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Abstract [Objectives] Effects of different processing techniques on the total alkaloid content in Toddalia asiatica Lam. were investigated by ultraviolet spectrophotometry.
[Methods] T. asiatica was processed by the water washing method， water boiling method， water steaming method， wine steaming method and wine processing method， and the total alkaloid content was determined by ultraviolet spectrophotometry.
[Results] Different processing methods can affect the total alkaloid content in the medicinal materials. Among them， the water steaming method and the wine processing method can increase the total alkaloid content of the medicinal materials， and the degree of improvement was higher in the water steaming method; and the water boiling method， water washing method and wine steaming method can reduce the total alkaloid content of the materials， and showed a reduction order of water boiling method > water washing method > wine steaming method.
[Conclusions] The water steaming method and the wine processing method can increase the total alkaloid content in T. asiatica， and the water boiling method， water washing method and wine steaming method can reduce the total alkaloid content of the medicinal material.
Key words Toddalia asiatica Lam.; Processing technique; Total alkaloids; Ultraviolet spectrophotometry
Toddalia asiatica Lam.， belonging to Rutaceae， also known as Sanbaibang， Feilongzhanxue， Jianxuefei， etc.， refers to its dry roots or root bark in Chinese pharmacy， and was first published in An Illustrated Book of Plants[1]. Later， it was recorded in Classified Herbality， Chinese Materia Medica， Chinese Materia Medica Dictionary， National Compilation of Chinese Herbal Medicine and local materia medica. As a common Chinese herbal medicine in China， T. asiatica is recorded in many minority books of Chinese medicine such as Zhuang， Yao， Miao， Dai， Dong and Mulao， mainly distributed in Guangxi， Sichuan， Guizhou， Yunnan， Shaanxi and other places. It is mild in nature， slightly toxic， and tastes slightly bitter， and has the effects of dispelling wind and relieving pain， dissipating blood stasis， stopping bleeding， and removing toxicity for detumescence[1]. Modern pharmacological research has shown that it has analgesic， antiinflammatory， antibacterial， antiviral， antioxidant， antitumor and cardiovascular protection activity[2-4]， clinically used mainly for the treatment of rheumatoid arthralgia， bruises， traumatic bleeding， amenorrhea due to stagnation of blood and other diseases[1]. T. asiatica is rich in alkaloids[5]， such as chelerythrine， dihydrochelerythrine， skimmianine， berberine and toddalidimerine， which are active antiinflammatory， analgesic and antitumor ingredients. However， studies have shown that chelerythrine contained in T. asiatica is an antitumor active ingredient， but it is easy to cause toxic reactions， and may be a neuromuscular toxin and toxic to the heart also [1-6]， thus bringing safety risks in the clinical use of T. asiatica.
Chinese Materia Medica records： T. asiatica is used for treating rheumatism， injuries from falls， fractures， contusions and strains， amenorrhea due to stagnation of blood， internal lesion caused by overexertion， pain and other diseases， mostly in the manner of immersing in or guiding by wine， which limits the wide application of T. asiatica to a certain extent. In this study， the content of chelerythrine in T. asiatica was changed by the method of Chinese medicine processing， and the effects of different processing methods on the total alkaloid content in T. asiatica were investigated by ultraviolet spectrophotometry， so as to provide reference for the research on the processing of T. asiatica and the safety of clinical medication.
Instruments， Reagents and Herbs
UV600 UVvisible spectrophotometer （Shimadzu Instrument （Suzhou） Co.， Ltd.）; DHG9240A electric blast drying oven （Shanghai Yiheng Scientific Instrument Co.， Ltd.）; MilliQ Advantage A10 pure water meter （Millipore ultra pure water system）; SQP electronic balance （Sedolis Scientific Instrument Co.， Ltd.）; KQ5200B ultrasonic cleaner （Kunshan Ultrasonic Instrument Co.， Ltd.）; DFY300C type swing highspeed pulverizer （Wenling Linda Machinery Limited）.
Yellow wine （alcohol content： 7.12%， China Shaoxing Yellow Rice Wine Group Co.， Ltd.）; methanol， chloroform， 95% ethanol， petroleum ether， ethanol， thymol blue， sodium hydroxide， and disodium hydrogen phosphate dodecahydrate （all analytically pure， Sinopharm Chemical Reagent Co.， Ltd.）.
