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Abstract ［Objectives］ This study was conducted to explore the best extraction technology of total flavonoids from Morinda citrifolia L.
［Methods］An orthogonal test was carried out to select the best extraction process with the yield of total flavonoids in the extract as the investigation index.
［Results］ Among the four factors of experimental design, the extraction time had a very significant impact on the results, the ethanol concentration had a significant impact on the results, and the extraction temperature and ethanol dosage had no significant impacts on the results. The optimal extraction process conditions for the total flavonoids of M. citrifolia were as follows: extraction time 1.5 h, ethanol concentration 90%, extraction temperature 60-65 ℃, and ethanol dosage 1∶20 (g/ml). The total flavonoids in M. citrifolia fruit were extracted according to the optimal extraction process conditions, and the yield was 2.936%.
［Conclusions］This study provides a theoretical basis for obtaining the optimal extraction process conditions for total flavonoids of M. citrifolia.
Key words Orthogonal test; Morinda citrifolia L.; Total flavonoids; Ultraviolet spectrophotometry
Received: Feburary 22, 2021 Accepted: April 20, 2021
Supported by 2020 National Undergraduate Innovation and Enterpreneurship Training Program (202011430133).
Mengxi ZHANG (1998-), female, P. R. China, devoted to research about pharmaceutical engineering.
*Corresponding author.
Morinda citrifolia L.［1-2］, also known as Indian mulberry, Noni, is a tropical evergreen shrub or small arbor in Morinda of Rubiaceae［3-4］. It is native to tropical islands in the South Pacific, and distributed in Chinas Hainan Island, Xisha Islands and Taiwan Island［5］. It is an important traditional medicinal plant for the indigenous peoples of Polynesia. Its leaves, roots, bark and immature fruits can all be used for treating diseases［5-7］. The main chemical components of M. citrifolia are flavonoids, coumarins, terpenes, alkaloids, anthraquinones, etc., which have antibacterial, anti-HIV, parasite-expelling and pain-relieving, blood pressure-lowering, immunity-enhancing and anti-cancer effects［8］. In this study, factors affecting the extraction of total flavonoids from M. citrifolia were investigated by an orthogonal test, aiming to provide an experimental basis for obtaining the optimal extraction process conditions.
Materials and Methods Materials
M. citrifolia L. fruit was provided by the Pharmaceutical Factory of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. The fruit was air-dried naturally, pulverized and sieved through a 50-mesh sieve for later use.
Instrument and reagents
Main instruments: TU-1810DSPC UV-Vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.); rotary evaporator (Buchi, Switzerland); SHZ-D circulating water vacuum pump (Yingyu Yuhua Instrument Factory, Gongyi City, Henan Province); electric thermostat water bath HH.S11-2 (Beijing Changan Scientific Instrument Factory).
Reagents: Rutin reference substance (National Institute for the Control of Pharmaceutical and Biological Products); reagents such as anhydrous ethanol, NaNO2, Al(NO3)3 and NaOH, all of which were analytically pure.
Experimental methods
Sample extraction
A total of 9 parts of M. citrifolia fruit powder (2 g) were precisely weighed, and reflux-extracted referring to literatures［9-11］ according to the L9(34) orthogonal design (Table 3, Table 4). Each part was extracted and vacuum filtered for 3 times, and the three filtrates were merged, and distilled under reduced pressure to recover ethanol. The extract was diluted with anhydrous ethanol to constant weight in a 100 ml volumetric flask and shaken to give the original solution to be tested.
A certain amount of the above-mentioned original solution to be tested (5 ml) was accurately measured, added into a 25 ml volumetric flask, and added with an appropriate amount of anhydrous ethanol. After ultrasonically dissolving the extract, the solution was diluted to constant volume, shaken and stood. A certain amount of the supernatant (5.0 ml) was placed in an evaporating dish, and added with 2.0 g of polyamide for adsorption. Ethanol was evaporated in a 50 ℃ water bath, and the rest was transferred into a chromatographic column. Elution was first performed with 25 ml of petroleum ether, obtaining the eluate which was discarded, and then with anhydrous ethanol, obtaining the eluate which was collected (until the hydrochloric acid-magnesium powder reaction was negative). The eluate was put in a 25 ml volumetric flask, and added with anhydrous ethanol to constant volume, obtaining the sample solution to be tested.
