaria also resulted in the differences in the diffraction patterns. All of these differences could be utilized for the discrimination and identification of different Fritillaria Traditional Chinese Medicine. In the present research, we conducted a systematic study on eight kinds of Fritillaria traditional Chinese medicines by X-ray power diffraction. X-ray diffraction is proved to be a new and powerful method to discriminate Fritillaria traditional Chinese medicines from different geographical origins.
Introduction
Fritillaria (Chinese name Beimu), the bulbs of various species of the genus Fritillaria (Liliaceae), is a very useful Traditional Chinese Medicine (TCM) with antitussive and expectorant functions (Cheetham et al., 1998; Chen and Liu, 2004; Gao and Li, 1994; Gao et al., 1997). Officially, herbal Beimu is derived from the bulbs of five Fritillaria species documented in China pharmacopoeia (Hua et al., 2003). These species include Fritillaria thunbergii Miq., Fritillaria ussurensis Maxim., Fritillaria pallidifloca Schrenk, Fritillaria cirrhosa D. Don and Fritillaria hupehensis Hsiao et K.C. Hsia which are all quality goods. Moreover, F. cirrhosa consists of F. cirrhosa D. Don, F. unibracteata Hsiao et K.C. Hsia, F. przewalskii Maxim., F. delavayi Franch. The former three are usually named as Song Beimu and Qing Beimu, whereas the later one is named as Lu Beimu according to their different characters. China has a wide range of Fritillaria resources which are distributed extensively. The Fritillaria Chinese medicinal materials are very easy to confuse, in addition, the size of Fritillaria bulb is much relevant to the growing conditions (Li et al., 1999). All of these result in the difficulty in original identification of Fritillaria.
By far, chromatographic method is mainly utilized for the identification of different Fritillaria Chinese medicinal materials. Since there are tens of major bioactive components, which are slightly different due to different growing conditions and geographical origins, we can not select only a limited number of specific constituents as essential evaluative criteria. Indeed, there are some contradicting results concerning the contents of some ingredients contained in the Fritillaria in the literature (Li et al., 2000; 2001). In the holistic theory of traditional Chinese medicine, the medicinal materials take effects in curing diseases as a whole. Any method or technique which destroys the wholeness of the traditional Chinese medicine will not be primarily accepted. Recently, there are some methods which keep the integrity of traditional Chinese medicine for discrimination of Fritillaria such as Fourier Transform Infrared spectroscopy (FTIR), differential thermal analysis (DTA) and X-ray diffraction analysis (Lin et al., 2001; Lu et al., 1997; Song and Zhao, 1997).
X-ray diffraction has the advantages of good fingerprint character and non-destruction. As we all know, the main component in the bulbs of Fritillaria is starch which occupied approximately 80% content in the total biomass (Wang et al., 2005). Starches separated form different Fritillaria showed different physicochemical, morphological, thermal and crystal properties in our previous studies (Wang et al., 2006a, b; Xiao, 2002). Starch is a good semicrystalline polymer which shows obvious diffraction peaks in the X-ray diffraction patterns. So, diffraction peaks of starch predominated in the diffraction pattern of Fritillaria powder. In order to discriminate the Fritillaria Traditional Chinese Medicine, in this study, different Fritillaria were studied by using X-ray powder diffraction. By analyzing the differences in the X-ray diffraction data of the Fritillaria, we could easily discriminate the Fritillaria Chinese medicinal materials.
Materials and Methods
Apparatus
X-ray powder diffraction measurements were done using Panalytical X’Pert
Pro diffractometer (PANalytical, Holand).
Samples
Fritillaria thunbergii Miq., Fritillaria ussurensis Maxim., Fritillaria
pallidifloca Schrenk, Fritillaria cirrhosa D.Don, Fritillaria
hupehensis Hsiao et K.C., Song Beimu, Qing Beimu, Lu Beimu were identified
and provided by National Institutes for the control of Pharmaceutical and Biological
Products.
Procedure
Firstly, all the eight Fritillaria bulbs were purified, comminuted
to powders which were sieved with 160 mesh sifter and then kept in a desiccator.
The eight Fritillaria powders were placed in the air for two weeks (in
order to balance the water content of Fritillaria powder) and then would
be tested. The dried Fritillaria powders were extracted with 95% ethanol
by cold immersion method for 24 h. The supernatant was removed and the settled
solid layer was resuspended in 95% ethanol and the cold immersion lasted for
24 h once again. The resulting suspension was filtrated with a G4 type anti-acid
filter. The residue was washed with 95% alcohol for several times, allowed to
dry at room temperature. The dried residue powder was resuspended with distilled
water again and again till the supernatant was transparent. The starch was then
collected and dried at room temperature for further use.