Chelerythrine reference substance （National Institutes for Food and Drug Control， batch number： 111718201402）. 111718201402）.
T. asiatica was purchased from Yaodu Square， Jinxiu Town， Laibin City， Guangxi， and was identified as the roots of T. asiatica Lam. by Professor Wei from Guangxi University of Traditional Chinese Medicine.
Experimental Methods and Results
Sample treatment
Water washing： Three parts of T. asiatica were， respectively， added with water to infiltrate the medicinal materials until the medicinal materials became soft， followed by ovendrying， pulverizing and sieving， obtaining the powder for later use. Water boiling： Three parts of T. asiatica were， respectively， added with water to boil the medicinal materials until the medicinal materials became soft， followed by fishing out， ovendrying， pulverizing and sieving， obtaining the powder for later use.
Water steaming： Three parts of T. asiatica， were， respectively， steamed until the medicinal materials became soft， followed by fishing out， ovendrying， pulverizing and sieving， obtaining the powder for later use.
Wine steaming： Three parts of T. asiatica， were， respectively， steamed on a steamer added with the yellow rice wine until the medicinal materials became soft， followed by fishing out， ovendrying， pulverizing and sieving， obtaining the powder for later use.
Wine processing： Three parts of T. asiatica， were， respectively， added with an appropriate amount of yellow rice wine and then water to the level above the medicinal materials， which were then fried after the surface of the medicinal materials became dry brown and showed some burned dots， and pulverized and sieved， obtaining the powder for later use.
Preparation of solutions
Preparation of reference solution
A certain amount of chelerythrine reference substance （2.63 mg） was weighed accurately to a 50 ml volumetric flask， added with methanol and diluted to constant volume， giving a standard reference stock solution.
Preparation of test solution
A certain amount of T. asiatica powder （0.1 g） was accurately weighed， placed in a 50 ml conical flask， added with 20 ml of methanol and weighed. The powder was ultrasonically extracted for 1 h， compensated to the above measured weight， and filtered， obtaining the filtrate for later use.
Preparation of reagent solutions
Preparation of acid dye： A certain amount of thymol blue （62.5 mg） was weighed into a mortar， added with 2.0 ml of NaOH （0.05 mol/L） solution， and ground to powder， which was then added into 250 ml volumetric flask， and diluted with distilled water to constant volume.
Preparation of buffer solution： A certain amount of disodium hydrogen phosphate dodecahydrate （21.488 4 g） was weighed， and added with 300 ml of distilled water to dissolve it， giving the disodium hydrogen phosphate dodecahydrate solution. A certain amount of citric acid monohydrate （6.304 2 g） was weighed and added with 300 ml of distilled water to dissolve it. The citric acid monohydrate solution was then added with the disodium hydrogen phosphate dodecahydrate solution to a pH of 4， obtaining a buffer solution. Developing method
A certain amount of the filtrate （10 ml） was added into a 60 mlseparating funnel， into which 2 ml of the acid dye and 15 ml of the buffer solution were then added， followed by mixing well. The mixed solution was extracted with chloroform for three times （10 ml of chloroform each time）. The chloroform layers were combined to a 50 ml volumetric flask， and diluted with chloroform to constant weight. The absorbance was measured at the maximum absorption wavelength.
Selection of maximum absorption wavelength
A certain amount of the reference solution and the test solution （10 ml） were， respectively， added with 2 ml of the acid dye and 15 ml of the buffer solution， followed by mixing well. The obtained two solutions were extracted with chloroform for three times （10 ml of chloroform each time）. The chloroform layers of each solution were merged into a 50 ml volumetric flask， and diluted with chloroform to constant volume. The obtained solution was subjected to UVvisible fullwavelength scanning， and the maximum absorption wavelength was determined to be 420 nm， as shown in Fig. 1.