Standard curve drawing［10］
A certain amount of rutin standard substance (5.0 mg) was accurately weighed and dried at 120 ℃ to a constant weight. The dried standard substance was dissolved with anhydrous ethanol, and diluted to 100 ml to obtain a standard solution with a concentration of 50 μg/ml. Certain amounts of the above rutin standard substance (0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml) were transferred into 10 ml volumetric flasks, which were then added with 0.3 ml of 5% sodium nitrite, respectively, obtaining solutions, which were stood for 6 min, and added with 0.3 ml of 10% aluminium nitrate. The solutions were then stood for 6 min, added with 4 ml of 4% sodium hydroxide, and added with distilled water to constant volume, followed by shaking and standing for 15 min. The absorbance was measured at 510 nm on a spectrophotometer, with anhydrous ethanol as a blank reference. With the absorbance A as the ordinate and the concentration C as the abscissa, regression analysis was performed, and a regression curve was drawn. As a result, in the range of 0-5 μg/ml, there was a good linear relationship between concentration and absorbance. The regression equation obtained was A=0.027 7C+0.001 (C: μg/ml), r=0.999 8.
Determination of total flavonoids in samples［10］
A certain amount of the above-mentioned sample solution to be tested (1.0 ml) was precisely transferred into 10 ml volumetric flasks and measured for the absorbance A at 510 nm according to the method under "Standard curve drawing", and the total flavonoid content in the extract was calculated according to the regression equation.
Methodological investigation
Precision investigation
A certain amount of the sample powder (2 g) was accurately weighed, and prepared into the sample solution to be tested according to the sample preparation method. A certain amount of the solution to be tested (1.0 ml) was accurately transferred into a 10 ml volumetric flask, and added with distilled water to 10 ml. The absorbance A was measured at 510 nm according to the method in "Standard curve drawing". The average absorbance of 5 measurements was 0.057 2, and the RSD was 0.67%, indicating that the precision of the instrument was good.
Stability study
The absorbance of the above solution was measured every 10 min. The average absorbance of 5 measurements was 0.056 1, and the RSD was 1.34% (n=5), indicating that the sample solution to be tested was stable within 1 h.
Reproducibility study
Five parts of the above solution were prepared in parallel, and measured for absorbance. The average yield of total flavonoids in 5 determinations was 2.936%, and the RSD was 0.883%, indicating that the method had good reproducibility.
Recovery test Certain amounts of sample solution to be tested were precisely transferred to 10 ml volumetric flasks, and the absorbance was determined according to the above method. The recoveries were calculated, and the average recovery was 100.64% (Table 1), indicating that the recovery of the method was good.
Results and Analysis
Selection of extraction method of total flavonoids
Considering that the total flavonoids of Morinda officinalis in the same genus have better solubility in ethanol, and the amount of dissolution is larger at the extraction time of 1 h［9］, so two extraction methods of M. citrifolia were investigated:
Immersion method: A certain amount of M. citrifolia fruit powder (3 g) was immersed with 15 time of 95% ethanol for 3 times, 1 h each time. The filtrates obtained by vacuum filtration were merged.
Reflux method: A certain amount of M. citrifolia fruit powder (3 g) was extracted with 15 time of 95% ethanol for 3 times, 1 h each time. The filtrates obtained by vacuum filtration were merged.
The total flavonoids of M. citrifolia were extracted by the above two extraction methods, and the yield of total flavonoids was determined sequentially. It was concluded that the reflux method had the highest total flavonoid content (1.960 2%), followed by the immersion method (1.325 8%). Therefore, the reflux method was selected as the extraction method for the total flavonoids of M. citrifolia, and the orthogonal test in Table 2 was carried out to obtain the optimal extraction conditions.
Orthogonal test for extraction of total flavonoids
On the basis of selecting reflux as the extraction method, the ethanol concentration, ethanol dosage, extraction temperature, and extraction time were considered as investigating factors. Each factor was set with 3 levels, and the number of extraction times was fixed at 3 times. The L9(34) orthogonal table was adopted to carry out the orthogonal test. The factor levels are shown in Table 2, and the yield of total flavonoids in the extract was used as an investigation index. The results are shown in Table 3.
For the extraction of total flavonoids, through intuitive analysis, the effect of factor B was not significant, so factor B was used as the error for variance analysis. The results are shown in Table 4.
