Phase Composition and Analysis of Surface Micromorphology of Animal Medicine Concha Arcae and Calcined Concha Arcae

The components of concha arcae and calcined concha arcae were analyzed by X-ray powder diffraction and calculated their contents, and their surface morphology was observed by scanning electron microscope. The experimental results showed that the concha arcae was composed of aragonite type and calcite type calcium carbonate, and calcined concha arcae was composed of calcium oxide, calcium hydroxide and calcium carbonate. The surface micromorphology of them were significantly different. Rietveld refinement can accurately and quickly complete the phase identification and quantitative analysis of raw and calcined products of concha arcae by Powder X-ray diffraction, it is more convenient and fast compared with chemical titration method.


Introduction
The marine animal traditional Chinese medicine concha arcae is the shell of Arca subcrenataLischke, Acragranosa Linnaeus or Acrainflata Reeve, also known as clam shell, tile house, ark shell, etc [1].Concha arcae is the first Marine medicine in Shennong Herbal Classic.Its name is derived from sea clam, Quebec clam, KuiLu, cockle, clam, ark bell, etc [2].The processing of concha arcae has a long history.There are five kinds of processing methods of herbs collected in previous dynasties, including raw product grinding, fire calcination, calcined vinegar quenching, calcined vinegar boiling and calcined brine boiling, among which calcined method is still used today.In the book "Lei Gong's Processing Theory", the processing method of "intermediates should be burned on fire and calcined until red" was first put forward.The processing method of calcined concha arcae in Chinese Pharmacopoeia 2020 is as follows: clean the concha arace, calcine it to crispness according to the calcination method, and specify that calcium carbonate is used as the indicator component for the content determination of concha arcae [3].
Marine traditional Chinese medicine is an important part of Chinese traditional medicine resources, which has been used to prevent and treat diseases for thousands of years.Concha arace tastes sweet and salty, and its nature is flat; It belongs to the lung, stomach and liver meridians.It has the effects of eliminating phlegm, removing blood stasis, softening and firmness, relieving acid and relieving pain, and can be used to treat scrofulous gall tumors, persistent phlegm and cough, stomach pain and acid vomiting, dental chancre, traumatic bleeding, chilblain, scald and fire injuries, etc [4,5].Modern pharmacological studies have proved that the mechanism of the effect of concha arace on acid inhibition and anti-acid is the neutralization of the active ingredient calcium carbonate with acid.However, after calcination, the acid making, pain relieving and astringent effects of concha arcae are enhanced, because alkaline calcium oxide and calcium hydroxide are generated in the calcination process, and the ability to neutralize gastric acid is enhanced, so the acid making and pain relieving effects are stronger.Yongping Liu et al. found that when treating small area burns, the concha arcae ointment not only had less dosage, rapid onset, shortened exudation time, accelerated wound healing, but also had fewer systemic adverse reactions and convenient clinical operation [6].In addition, Hu Ding et al. used concha arcae powder combined with mesalazine to treat ulcerative colitis, and the experimental results showed that the effect was significant [7].It was speculated that such compatibility could improve immunity, reduce inflammatory reaction, and improve oxygen free radical scavenging capacity.Oh Y et al. found that the concha arcae protein derived peptide can up regulate the typical Wnt/ β-Catenin signaling pathway can stimulate osteoblast differentiation of human bone marrow mesenchymal stem cells, which can be used as a potential drug to treat osteoporosis [8].
Concha arcae has high medicinal value.The crystal structure changes after calcination, which can produce the effect of enhancing efficiency and reducing toxicity.The effective ingredients of traditional Chinese medicine are the material basis to play a therapeutic role, and the effective ingredient of concha arcae is calcium carbonate.At present, EDTA complexometric titration method is mainly used for the analysis of macro components in concha arcae, while Rietveld refined quantitative calculation has not been reported.In this study, powder X-ray diffraction analysis combined with Rietveld refinement was used to analyze and calculate the macro components and their content percentage in concha arcae and calcined concha arcae, and a fast and effective quantitative calculation method was established, which has important research significance for the analysis of marine animal traditional Chinese medicine; The morphology and structure of the two were characterized by scanning electron microscope, and their microscopic differences were observed, which can be used to evaluate the quality of concha arcae products and help to better play the clinical medicinal value of concha arcae products.The experiment confirmed the feasibility of Rietveld refined quantitative calculation of macro components in raw and calcined concha arcae products, and provided a new method for quantitative calculation of concha arcae components.This method can quickly realize quantitative analysis of concha arcae, judge whether the content of its effective components is qualified, and optimize the quality control methods of shellfish medicines such as concha arcae.