A. Reference solution; B. test solution.
Fig. 1 Ultravioletvisible fullwavelength scanning spectrum
Drawing of standard curve
Certain amounts of the reference solution under "Preparation of reference solution" （1， 2， 4， 6， 8 and 10 ml） were accurately weighed into a 60 ml separating funnel， respectively. Development was then performed according to the method of "Developing method". The absorbance value was measured with a UV spectrophotometer at the wavelength of 420 nm. With absorbance value as the ordinate and the concentration as the abscissa， the regression equationof chelerythrine was obtained： y=75.27x-0.064 9， R2=0.999 1， and the linear range was 1.052-10.520 μg/ml， as shown in Table 1 and Fig. 2.
Methodology investigation
Precision test
The reference solution of chelerythrine under "Preparation of reference solution" was taken and developed according to the method under "Developing method". The absorbance was measured with an ultraviolet spectrophotometer at 420 nm for 6 times continuously. The RSD of chelerythrine absorbance was 0.43%（n=6）， indicating that the precision of the instrument was good.
Stability test
The test solution under "Preparation of reference solution" was taken and developed according to the method under "Developing method". The absorbance was measured at 0， 1， 2， 4， 6， 8， 16 and 24 h， respectively. The RSD of the chelerythrine absorbance was 2.31% （n=8）， indicating that the test solution was stable within 24 h. Repeatability test
The same batch of samples were weighed accurately， and prepared into the test solutions according to the method of "Preparation of reference solution"， in a total of 6 parts. The solutions were developed according to the method under "Developing method". The absorbance values of the solutions were measured with an ultraviolet spectrophotometer at 420 nm. As a result， the RSD of the chelerythrine absorbance was 3.47%（n=6）， indicating that the method has good repeatability.
Recovery test
Six parts of 0.25 mg/ml T. asiatica sample （5 ml each） were added into a separating funnel， respectively. Then， 1.052， 2.104， 4.208， 6.312， 8.416 and 10.52 μg/ml of chelerythrine reference solutions （5 ml each） were added into the six funnels， respectively， and prepared into test solutions according to the method of "Preparation of test solution". The test solutions were developed according to the method under "Developing method". Recovery values were finally calculated. The average recovery rate of the calculated values was 99.77%， and the RSD was 2.6% （n=6）， indicating that the accuracy of this method is good， as shown in Table 2.
Long CHEN et al. Effect of Different Processing Techniques on the Content of Total Alkaloids in Toddalia asiatica Lam.
Determination of total alkaloids in different processed products of T. asiatica
A certain amount of processed T. asiatica products （0.1 g each） were weighed， and prepared into test solutions according to the method of "Preparation of test solution". The obtained test solutions were subjected to development according to the color development method under "Developing method". The absorbance values were determined with an ultraviolet spectrophotometer at 420 nm. The total alkaloid contents in different processed products and raw product of T. asiatica ranked as the water boiling method， water washing method， wine steaming method， wine processing method， water steaming method and raw product from low to high， as shown in Table 3.
Conclusions and Discussion
The results show that the processed products can affect the total alkaloid content in the medicinal materials. It might be because that the different processing methods cause chemical changes of alkaloids or changes of the physical and chemical properties of alkaloids or changes of the solubility of alkaloids. Compared with raw materials， the water washing method can reduce the total alkaloid content， which might be due to that partial alkaloids are decomposed in water or the alkaloids which are easily dissolved in water on the surface of the medicinal materials are lost during the washing process. The water boiling method reduces the total alkaloid content of the medicinal materials， which might be because that the alkaloids are decomposed by water decoction， thereby reducing the total alkaloid content. The water steaming method can increase the total alkaloid content by softening the texture of the watersteamed materials， which allows the alkaloids on the surface of the materials to ooze out with water vapor， thereby improving the dissolution rate of the components. The reason why the total alkaloid content of the wine steaming method is lower than that of the water steaming method and lower than that of the raw product might be because the evaporation of wine is stronger than that of water， but the penetration ability is weaker than water， and the total alkaloids are dissipated with the transpiration of wine and finally show a content overall decreased. The wine processing method is slightly higher than the raw product in total alkaloid content， which might be due to that the alkaloids slowly rise out with wine after the infiltration， which increases the alkaloid dissolution rate， and the total alkaloid content is increased. On the whole， the effects of the processing techniques on the medicinal materials are basically consistent with the traditional processing purposes. Water decoction can alleviate the sturdiness of raw medicinal materials， reduce the toxic component content of medicinal materials， and ensure the safety of medicinal materials for oral administration. The wine processing method can increase the total alkaloid content and enhance the antiinflammatory， antibacterial and analgesic activity， which conforms with its use for the treatment of rheumatic pain and bruises and its effect of promoting blood circulation to arrest pain after being steeped in wine or subjected to alcohol processing. The water washing method can reduce the active ingredients of the medicinal materials and should be washed with less water， and the experimental results are also consistent with the traditional usage experience. The water steaming method can increase the total alkaloid content of the medicinal materials， while the wine steaming method can reduce the total alkaloid content. The ability of them to increase or decrease the content is equivalent， which provides the possibility of expanding the ranges of clinical use methods and indications of T. asiatica. Meanwhile， it provides a research idea to alleviate the inconvenience of traditional wine steeping or alcohol processing.