Secondly, each sample of Fritillaria powder and Fritillaria starch was packed tightly in a rectangular glass cell and put the glass cell in the diffractometer. The operating conditions included: the CoK|αtube operated at a fixed power source(40KV, 40mA), Scanning range of [4-60°] for Fritillaria powders or [4-40°] for Fritillaria starch, λ = 1.78901, step size of 0.0330, scan step time 30.8451. All X-ray diffraction measurements were done in air, at room temperature. The same measurements were made at room temperature for three times.
Thirdly, the degree of crystallinity of samples was quantitatively. A smooth curve which connected peak baselines was computer-plotted on the diffractograms (Fig. 1). The area above the smooth curve was taken as the crystalline portion and the lower area between smooth curve and the linear baseline which connected the two points of the intensity 2θ of 30° and 10° in the samples was taken as the amorphous section.
| Fig. 1: | Calculation of the relative degree of the crystallinity |
The upper diffraction peak area and the total diffraction area over the diffraction angle 10-30°2θ were integrated using Smadchrom software (Morgan and Kennedy Research, Australia). The ratio of upper area to total diffraction was taken as the degree of crystallinity. For this evaluation, we utilized the powders which had almost identical moisture contents (~12%) in order to minimize the effect of different moisture contents on crystallinity.
The Equation of the degree of crystallinity is as follows:
Xc = Ac / (Ac + Aa) |
Where:
Xc refers to the degree of crystallinity; Ac refers to the crystallized area on the X-ray diffractogram; Aa refers to the amorphous area on the X-ray diffractogram.
Lastly, the data reported in all the tables are average of triplicate observations. Statistical comparison of means was conducted using the Student’s t-test in a General Linear Model (GLM) procedure on an SAS system (release 8.2, SAS Institute, Cary, NC).
Results and Discussion
X-ray Diffraction Patterns of Fritillaria Powders
X-ray diffraction is an important method to study phase and crystal structure
of substance. With the development of X-ray diffraction technology, it is now
used for the identification of the Traditional Chinese Medicine (TCM) and TCM
preparation. Spectra obtained by this method possess the characteristics of
much information, strong fingerprint and stability and reliability. Some of
them were utilized to analyze qualitatively according to a set of powder diffraction
data cards published by international powder X-ray association board.
The X-ray powder diffractograms of F. thunbergii, F. ussurensis, F. palldifloca, F. cirrhosa and F. hupehensis were presented in Fig. 2.
All the five Fritillaria powders gave the strongest diffraction peak at 20.0°2θ and a few small peaks at around 2θ values of 6.6°, 16.6°, 23.1°, 26.1°, 28.3°, 30.1°. These diffraction peaks were mainly attributed to the semicrystalline starch contained in the Fritillaria. This result revealed that crystal type of starches contained in the five Fritillaria was a characteristic B-type. As for the F. thunbergii, the intensity of diffraction peak at 6.7°2θ was weaker than that of the other Fritillaria.
| Fig. 2: | X-ray diffraction patterns of five Fritillaria powders. a: F. thunbergii; b: F. ussurensis; c: F. pallidifloca; d: F. cirrhosa and e: F. hupehensis |
| Table 1: | Degree of crystallinity of the five Fritillaria powders |
| Mean values with the same letters are not significantly different | |
F. thunbergii and F. cirrhosa only showed a small single-diffraction peak at 16.8°2θ, whereas the single-diffraction peak at 16.8°2θ was divided into two diffraction peaks at 16.4° and 17.3° 2θ for F. ussurensis, F. pallidifloca and F. hupehensis. F. hupehensis showed two additional diffraction peaks at around 2θ values of 36.4°and 40.4° while it was only one diffraction peak at about 40.4°2θ for the other Fritillaria.
According to the above analysis, the five Fritillaria can be sorted into three classes: F. ussurensis and F. pallidifloca belong to one class, F. thunbergii and F. cirrhosa belongs to another class, F. hupehensis belongs to the third class.
The degree of crystallinity of five kinds of Fritillaria powders calculated from the above Fig. 2 were shown in Table 1.
In the light of the degree of crystallinity in Table 1, the five Fritillaria can also be divided into three classes: F. thunbergii belongs to one class, F. ussurensis and F. cirrhosa belong to another class, F. pallidifloca and F. hupehensis belong to the third class. If the differences in characteristic diffraction peaks of Fritillaria powders were considered, the five Fritillaria could clearly be discriminated.