According to Table 2 and Table 3, it can be seen that ethanol dosage and extraction temperature had no significant impacts on the index, while ethanol concentration had a significant impact on the index. The most significant factor was extraction time. From the range calculation, it can be seen that RD>RA>RC>RB, and therefore, the primary and secondary order of the factors affecting the total flavonoids from M. citrifolia was D>A>C>B. Analyzing the comprehensive effect of various factors on total flavonoids, and taking into account the extraction cost, the best extraction scheme was A2B2C1D3. Mengxi ZHANG et al. Study on the Extraction Process of Total Flavonoids from Morinda citrifolia L.
Verification of optimal extraction conditions
According to the above-determined optimal conditions, 5 parallel tests were performed. The total flavonoid content obtained was higher than the yield of any test in the orthogonal test, and the average yield and RSD were 2.936% and 0.883%, respectively, which were comparable with the results in the orthogonal table. It further showed that the above-mentioned optimal conditions obtained by the orthogonal test were indeed the best process for extracting total flavonoids from M. citrifolia.
Conclusions and Discussion
Based on the reflux extraction method, a more reasonable four-factor and three-level orthogonal test was designed. Four factors were selected to study the effects on the extraction of total flavonoids from M. citrifolia fruit. The experimental results showed that the reflux extraction technology had the advantages of shorter extraction time and higher yield of total flavonoids compared with traditional immersion. According to the test results, the extraction process finally determined was taking an appropriate amount of crushed M. citrifolia powder, which was extracted with ethanol at a dosage of 1∶20 (g/ml) and a concentration of 90% at an extraction temperature in 60-65 ℃ for 1.5 h.
In this study, the orthogonal test method was adopted to optimize the experimental conditions, which greatly improved the extraction efficiency of total flavonoids in M. citrifolia. M. citrifolia fruit powder contains flavonoids, and thus provides another usable resource for the acquisition of natural flavonoids. This study provides a basis for the further development and utilization of medicinal ingredients of M. citrifolia.
References
［1］ WILL MC CLATCHEY. From polynesian healers to health food stores: Changing perspectives of Morinda citrifolia (Rubiaceae)［J］. Integrative Cancer Therapies, 2002, 1 (2): 110-120.
［2］ WANG MF, KIKUZAKI H, JIN Y, et al. Novel glycosides from Noni (Morinda citrifolia)［J］. Journal of Natural Products, 2000, 63: 1182-1183.
［3］ KAMIYA K, TANAKA Y, ENDANG H, et al. Chemical constituents of Morinda citrifolia fruits inhibit copper-induced low-density lipoprotein oxidation［J］. Journal of Agricultural and Food Chemistry, 2004, 52(19): 5843-5848.
［4］ ZIN Z M, HAMID AA, OSMAN A. Antioxidative activity of extracts from Mengkudu (Morinda citrifolia L.) root, fruit and leaf［J］. Food Chemistry, 2002, 78(2): 227-231. ［5］ WANG MY, SU C. Cancer preventive effect of Morinda citrifolia (Noni)［J］. Annals of the New York Academy of Sciences, 2001, 952: 161-168.
［6］ LIU G, BODE A, MA WY, et al. Two novel glycosides from the fruits of Morinda citrifolia (noni) inhibit AP-1 transactivation and cell transformation in the mouse epidermal JB6 Cell line［J］. Cancer Research, 2001, 61: 5749-5756.
［7］ SANG SM, CHENG XF, ZHU NQ, et al. Flavonol glycosides and novel iridoid glycoside from the leaves of Morinda citrifolia［J］. Journal of Agricultural and Food Chemistry, 2001, 49(9): 4478-4481.
［8］ WANG MY, WESTt BJ, JENSEN CJ, et al. Morinda citrifolia (Noni): A literature review and recent advances in Noni research［J］. Acta Pharmacologica Sinica, 2002, 23(12):1127-1141.
［9］ SUN ZS, LIN Q, PEI CH, et al. Study on the property of dissolving out for effective compositions in the powder of Morinda officinalis How［J］. Natural Science Journal of Hainan University, 2004, 22(2): 164-166. (in Chinese)
［10］ WANG JH, CHI RZ, SU GQ. The optimum extraction condition of general flavonoids from Glycyrrhiza uralensis Fisch with ethanol［J］. Chemical & Biological Engineering, 2005, 22(11): 45-46. (in Chinese)
［11］ WANG HW, LU XE, LIU YQ, et al. Study on the extracting technology of flavones from the leaves of Artocarpus heterophyllus Lam. by orthogonal design［J］. Guangdong Chemical Industry, 2006, 33(8): 26-29. (in Chinese)
［Methods］An orthogonal test was carried out to select the best extraction process with the yield of total flavonoids in the extract as the investigation index.