References
[1] Editorial committee of Chinese materia medica， State Administration of Traditional Chinese Medicine. Chinese materia medica[M]. Shanghai： Shanghai Science and Technology Press， 1999. （in Chinese）
[2] FANG SM， FANG G， SU J， et al. Advances in research on pharamacological effects of Toddalia asiatica Lam.[J]. Journal of International Pharmaceutical Research， 2016， 43（05）： 863-866. （in Chinese）
[3] SHI L， LI D， KANG WY. Advances in research on chemical constituents and pharmacological effects of Toddalia asiatica Lam.[J]. Chinese Pharmacy， 2011， 22（7）： 666-668. （in Chinese）
[4] YUN XL. Advances in pharmacological effects and clinical application of Miao medicine Toddalia asiatica Lam.[J]. Journal of Medicine & Pharmacy of Chinese Minorities， 2012， 18（07）： 58-60. （in Chinese）
[5] ZHOU W， SUN WB， ZENG QF， et al. Progress in pharmaceutical research of Toddalia asiatica Lam.[J]. China Journal of Traditional Chinese Medicine and Pharmacy， 2018， 33（8）： 3515-3522. （in Chinese）
[6] LUO FY， MA XQ， LIN F. Study on the longterm toxicity of anticancer drug in Chelidonium majus in rats[J]. Carcinogenesis， Teratogenesis & Mutagenesis， 2014， 26（6）： 459-462. （in Chinese）
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
[Methods] T. asiatica was processed by the water washing method， water boiling method， water steaming method， wine steaming method and wine processing method， and the total alkaloid content was determined by ultraviolet spectrophotometry.
[Results] Different processing methods can affect the total alkaloid content in the medicinal materials. Among them， the water steaming method and the wine processing method can increase the total alkaloid content of the medicinal materials， and the degree of improvement was higher in the water steaming method; and the water boiling method， water washing method and wine steaming method can reduce the total alkaloid content of the materials， and showed a reduction order of water boiling method > water washing method > wine steaming method.
[Conclusions] The water steaming method and the wine processing method can increase the total alkaloid content in T. asiatica， and the water boiling method， water washing method and wine steaming method can reduce the total alkaloid content of the medicinal material.
Key words Toddalia asiatica Lam.; Processing technique; Total alkaloids; Ultraviolet spectrophotometry
Toddalia asiatica Lam.， belonging to Rutaceae， also known as Sanbaibang， Feilongzhanxue， Jianxuefei， etc.， refers to its dry roots or root bark in Chinese pharmacy， and was first published in An Illustrated Book of Plants[1]. Later， it was recorded in Classified Herbality， Chinese Materia Medica， Chinese Materia Medica Dictionary， National Compilation of Chinese Herbal Medicine and local materia medica. As a common Chinese herbal medicine in China， T. asiatica is recorded in many minority books of Chinese medicine such as Zhuang， Yao， Miao， Dai， Dong and Mulao， mainly distributed in Guangxi， Sichuan， Guizhou， Yunnan， Shaanxi and other places. It is mild in nature， slightly toxic， and tastes slightly bitter， and has the effects of dispelling wind and relieving pain， dissipating blood stasis， stopping bleeding， and removing toxicity for detumescence[1]. Modern pharmacological research has shown that it has analgesic， antiinflammatory， antibacterial， antiviral， antioxidant， antitumor and cardiovascular protection activity[2-4]， clinically used mainly for the treatment of rheumatoid arthralgia， bruises， traumatic bleeding， amenorrhea due to stagnation of blood and other diseases[1]. T. asiatica is rich in alkaloids[5]， such as chelerythrine， dihydrochelerythrine， skimmianine， berberine and toddalidimerine， which are active antiinflammatory， analgesic and antitumor ingredients. However， studies have shown that chelerythrine contained in T. asiatica is an antitumor active ingredient， but it is easy to cause toxic reactions， and may be a neuromuscular toxin and toxic to the heart also [1-6]， thus bringing safety risks in the clinical use of T. asiatica.