Experimental instruments and materials
The phase test was completed on the intelligent high-power Smart lab 2006 X-ray diffractometer (Nihon Nihon Co., Ltd.).The test voltage was 30 kV, the acceleration voltage was 5 kV, and the current was 20 mA.The scanning mode was continuous scanning.The scanning speed was 4º/min, and the scanning range was 10 o ~80 o .The copper target Cu Ka radiation was used as the line source (λ = 1.54184Å).The morphology was observed using SU-5000 field emission scanning electron microscope (Hitachi, Japan), electronic balance (JA5003N, Shanghai Precision Scientific Instrument Co., Ltd.); Mafu Furnace (dl-10251, Nanjing Prade Machinery Manufacturing Co., Ltd.); Ceramic mortar; 100 mL beaker; 200 mesh sieve; Distilled water; Concha arcae (purchased from the Chinese Herbal Medicine Slices Factory of Guangxi Guigang Lvzhiyuan Seeding and Breeding Development Co., Ltd., and identified by Associate Professor Xiaohua Wang , Teaching and Research Section of Traditional Chinese Medicine, Guilin Medical University).

Experimental methods
The preparation process of concha arcae powder is as follows: Weigh 30g raw concha arcae, wash, dry, grind, pass 200 mesh sieve, and then dry.The concha arcae powder is almost white.The preparation process of calcined concha arcae powder is as follows: Weigh 30g of raw concha arcae powder and place it in a crucible, then put it into a muffle furnace, heat it to 750℃ and calcine it for two hours, cool it naturally, take it out, crush it and pass it through a 200 mesh sieve.The calcined concha arcae powder is grayish white and crisp.Take the above two kinds of powders for X-ray diffraction analysis, explore their phase composition and carry out quantitative analysis; The surface microstructures of the two were observed by scanning electron microscopy to see if they were different.

Figure 1. XRD patterns of concha arcae
Figure 1 shows the X-ray diffraction pattern of concha arcae.Calcium carbonate has three isomers, which are orthorhombic aragonite, trigonal calcite and hexagonal vaterite.It can be seen from the Xray diffraction pattern that the sample at 2θ to 26.26 °, 27.28 °, 33.14 °, 37.9 °, 45.88 ° and 52.44 °, there are obvious diffraction peaks.The positions of these diffraction peaks correspond to the atlas of aragonite type calcium carbonate standard sample (PDF # 76-0606), indicating that the raw concha arcae is mainly composed of aragonite type calcium carbonate; In addition, the sample also had peak responses at 29.46 ° and 39.44 °, which corresponded to the atlas of calcite calcium carbonate standard (PDF # 83-0577), indicating that the secondary component of raw concha arcae was calcite calcium carbonate.From the above analysis, we can see that concha arcae stone is composed of two phases, namely aragonite and calcite.
. XRD patterns of calcined concha arcae Figure 2 shows the X-ray diffraction pattern of calcined corrugated.It can be seen from the X-ray diffraction pattern that the sample at 2θ to 32.22 °, 37.36 °, 53.86 °, 64.14 ° and 67.38 °, there are obvious diffraction peaks, and the positions of most of the diffraction peaks correspond to the atlas of calcium oxide standard (PDF # 99-0070), which indicates that most of the ingredients in the calcined concha arcae are calcium oxide.In addition, the sample also has a high peak response at 23.14 °, 29.40 °, 36.03°, 39.45 °, 43.16 ° and 48.53 °, which basically corresponds to the calcium carbonate standard (PDF# 86-0174), indicating that the calcined concha arcae contains a small amount of calcium carbonate.Finally, when corresponding to the standard spectrogram of calcium hydroxide (PDF# 72-0156), there are also peak responses at 28.73 °, 34.17 °, 47.25 °, 50.89 ° and 54.46 °, but the response value is not high, indicating that the calcined concha arcae contains a small amount of calcium hydroxide at the same time.From the above analysis, it can be seen that calcined concha arcae is composed of three components, namely calcium oxide, calcium carbonate and calcium hydroxide.The micro-morphology of concha arcae powder was characterized by SEM, as shown in Figure 3.It can be seen from SEM image 3a that the surface of the concha arcae is not smooth, and is full of uneven holes, which are unevenly distributed, of different sizes, and irregular.Figure 3b shows the microstructure magnified to 1.5 thousand times.It can be seen that the surface of the concha arcae is rough, with many irregular particles with different shapes.In Figure 3c, it can be seen that there are a large number of blocky particles on the surface of the concha arcae, which look like undulating hills.Figure 3d shows that the particles on the surface of the concha arcae are spherical and blocky, and the particles are tightly stacked together, forming an uneven channel structure.Figure 3e shows the micromorphology magnified by 8000 times.It can be seen that there are gullies of different depths on the surface of the concha arcae.It can also be seen that there are large and regular elliptical particles on the surface of the concha arcae.The surface of the particles is full of round and strip holes, which are arranged regularly.When this regular particle is magnified, it can be seen from Figure 3f that the particle is patterned and covered with oval holes of similar shape, of which the small hole has 91.3 nm and the length diameter ratio is 2.33; the macropore is 137 nm and the aspect ratio is 3.15.4 shows the micro morphology of the concha arcae after calcination at 750℃ for 2 hours.It can be seen from Figure 4a of the scanning electron microscope picture that the calcined concha arcae fragments have regular, obtuse angle like layered lines, many uneven irregular protrusions on the section, and some layered structures have tiny holes on them; Although the surface of Figure 4b is rough, many sections are relatively flat and have many cracks.It can be seen from Figure 4c of the scanning electron microscope picture that the surface of the calcined concha arcae has a regular, obtuse angle shaped layer pattern, which looks like a layered structure stacked with bricks, and some layered structures have holes on them; Figure 6d shows the enlarged layered pattern.It can be clearly seen that many particles similar to small tree nodules are gathered at the edge of the pattern.Figure 4e shows the microscopic view of the calcined concha arcae hole after being magnified by 5000 times.It can be observed in the scanning electron microscope that the fragments are relatively flat and uniform in thickness.The four circles with luminous edges on the surface are the calcined concha arcae hole, and the maximum hole diameter is 1.3 μ m.The minimum is 500 nm; Figure 4f shows a small hole magnified by 45000 times.It can be clearly seen that the boundary of the hole is not smooth, the surface of the hole is rough, and there are uneven depressions.