In order to discriminate the different F. cirrhosa better, they were studied by X-ray diffraction analysis. The X-ray diffraction patterns of three F. cirrhosa were shown in Fig. 3.
Three F. cirrhosa also gave the strongest diffraction peak at 20.1°2θ and a few small peaks at around 2θ values of 6.6°, 16.6°, 23.1°, 26.1°, 28.3°, 30.1°. As for Song Beimu, it showed two obvious diffraction peaks at 16.6°2θ while there was not obvious double peaks for Qing Beimu and Lu Beimu in the X-ray diffraction patterns. The degree of crystallinity of three F. cirrhosa calculated from Fig. 3 was shown in Table 2.
The degree of crystallinity of the three F. cirrhosa differed significantly (Table 2), Song Beimu showed the highest degree of crystallinity, whereas Qing Beimu showed the lowest degree of crystallinity. According to the difference in the degree of crystallnity of three F. cirrhosa, they can be separated into three classes clearly.
| Table 2: | Degree of crystallinity of the three F. cirrhosa powders |
| Mean values with the same letters are not significantly different | |
| Table 3: | X-ray diffraction data of starches from different Fritillaria |
| a Means with the same letter are not significantly different (p<0.05) | |
| Fig. 3: | X-ray diffraction patterns of different F. cirrhosa powders. a: Qing Beimu; b: Song Beimu; c: Lu Beimu |
X-ray Diffraction Measurements of Fritillaria Starches
In order to further discriminate the different Fritillaria, the X-ray
diffraction patterns of starch contained in the Fritillaria was obtained
(Fig. 4).
The corresponding X-ray diffraction parameters and crystallinity level calculated from the ration of diffraction peak area and total diffraction area were given in Table 3. The scattering angle, at which the diffraction intensities can be observed was 2θ and the d spacing was used to discriminate the planes of different sites. The five Fritillaria starches showed the highly similar X-ray diffraction patterns. They gave the strongest diffraction peaks at 6.6°and 20°2θ and a few small peaks at around 2θ values of 17°, 23°, 26°, 28° and 30°. These diffraction patterns were basically the same as that of potato starch. This analysis indicated that the crystal type of starches separated from the five Fritillaria was a characteristic B-type.
Due to the different origins of Fritillaria, starch contained in the bulb of Fritillaria had the different crystal properties such as diffraction angle, d spacing and degree of crystallinity. For example, although the crystal type of all the five Fritillaria starches was characteristic B-type, diffraction angles at 6°, 20° 2θ and corresponding d spacing were different from each other.
| Fig. 4: | X-ray diffraction spectra of the five Fritillaria starches. a: F. thubergii; b: F. ussurensis; c: F. crrhosa; d: F. pallidifloca; e: F. huphensis |
| Fig. 5: | X-ray diffraction spectra of the three Fritillaria starches, a: Qing Beimu., b: Song Beimu, c: Lu Beimu |
| Table 4: | X-ray diffraction data of starches from different F. cirrhosa |
| a Means with the same letter are not are not significantly different (p<0.05) | |
The crystallinity level also differed significantly each other. According to the above detailed diffraction parameters and crystallinity level of starch from different Fritillaria, the five Fritillaria could be easily separated.
Although three Cirrhosa F. starches showed the highly similar X-ray diffraction patterns (Fig. 5), they still showed the different X-ray diffraction parameters and crystallinity level (Table 4). For example, d spacing at 20° 2θ of Qing Beimu starch was 5.07 Å, whereas they were 5.13 Å and 5.17 Å for Song Beimu and Lu Beimu starches. Further more, degree of crystallinity of starches from different F. cirrhosa differed significantly. All of these could provide us information to discriminate different F. cirrhosa.
Conclusions
According to the above analysis, we could conclude that different Fritillaria from different geographical origins could be identified by X-ray powder diffraction patterns. Due to the differences in types and content of chemical constituents in different Fritillaria, they showed the characteristic X-ray diffraction patterns and parameters which could help us discriminated the different Fritillaria from different geographical origins. In addition, starch from different Fritillaria also showed the different parameters and degree of crystallinity. These differences in the crystalline properties of starch also could provide us the information to discriminate different Fritillaria. Because this technology is very advanced and simple, it could be recommended for the discrimination and identification of other Chinese medicine which were very difficult to identify.