Chinese Materia Medica records： T. asiatica is used for treating rheumatism， injuries from falls， fractures， contusions and strains， amenorrhea due to stagnation of blood， internal lesion caused by overexertion， pain and other diseases， mostly in the manner of immersing in or guiding by wine， which limits the wide application of T. asiatica to a certain extent. In this study， the content of chelerythrine in T. asiatica was changed by the method of Chinese medicine processing， and the effects of different processing methods on the total alkaloid content in T. asiatica were investigated by ultraviolet spectrophotometry， so as to provide reference for the research on the processing of T. asiatica and the safety of clinical medication.
Instruments， Reagents and Herbs
UV600 UVvisible spectrophotometer （Shimadzu Instrument （Suzhou） Co.， Ltd.）; DHG9240A electric blast drying oven （Shanghai Yiheng Scientific Instrument Co.， Ltd.）; MilliQ Advantage A10 pure water meter （Millipore ultra pure water system）; SQP electronic balance （Sedolis Scientific Instrument Co.， Ltd.）; KQ5200B ultrasonic cleaner （Kunshan Ultrasonic Instrument Co.， Ltd.）; DFY300C type swing highspeed pulverizer （Wenling Linda Machinery Limited）.
Yellow wine （alcohol content： 7.12%， China Shaoxing Yellow Rice Wine Group Co.， Ltd.）; methanol， chloroform， 95% ethanol， petroleum ether， ethanol， thymol blue， sodium hydroxide， and disodium hydrogen phosphate dodecahydrate （all analytically pure， Sinopharm Chemical Reagent Co.， Ltd.）.
Chelerythrine reference substance （National Institutes for Food and Drug Control， batch number： 111718201402）. 111718201402）.
T. asiatica was purchased from Yaodu Square， Jinxiu Town， Laibin City， Guangxi， and was identified as the roots of T. asiatica Lam. by Professor Wei from Guangxi University of Traditional Chinese Medicine.
Experimental Methods and Results
Sample treatment
Water washing： Three parts of T. asiatica were， respectively， added with water to infiltrate the medicinal materials until the medicinal materials became soft， followed by ovendrying， pulverizing and sieving， obtaining the powder for later use. Water boiling： Three parts of T. asiatica were， respectively， added with water to boil the medicinal materials until the medicinal materials became soft， followed by fishing out， ovendrying， pulverizing and sieving， obtaining the powder for later use.
Water steaming： Three parts of T. asiatica， were， respectively， steamed until the medicinal materials became soft， followed by fishing out， ovendrying， pulverizing and sieving， obtaining the powder for later use.
Wine steaming： Three parts of T. asiatica， were， respectively， steamed on a steamer added with the yellow rice wine until the medicinal materials became soft， followed by fishing out， ovendrying， pulverizing and sieving， obtaining the powder for later use.
Wine processing： Three parts of T. asiatica， were， respectively， added with an appropriate amount of yellow rice wine and then water to the level above the medicinal materials， which were then fried after the surface of the medicinal materials became dry brown and showed some burned dots， and pulverized and sieved， obtaining the powder for later use.
Preparation of solutions
Preparation of reference solution
A certain amount of chelerythrine reference substance （2.63 mg） was weighed accurately to a 50 ml volumetric flask， added with methanol and diluted to constant volume， giving a standard reference stock solution.
Preparation of test solution
A certain amount of T. asiatica powder （0.1 g） was accurately weighed， placed in a 50 ml conical flask， added with 20 ml of methanol and weighed. The powder was ultrasonically extracted for 1 h， compensated to the above measured weight， and filtered， obtaining the filtrate for later use.