［Results］ Among the four factors of experimental design, the extraction time had a very significant impact on the results, the ethanol concentration had a significant impact on the results, and the extraction temperature and ethanol dosage had no significant impacts on the results. The optimal extraction process conditions for the total flavonoids of M. citrifolia were as follows: extraction time 1.5 h, ethanol concentration 90%, extraction temperature 60-65 ℃, and ethanol dosage 1∶20 (g/ml). The total flavonoids in M. citrifolia fruit were extracted according to the optimal extraction process conditions, and the yield was 2.936%.
［Conclusions］This study provides a theoretical basis for obtaining the optimal extraction process conditions for total flavonoids of M. citrifolia.
Key words Orthogonal test; Morinda citrifolia L.; Total flavonoids; Ultraviolet spectrophotometry
Received: Feburary 22, 2021 Accepted: April 20, 2021
Supported by 2020 National Undergraduate Innovation and Enterpreneurship Training Program (202011430133).
Mengxi ZHANG (1998-), female, P. R. China, devoted to research about pharmaceutical engineering.
*Corresponding author.
Morinda citrifolia L.［1-2］, also known as Indian mulberry, Noni, is a tropical evergreen shrub or small arbor in Morinda of Rubiaceae［3-4］. It is native to tropical islands in the South Pacific, and distributed in Chinas Hainan Island, Xisha Islands and Taiwan Island［5］. It is an important traditional medicinal plant for the indigenous peoples of Polynesia. Its leaves, roots, bark and immature fruits can all be used for treating diseases［5-7］. The main chemical components of M. citrifolia are flavonoids, coumarins, terpenes, alkaloids, anthraquinones, etc., which have antibacterial, anti-HIV, parasite-expelling and pain-relieving, blood pressure-lowering, immunity-enhancing and anti-cancer effects［8］. In this study, factors affecting the extraction of total flavonoids from M. citrifolia were investigated by an orthogonal test, aiming to provide an experimental basis for obtaining the optimal extraction process conditions.
Materials and Methods Materials
M. citrifolia L. fruit was provided by the Pharmaceutical Factory of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences. The fruit was air-dried naturally, pulverized and sieved through a 50-mesh sieve for later use.
Instrument and reagents
Main instruments: TU-1810DSPC UV-Vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.); rotary evaporator (Buchi, Switzerland); SHZ-D circulating water vacuum pump (Yingyu Yuhua Instrument Factory, Gongyi City, Henan Province); electric thermostat water bath HH.S11-2 (Beijing Changan Scientific Instrument Factory).
Reagents: Rutin reference substance (National Institute for the Control of Pharmaceutical and Biological Products); reagents such as anhydrous ethanol, NaNO2, Al(NO3)3 and NaOH, all of which were analytically pure.
Experimental methods
Sample extraction
A total of 9 parts of M. citrifolia fruit powder (2 g) were precisely weighed, and reflux-extracted referring to literatures［9-11］ according to the L9(34) orthogonal design (Table 3, Table 4). Each part was extracted and vacuum filtered for 3 times, and the three filtrates were merged, and distilled under reduced pressure to recover ethanol. The extract was diluted with anhydrous ethanol to constant weight in a 100 ml volumetric flask and shaken to give the original solution to be tested.
A certain amount of the above-mentioned original solution to be tested (5 ml) was accurately measured, added into a 25 ml volumetric flask, and added with an appropriate amount of anhydrous ethanol. After ultrasonically dissolving the extract, the solution was diluted to constant volume, shaken and stood. A certain amount of the supernatant (5.0 ml) was placed in an evaporating dish, and added with 2.0 g of polyamide for adsorption. Ethanol was evaporated in a 50 ℃ water bath, and the rest was transferred into a chromatographic column. Elution was first performed with 25 ml of petroleum ether, obtaining the eluate which was discarded, and then with anhydrous ethanol, obtaining the eluate which was collected (until the hydrochloric acid-magnesium powder reaction was negative). The eluate was put in a 25 ml volumetric flask, and added with anhydrous ethanol to constant volume, obtaining the sample solution to be tested.