Conclusions
In this study, powder X-ray diffraction method and Rietveld refinement were used to quantitatively analyze the phases of raw and calcined concha arcae products to determine the percentage content of macro components.The traditional chemical titration method can only measure the total amount of calcium carbonate in concha arcae, and cannot analyze the specific phase of calcium carbonate.The method used by the author is more rapid and simple than chemical analysis, and can also quantify the phase of calcium carbonate, which can provide a reference for the quality evaluation of concha arcae and calcined concha arcae.To sum up, the composition and surface microstructure of concha arcae and calcined concha arcae are quite different.This study can provide a basis for the safe and rational use of concha arcae.

Figure 3 .
Figure 3. SEM images of concha arcae Magnification times (a: 600 b: 1500 c: 5000 d: 10000 e: 8000 f: 35000 )The micro-morphology of concha arcae powder was characterized by SEM, as shown in Figure3.It can be seen from SEM image 3a that the surface of the concha arcae is not smooth, and is full of uneven holes, which are unevenly distributed, of different sizes, and irregular.Figure3bshows the microstructure magnified to 1.5 thousand times.It can be seen that the surface of the concha arcae is rough, with many irregular particles with different shapes.In Figure3c, it can be seen that there are a large number of blocky particles on the surface of the concha arcae, which look like undulating hills.Figure3dshows that the particles on the surface of the concha arcae are spherical and blocky, and the particles are tightly stacked together, forming an uneven channel structure.Figure3eshows the micromorphology magnified by 8000 times.It can be seen that there are gullies of different depths on the surface of the concha arcae.It can also be seen that there are large and regular elliptical particles on the surface of the concha arcae.The surface of the particles is full of round and strip holes, which are arranged regularly.When this regular particle is magnified, it can be seen from Figure3fthat the particle is patterned and covered with oval holes of similar shape, of which the small hole has 91.3 nm and the length diameter ratio is 2.33; the macropore is 137 nm and the aspect ratio is 3.15.

Figure 4 .
Figure 4. SEM images of calcined concha arcae Magnification times (a: 1800 b: 6000 c: 10000 d: 18000 e: 5000 f: 45000) Figure4shows the micro morphology of the concha arcae after calcination at 750℃ for 2 hours.It can be seen from Figure4aof the scanning electron microscope picture that the calcined concha arcae fragments have regular, obtuse angle like layered lines, many uneven irregular protrusions on the section, and some layered structures have tiny holes on them; Although the surface of Figure4bis rough, many sections are relatively flat and have many cracks.It can be seen from Figure4cof the scanning electron microscope picture that the surface of the calcined concha arcae has a regular, obtuse angle shaped layer pattern, which looks like a layered structure stacked with bricks, and some layered structures have holes on them; Figure6dshows the enlarged layered pattern.It can be clearly seen that many particles similar to small tree nodules are gathered at the edge of the pattern.Figure4eshows the microscopic view of the calcined concha arcae hole after being magnified by 5000 times.It can be observed in the scanning electron microscope that the fragments are relatively flat and uniform in thickness.The four circles with luminous edges on the surface are the calcined concha arcae hole, and the maximum hole diameter is 1.3 μ m.The minimum is 500 nm; Figure4fshows a small hole magnified by 45000 times.It can be clearly seen that the boundary of the hole is not smooth, the surface of the hole is rough, and there are uneven depressions.