Preparation of reagent solutions
Preparation of acid dye： A certain amount of thymol blue （62.5 mg） was weighed into a mortar， added with 2.0 ml of NaOH （0.05 mol/L） solution， and ground to powder， which was then added into 250 ml volumetric flask， and diluted with distilled water to constant volume.
Preparation of buffer solution： A certain amount of disodium hydrogen phosphate dodecahydrate （21.488 4 g） was weighed， and added with 300 ml of distilled water to dissolve it， giving the disodium hydrogen phosphate dodecahydrate solution. A certain amount of citric acid monohydrate （6.304 2 g） was weighed and added with 300 ml of distilled water to dissolve it. The citric acid monohydrate solution was then added with the disodium hydrogen phosphate dodecahydrate solution to a pH of 4， obtaining a buffer solution. Developing method
A certain amount of the filtrate （10 ml） was added into a 60 mlseparating funnel， into which 2 ml of the acid dye and 15 ml of the buffer solution were then added， followed by mixing well. The mixed solution was extracted with chloroform for three times （10 ml of chloroform each time）. The chloroform layers were combined to a 50 ml volumetric flask， and diluted with chloroform to constant weight. The absorbance was measured at the maximum absorption wavelength.
Selection of maximum absorption wavelength
A certain amount of the reference solution and the test solution （10 ml） were， respectively， added with 2 ml of the acid dye and 15 ml of the buffer solution， followed by mixing well. The obtained two solutions were extracted with chloroform for three times （10 ml of chloroform each time）. The chloroform layers of each solution were merged into a 50 ml volumetric flask， and diluted with chloroform to constant volume. The obtained solution was subjected to UVvisible fullwavelength scanning， and the maximum absorption wavelength was determined to be 420 nm， as shown in Fig. 1.
A. Reference solution; B. test solution.
Fig. 1 Ultravioletvisible fullwavelength scanning spectrum
Drawing of standard curve
Certain amounts of the reference solution under "Preparation of reference solution" （1， 2， 4， 6， 8 and 10 ml） were accurately weighed into a 60 ml separating funnel， respectively. Development was then performed according to the method of "Developing method". The absorbance value was measured with a UV spectrophotometer at the wavelength of 420 nm. With absorbance value as the ordinate and the concentration as the abscissa， the regression equationof chelerythrine was obtained： y=75.27x-0.064 9， R2=0.999 1， and the linear range was 1.052-10.520 μg/ml， as shown in Table 1 and Fig. 2.
Methodology investigation
Precision test
The reference solution of chelerythrine under "Preparation of reference solution" was taken and developed according to the method under "Developing method". The absorbance was measured with an ultraviolet spectrophotometer at 420 nm for 6 times continuously. The RSD of chelerythrine absorbance was 0.43%（n=6）， indicating that the precision of the instrument was good.
Stability test
The test solution under "Preparation of reference solution" was taken and developed according to the method under "Developing method". The absorbance was measured at 0， 1， 2， 4， 6， 8， 16 and 24 h， respectively. The RSD of the chelerythrine absorbance was 2.31% （n=8）， indicating that the test solution was stable within 24 h. Repeatability test
The same batch of samples were weighed accurately， and prepared into the test solutions according to the method of "Preparation of reference solution"， in a total of 6 parts. The solutions were developed according to the method under "Developing method". The absorbance values of the solutions were measured with an ultraviolet spectrophotometer at 420 nm. As a result， the RSD of the chelerythrine absorbance was 3.47%（n=6）， indicating that the method has good repeatability.
Recovery test
Six parts of 0.25 mg/ml T. asiatica sample （5 ml each） were added into a separating funnel， respectively. Then， 1.052， 2.104， 4.208， 6.312， 8.416 and 10.52 μg/ml of chelerythrine reference solutions （5 ml each） were added into the six funnels， respectively， and prepared into test solutions according to the method of "Preparation of test solution". The test solutions were developed according to the method under "Developing method". Recovery values were finally calculated. The average recovery rate of the calculated values was 99.77%， and the RSD was 2.6% （n=6）， indicating that the accuracy of this method is good， as shown in Table 2.
Long CHEN et al. Effect of Different Processing Techniques on the Content of Total Alkaloids in Toddalia asiatica Lam.