Standard curve drawing［10］
A certain amount of rutin standard substance (5.0 mg) was accurately weighed and dried at 120 ℃ to a constant weight. The dried standard substance was dissolved with anhydrous ethanol, and diluted to 100 ml to obtain a standard solution with a concentration of 50 μg/ml. Certain amounts of the above rutin standard substance (0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml) were transferred into 10 ml volumetric flasks, which were then added with 0.3 ml of 5% sodium nitrite, respectively, obtaining solutions, which were stood for 6 min, and added with 0.3 ml of 10% aluminium nitrate. The solutions were then stood for 6 min, added with 4 ml of 4% sodium hydroxide, and added with distilled water to constant volume, followed by shaking and standing for 15 min. The absorbance was measured at 510 nm on a spectrophotometer, with anhydrous ethanol as a blank reference. With the absorbance A as the ordinate and the concentration C as the abscissa, regression analysis was performed, and a regression curve was drawn. As a result, in the range of 0-5 μg/ml, there was a good linear relationship between concentration and absorbance. The regression equation obtained was A=0.027 7C+0.001 (C: μg/ml), r=0.999 8.
Determination of total flavonoids in samples［10］
A certain amount of the above-mentioned sample solution to be tested (1.0 ml) was precisely transferred into 10 ml volumetric flasks and measured for the absorbance A at 510 nm according to the method under "Standard curve drawing", and the total flavonoid content in the extract was calculated according to the regression equation.
Methodological investigation
Precision investigation
A certain amount of the sample powder (2 g) was accurately weighed, and prepared into the sample solution to be tested according to the sample preparation method. A certain amount of the solution to be tested (1.0 ml) was accurately transferred into a 10 ml volumetric flask, and added with distilled water to 10 ml. The absorbance A was measured at 510 nm according to the method in "Standard curve drawing". The average absorbance of 5 measurements was 0.057 2, and the RSD was 0.67%, indicating that the precision of the instrument was good.
Stability study
The absorbance of the above solution was measured every 10 min. The average absorbance of 5 measurements was 0.056 1, and the RSD was 1.34% (n=5), indicating that the sample solution to be tested was stable within 1 h.
Reproducibility study
Five parts of the above solution were prepared in parallel, and measured for absorbance. The average yield of total flavonoids in 5 determinations was 2.936%, and the RSD was 0.883%, indicating that the method had good reproducibility.
Recovery test Certain amounts of sample solution to be tested were precisely transferred to 10 ml volumetric flasks, and the absorbance was determined according to the above method. The recoveries were calculated, and the average recovery was 100.64% (Table 1), indicating that the recovery of the method was good.
Results and Analysis
Selection of extraction method of total flavonoids
Considering that the total flavonoids of Morinda officinalis in the same genus have better solubility in ethanol, and the amount of dissolution is larger at the extraction time of 1 h［9］, so two extraction methods of M. citrifolia were investigated:
Immersion method: A certain amount of M. citrifolia fruit powder (3 g) was immersed with 15 time of 95% ethanol for 3 times, 1 h each time. The filtrates obtained by vacuum filtration were merged.
Reflux method: A certain amount of M. citrifolia fruit powder (3 g) was extracted with 15 time of 95% ethanol for 3 times, 1 h each time. The filtrates obtained by vacuum filtration were merged.
The total flavonoids of M. citrifolia were extracted by the above two extraction methods, and the yield of total flavonoids was determined sequentially. It was concluded that the reflux method had the highest total flavonoid content (1.960 2%), followed by the immersion method (1.325 8%). Therefore, the reflux method was selected as the extraction method for the total flavonoids of M. citrifolia, and the orthogonal test in Table 2 was carried out to obtain the optimal extraction conditions.
Orthogonal test for extraction of total flavonoids
On the basis of selecting reflux as the extraction method, the ethanol concentration, ethanol dosage, extraction temperature, and extraction time were considered as investigating factors. Each factor was set with 3 levels, and the number of extraction times was fixed at 3 times. The L9(34) orthogonal table was adopted to carry out the orthogonal test. The factor levels are shown in Table 2, and the yield of total flavonoids in the extract was used as an investigation index. The results are shown in Table 3.
For the extraction of total flavonoids, through intuitive analysis, the effect of factor B was not significant, so factor B was used as the error for variance analysis. The results are shown in Table 4.
According to Table 2 and Table 3, it can be seen that ethanol dosage and extraction temperature had no significant impacts on the index, while ethanol concentration had a significant impact on the index. The most significant factor was extraction time. From the range calculation, it can be seen that RD>RA>RC>RB, and therefore, the primary and secondary order of the factors affecting the total flavonoids from M. citrifolia was D>A>C>B. Analyzing the comprehensive effect of various factors on total flavonoids, and taking into account the extraction cost, the best extraction scheme was A2B2C1D3. Mengxi ZHANG et al. Study on the Extraction Process of Total Flavonoids from Morinda citrifolia L.
Verification of optimal extraction conditions
According to the above-determined optimal conditions, 5 parallel tests were performed. The total flavonoid content obtained was higher than the yield of any test in the orthogonal test, and the average yield and RSD were 2.936% and 0.883%, respectively, which were comparable with the results in the orthogonal table. It further showed that the above-mentioned optimal conditions obtained by the orthogonal test were indeed the best process for extracting total flavonoids from M. citrifolia.
Conclusions and Discussion
Based on the reflux extraction method, a more reasonable four-factor and three-level orthogonal test was designed. Four factors were selected to study the effects on the extraction of total flavonoids from M. citrifolia fruit. The experimental results showed that the reflux extraction technology had the advantages of shorter extraction time and higher yield of total flavonoids compared with traditional immersion. According to the test results, the extraction process finally determined was taking an appropriate amount of crushed M. citrifolia powder, which was extracted with ethanol at a dosage of 1∶20 (g/ml) and a concentration of 90% at an extraction temperature in 60-65 ℃ for 1.5 h.
In this study, the orthogonal test method was adopted to optimize the experimental conditions, which greatly improved the extraction efficiency of total flavonoids in M. citrifolia. M. citrifolia fruit powder contains flavonoids, and thus provides another usable resource for the acquisition of natural flavonoids. This study provides a basis for the further development and utilization of medicinal ingredients of M. citrifolia.
References
［1］ WILL MC CLATCHEY. From polynesian healers to health food stores: Changing perspectives of Morinda citrifolia (Rubiaceae)［J］. Integrative Cancer Therapies, 2002, 1 (2): 110-120.
［2］ WANG MF, KIKUZAKI H, JIN Y, et al. Novel glycosides from Noni (Morinda citrifolia)［J］. Journal of Natural Products, 2000, 63: 1182-1183.
［3］ KAMIYA K, TANAKA Y, ENDANG H, et al. Chemical constituents of Morinda citrifolia fruits inhibit copper-induced low-density lipoprotein oxidation［J］. Journal of Agricultural and Food Chemistry, 2004, 52(19): 5843-5848.
［4］ ZIN Z M, HAMID AA, OSMAN A. Antioxidative activity of extracts from Mengkudu (Morinda citrifolia L.) root, fruit and leaf［J］. Food Chemistry, 2002, 78(2): 227-231. ［5］ WANG MY, SU C. Cancer preventive effect of Morinda citrifolia (Noni)［J］. Annals of the New York Academy of Sciences, 2001, 952: 161-168.
［6］ LIU G, BODE A, MA WY, et al. Two novel glycosides from the fruits of Morinda citrifolia (noni) inhibit AP-1 transactivation and cell transformation in the mouse epidermal JB6 Cell line［J］. Cancer Research, 2001, 61: 5749-5756.
［7］ SANG SM, CHENG XF, ZHU NQ, et al. Flavonol glycosides and novel iridoid glycoside from the leaves of Morinda citrifolia［J］. Journal of Agricultural and Food Chemistry, 2001, 49(9): 4478-4481.
［8］ WANG MY, WESTt BJ, JENSEN CJ, et al. Morinda citrifolia (Noni): A literature review and recent advances in Noni research［J］. Acta Pharmacologica Sinica, 2002, 23(12):1127-1141.
［9］ SUN ZS, LIN Q, PEI CH, et al. Study on the property of dissolving out for effective compositions in the powder of Morinda officinalis How［J］. Natural Science Journal of Hainan University, 2004, 22(2): 164-166. (in Chinese)
［10］ WANG JH, CHI RZ, SU GQ. The optimum extraction condition of general flavonoids from Glycyrrhiza uralensis Fisch with ethanol［J］. Chemical & Biological Engineering, 2005, 22(11): 45-46. (in Chinese)
［11］ WANG HW, LU XE, LIU YQ, et al. Study on the extracting technology of flavones from the leaves of Artocarpus heterophyllus Lam. by orthogonal design［J］. Guangdong Chemical Industry, 2006, 33(8): 26-29. (in Chinese)