Determination of total alkaloids in different processed products of T. asiatica
A certain amount of processed T. asiatica products （0.1 g each） were weighed， and prepared into test solutions according to the method of "Preparation of test solution". The obtained test solutions were subjected to development according to the color development method under "Developing method". The absorbance values were determined with an ultraviolet spectrophotometer at 420 nm. The total alkaloid contents in different processed products and raw product of T. asiatica ranked as the water boiling method， water washing method， wine steaming method， wine processing method， water steaming method and raw product from low to high， as shown in Table 3.
Conclusions and Discussion
The results show that the processed products can affect the total alkaloid content in the medicinal materials. It might be because that the different processing methods cause chemical changes of alkaloids or changes of the physical and chemical properties of alkaloids or changes of the solubility of alkaloids. Compared with raw materials， the water washing method can reduce the total alkaloid content， which might be due to that partial alkaloids are decomposed in water or the alkaloids which are easily dissolved in water on the surface of the medicinal materials are lost during the washing process. The water boiling method reduces the total alkaloid content of the medicinal materials， which might be because that the alkaloids are decomposed by water decoction， thereby reducing the total alkaloid content. The water steaming method can increase the total alkaloid content by softening the texture of the watersteamed materials， which allows the alkaloids on the surface of the materials to ooze out with water vapor， thereby improving the dissolution rate of the components. The reason why the total alkaloid content of the wine steaming method is lower than that of the water steaming method and lower than that of the raw product might be because the evaporation of wine is stronger than that of water， but the penetration ability is weaker than water， and the total alkaloids are dissipated with the transpiration of wine and finally show a content overall decreased. The wine processing method is slightly higher than the raw product in total alkaloid content， which might be due to that the alkaloids slowly rise out with wine after the infiltration， which increases the alkaloid dissolution rate， and the total alkaloid content is increased. On the whole， the effects of the processing techniques on the medicinal materials are basically consistent with the traditional processing purposes. Water decoction can alleviate the sturdiness of raw medicinal materials， reduce the toxic component content of medicinal materials， and ensure the safety of medicinal materials for oral administration. The wine processing method can increase the total alkaloid content and enhance the antiinflammatory， antibacterial and analgesic activity， which conforms with its use for the treatment of rheumatic pain and bruises and its effect of promoting blood circulation to arrest pain after being steeped in wine or subjected to alcohol processing. The water washing method can reduce the active ingredients of the medicinal materials and should be washed with less water， and the experimental results are also consistent with the traditional usage experience. The water steaming method can increase the total alkaloid content of the medicinal materials， while the wine steaming method can reduce the total alkaloid content. The ability of them to increase or decrease the content is equivalent， which provides the possibility of expanding the ranges of clinical use methods and indications of T. asiatica. Meanwhile， it provides a research idea to alleviate the inconvenience of traditional wine steeping or alcohol processing.
References
[1] Editorial committee of Chinese materia medica， State Administration of Traditional Chinese Medicine. Chinese materia medica[M]. Shanghai： Shanghai Science and Technology Press， 1999. （in Chinese）
[2] FANG SM， FANG G， SU J， et al. Advances in research on pharamacological effects of Toddalia asiatica Lam.[J]. Journal of International Pharmaceutical Research， 2016， 43（05）： 863-866. （in Chinese）
[3] SHI L， LI D， KANG WY. Advances in research on chemical constituents and pharmacological effects of Toddalia asiatica Lam.[J]. Chinese Pharmacy， 2011， 22（7）： 666-668. （in Chinese）
[4] YUN XL. Advances in pharmacological effects and clinical application of Miao medicine Toddalia asiatica Lam.[J]. Journal of Medicine & Pharmacy of Chinese Minorities， 2012， 18（07）： 58-60. （in Chinese）
[5] ZHOU W， SUN WB， ZENG QF， et al. Progress in pharmaceutical research of Toddalia asiatica Lam.[J]. China Journal of Traditional Chinese Medicine and Pharmacy， 2018， 33（8）： 3515-3522. （in Chinese）
[6] LUO FY， MA XQ， LIN F. Study on the longterm toxicity of anticancer drug in Chelidonium majus in rats[J]. Carcinogenesis， Teratogenesis & Mutagenesis， 2014， 26（6）： 459-462. （in Chinese）
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU