CN101530193B - Method for comprehensively extracting and purifying oxidation-resistant active ingredient in sugarcane shoots or slag - Google Patents
Method for comprehensively extracting and purifying oxidation-resistant active ingredient in sugarcane shoots or slag Download PDFInfo
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Abstract
The invention discloses a method for comprehensively extracting and purifying anthocyanin and general flavone which does not comprise the anthocyanin in sugarcane toppers or slag. The method comprises the steps of firstly extracting the sugarcane toppers or slag by an organic solvent, concentrating the extract and passing the extract through a chromatographic column filled with a macro-porous adsorptive resin, removing water-soluble impurities such as glucide by a gradient elution method, respectively collecting the ethanol eluent with a low concentration and the ethanol eluent with a high concentration to obtain two ingredients of the anthocyanin and the general flavone which does not comprise the anthocyanin, and respectively enriching and purifying the ingredients of the anthocyanin and the general flavone which does not comprise the anthocyanin by a solvent extraction method and an acid and alkali treatment method so as to obtain the anthocyanin and the general flavone which does not comprise the anthocyanin. Compared with the reported methods for extraction and separation of similar ingredients, the method of the invention can not only completely extract two main oxidation-resistant ingredients in the sugarcane toppers or slag, also the reagents consumed in the technological process can be recycled. The method has low production cost, high purity and mature technology, and is easily controlled and in favour of industrial production.
Description
Technical field
The invention belongs to the method and technology field of main oxidation-resistant active ingredient in comprehensive extraction separation and purification sugarcane toppers or the slag, be specifically related to main oxidation-resistant active ingredient---the method for total flavones, anthocyanidin in a kind of comprehensive extraction and purifying sugarcane toppers or the slag.
Background technology
Sugarcane (Saccharum officenarum L.) is Gramineae saccharum plant, originates in tropical and subtropical region, and its light saturation point is high, CO2 compensation point is low, the photorespiration rate is low, and rate of photosynthisis is large, and the cultivated area of sugarcane 90% is distributed between the tropic of north and south.China is the earliest the country of growing cane in the world, and history in 2500 is arranged so far approximately, and the sugarcane biological yield is high, and income is large, is the main raw material of sugaring.Sugar is one of human essential edible product, it also is the important source material of the foodstuffs industry such as candy, beverage, and sugarcane juice flavor cold in nature is sweet, have clearing heat and promoting fluid, moisturize the function of the long-pending therapeutic method to keep the adverse qi flowing downward of quenching the thirst, disappear, be applicable to pyreticosis Tianjin wound, restlessness and thirst, gastric disorder causing nausea vomiting, xeropulmonary cough, all diseases of constipation due to dry stool, be the good beverage of four seasons health care, China cures the disease with the sugar that sugarcane and sugarcane are made before more than 1,000 years.Simultaneously, the important source material of sugarcane or light industry, chemical industry and energy field.
Recent study finds that some have the composition than the high anti-oxidation activity in addition except containing a large amount of sucrose in the sugarcane.Studies show that antioxidant component mainly is some polyphenols such as flavones in the sugarcane, anthocyanidin etc., they all have been widely used in the industry such as food, makeup, medicine.Flavonoid compound has different physiological roles and pharmaceutical use, mainly contains: antitumor, anti-oxidant, suppress the pharmacological actions such as platelet aggregation, antiviral, estrogen regulating, preventing cardiovascular disease, hypoglycemic and neuroprotective system.Anthocyanidin is different from other antioxidant, can directly protect brain and neural system by hemato encephalic barrier, and therefore its pharmacologically active also becomes more powerful, wider than other oxidation inhibitor purposes; Its physiological action is mainly reflected in can remove human free radical, anticancer, and the enzyme inhibition of living protect the aspects such as cardiovascular and immunoregulatory activity.Therefore above two constituents have preferably use value and DEVELOPMENT PROSPECT.
The existing a small amount of report of the research of chromocor compound in the relevant bagasse, such as Renata Colombo (Renata Colombo, Fernando M.Lancas, Janete H.Yariwake.Determination of flavonoids in cultivated sugarcane leaves, bagasse, juice and in transgenic sugarcane by liquid chromatography-UV detection[J] .Journal of Chromatography A, 2006,118-124; Renata Colombo, Janete H.Yariwake, Emerson F.Queiroz, On-line ldentification of FurtherFlavone C-and O-Glycosides from Sugarcane (Saccharum officinarum L., Gramineae) by HPLC-UV-MS[J] .Phytochemical Analysis, 2006 (17): 337-343; Renata Colombo, Janete H.Yariwake, Emerson F.Queiroz etal, On-line identification of sugarcane (Saccharum officinarum L.) methoxyflavones by liquid chromatography-UVdetection using post-column derivatization and liquid chromatography-mass spectrometry[J] .Journal ofChromatography A, 2005,51-59) etc. use high performance liquid chromatography, liquid-the light such as matter coupling, the chromatogram means are to Caulis Sacchari sinensis leaf, sugar cane juice, part flavonoid glycoside monomer has carried out stratographic analysis and Structural Identification in the bagasse, and general flavone content is measured.But the research of flavones only rests on the trace analysis context of detection in the bagasse, its separation purifying technique method is not studied, and the simultaneously research for flavones in the sugarcane toppers had not also had bibliographical information.And about the research of anthocyanidin in the sugarcane toppers except the inventor, have not yet to see any correlation report.
The industrial purification method of flavonoid compound mainly contains supercritical fluid extraction, and (what expands, Li Yufeng, Zhang Xiuyuan is etc. the research [J] of supercritical fluid extraction ginko leaves flavone class material. Shanxi Food Industry, 2005 (4): 2-5), polymeric amide chromatography method (Guo Yanhua, Xu Jiangyang, the preliminary evaluation [J] of the extraction purifying of cordate houttuynia total flavones and flavones type. Food science, 2007,28 (9): 287-291) etc., above method equipment is complicated, and cost is higher.In addition, Wu Jianzhong (CN 1814695A) etc. discloses a kind of method of extracting polyphenoils in the Caulis Sacchari sinensis leaf take alcohol-water as solvent, the oxidation-resistant active ingredient in the heating reflux method lixiviate Caulis Sacchari sinensis leaf is used in this invention, can cause to a certain degree destruction to its activeconstituents like this, and n-butanol extraction purifying oxidation-resistant active ingredient is only adopted in this invention, and specific aim is not strong.And the raw material of this invention also is subject to certain limitation, and only adopting Caulis Sacchari sinensis leaf is raw material, and finds that according to the study flavones content in the sugarcane toppers is far above Caulis Sacchari sinensis leaf.The purification process of anthocyanidin mainly contains Thin-layer chromatography (Wu Chaoxia, Meng Xianjun, gold Lei. the preliminary study [J] that polyamide separation and purification grape pip procyanidin and portion of product component consist of. food research and development, 2007,28 (2): 71-74), macroporous absorption column chromatography (Li Runfeng, Lv Xiaoling, Wang Qinghua. Procyanidins in Grape Seed extracts and the preliminary study of separating [J]. grain and oil processing and food machinery, 2003 (6): 67-69), supercritical fluid extraction (Zheng Yongli. the analysis of Procyanidins in Grape Seed, extraction and purification [D]. Hebei University of Technology, 2004) etc.Sun Xiaoxue (Sun Xiaoxue, the extraction of aldehydes matter and applied research [D] in the sugarcane toppers. Guangxi University, 2007) the total aldehydes matter in the sugarcane toppers (comprising flavones, anthocyanidin and other phenols) qualitative and quantitative analysis and anti-oxidant and fresh-keeping applied research thereof have been carried out, polyphenol in this thesis polymeric amide chromatography method purifying sugarcane toppers, and polymeric amide is not strong to the selectivity of flavones in the sugarcane and anthocyanidin, these two kinds of compositions effectively can't be separated.At present, comparatively economical and practical Flavonoids by Macroporous Adsorption Resin not yet is useful on the report of anthocyanidin in the separation and purification sugarcane.The present invention is applied to acid-alkali treatment and macroporous absorption column chromatography in the separation and purification of total flavones and anthocyanidin in sugarcane toppers or the bagasse first, that its method is compared with existing document is with low cost, method is ripe, with strong points, by technical economical analysis as can be known, the rate of profit of technique of the present invention can reach 10%～25%, is easier to medium-sized and small enterprises and realizes industrialization.
Sugar degree in the sugarcane toppers is very low, and main component is polyphenols.It is squeezed the extraction syrup with the sugarcane mobile jib, then strengthened the content of coexistent impurity in the sugar, both having purified to sucrose has increased burden, also is unfavorable for the wherein development and use of effective constituent except sugar.If before entering press machines, simply the very low sugarcane toppers of sugar degree is cut down, effectively utilize---extract anti-oxidant activity composition wherein, then can substantially solve this contradiction.In addition, bagasse mainly as the fuel of steam boiler, make the main raw material(s) of low density and medium density fiber sheet material etc., anti-oxidant composition wherein goes out of use, resource is underutilized.As previously mentioned, research for effective active composition in the sugarcane only relates to wherein trace analysis detection, physico-chemical property or the preliminary activity research of a certain effective constituent, and mostly only rest on theoretical research stage, in sugarcane toppers or the slag in the extraction of a certain effective constituent, separation, purifying, the especially sugarcane the comprehensive extraction of effective constituent, separation, purifying bibliographical information is not also arranged.Therefore carry out the comprehensive utilization of Sugarcane Industry waste sugarcane toppers, bagasse resource, not only reduced environmental pollution, take full advantage of resource, but also can increase economic efficiency, have good social value and economic worth.
The present inventor has done first comprehensive analysis for the content of antioxidant in the various materials in the various raw materials in the cane sugar manufacture commercial run, the technological process (more than 10 plant) and has measured, and has established important basis for developing effective comprehensive utilization active substance wherein.
The present invention proposes comprehensively to extract anti-oxidation active substance (total flavones in the separation of sugarcane tip or the slag first, anthocyanidin) method, adopting source cane sugar manufacture industrial waste product sugarcane toppers, bagasse sufficient, cheap, that regenerative power is strong is raw material, the method of using repeatedly extraction to combine with the macroporous absorption column chromatography on the basis that does not affect original sugaring operation is extracted simultaneously and is isolated two kinds of high anti-oxidation activeconstituentss, the method technique is simple, with low cost, low in the pollution of the environment, possess good industrialization trend and wide application prospect.
Summary of the invention
The objective of the invention is to adopt source cane sugar manufacture industrial waste product sugarcane toppers, bagasse sufficient, cheap, that regenerative power is strong is raw material, the method of anti-oxidation active substance (total flavones, anthocyanidin) in a kind of comprehensive extraction separation of sugarcane tip or the slag is proposed.The method can not damage oxidation-resistant active ingredient contained in sugarcane toppers or the slag on the one hand, and can obtain simultaneously two kinds of effective constituent positions, improved resource utilization, adopted on the other hand the sugar industry waste products as raw material, it is with low cost, simple process, be easy to control, with strong points, extracting cycle is short, do not affect original sugaring operation, have good industrial advantages and exploitation meaning.
(the total flavones of anti-oxidation active substance in a kind of comprehensively extracting and purifying sugarcane toppers that reaches the object of the invention and provide or the slag, anthocyanidin) method, it is characterized in that the method is first with sugarcane toppers or bagasse raw material pulverizing, lixiviate, then with vat liquor by being filled with the chromatography column of macroporous adsorbent resin, with pure water wash away sugar and other water-soluble impurities after, re-use concentration of volume percent and be 10～100% ethanol-water solution and carry out gradient elution, concentrated, regulating concentrate eluant pH value with alkaline solution is alkalescence, use the extraction agent of 1～3 times of volume to extract 1～4 time, collect the buck layer, again the gained alkaline solution is transferred to acidity with pH adjusting agent, and then upward go on foot the gained acid solution with the extraction agent extraction, wash with water to neutrality, concentrated, vacuum-drying gets total flavones.The collected volume percentage concentration is 50～100% elutriant, re-uses the ethyl acetate extraction 1～3 time of 1～3 times of volume, and concentrated, lyophilize gets anthocyanidin.
In the aforesaid method concrete grammar of sugar cane juice raw material lixiviate be waste sugarcane toppers or the bagasse of the sugar industry after pulverizing be raw material, to use concentration of volume percent be 60～95% solvent extracts each 1～3 hour 2～4 times with the solid-liquid ratio of 1: 5～1: 15 (mass volume ratio).
The concrete grammar of vat liquor column chromatography is by being filled with the chromatography column of macroporous adsorbent resin with vat liquor in the aforesaid method, with pure water wash away sugar and other water-soluble impurities after, the ethanol-water solution that re-uses concentration of volume percent 10～100% carries out gradient elution, the collected volume percentage concentration is 50～100% elutriant, behind concentrating under reduced pressure, the pH value of regulating concentrate eluant with alkaline solution is 8～12, use the extraction agent of 1～3 times of volume to extract 1～4 time, collect the buck layer, again alkaline solution being regulated its pH value with pH adjusting agent is 3～6, extraction agent with 1～3 times of volume extracts acid solution 2～4 times, wash with water again to neutrality, then after reduction vaporization concentrates, vacuum-drying, through high-performance liquid chromatogram determination, namely get the total flavones amount and count 0.28～1.3mg/g take sugarcane raw material (the moisture sugarcane toppers after the squeezing or slag are as the basis).
The concrete grammar of vat liquor column chromatography is by being filled with the chromatography column of macroporous adsorbent resin with vat liquor in the aforesaid method, with pure water wash away sugar and other water-soluble impurities after, re-use the ethanol-water solution gradient elution of concentration of volume percent 10～100%, the collected volume percentage concentration is 10～40% elutriant, re-use the ethyl acetate extraction 1～3 time of 1～3 times of volume, after reduction vaporization is concentrated, vacuum-drying, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.053～0.63mg/g take sugarcane raw material (moisture sugarcane toppers or slag are as the basis), purity is 60～85%.
Used solvent is ethanol-water solution or methanol-water solution in total flavones, the anthocyanidin raw material leaching process, and extracting mode is that supersound extraction or pickling process are extracted or percolation extracts.The used pH adjusting agent of its vat liquor column chromatography purification process is hydrochloric acid or acid or glacial acetic acid or phosphoric acid or sulfuric acid, alkaline solution is sodium hydroxide or potassium hydroxide or yellow soda ash or sodium hydrogen carbonate solution, used macroporous adsorbent resin is D101 or D301 or AB-8 or D100 or D141 type macroporous adsorbent resin, and extraction agent is ethyl acetate or propyl carbinol.
Compared with the prior art the present invention has the following advantages:
1, because integrated extraction technique provided by the invention can all extract two kinds of main oxidation-resistant active ingredients in sugarcane toppers or the slag, and methodological science is efficient, and is with strong points, resource utilization significantly improves.
2, since the present invention to adopt the sugarcane trade waste be raw material, raw material sources are abundant, and are cheap, renewable is strong, production cost is low, does not affect the carrying out of original sugaring operation.
3, because product of the present invention is taken from natural phant, substantially have no side effect, can be used as in medicine or food or the cosmetics additive, security is good, has very high development and application values.
4, because the present invention adopts the sugar industry waste products, not only significantly reduce environmental pollution, made resource obtain more taking full advantage of, also increased the industry added value.
5, because the present invention sets up from the sugar industry waste products the comprehensive method of extracting multiple oxidation-resistant active ingredient first, the comprehensive utilizating research of later cane sugar manufacture industrial product high added value composition is laid the foundation.
6, because the present invention adopts is to extract or the purifying mode all is comparatively ripe, whole technical conditions is gentle in addition, each process procedure solvent for use is recyclable, thereby simple to operate, be easy to control, production cost is low, not only meets the resource that takes full advantage of that country vigorously advocates, the recycling economy of environmental contamination reduction requires and the Scientific Outlook on Development, and is conducive to realize suitability for industrialized production.
Embodiment
Below by embodiment the present invention is specifically described; be necessary to be pointed out that at this following examples are only for the invention will be further described; can not be interpreted as limiting the scope of the invention; the person skilled in the art in this field makes some nonessential improvement and adjustment according to the invention described above content to the present invention, still belongs to protection domain of the present invention.
Embodiment 1
The ethanol of the use of the sugarcane toppers raw material 100g after the chopping 60% is extracted each 1 hour 2 times with the solid-liquid ratio pickling process of 1: 15 (mass volume ratio).Then with the raw material extracting solution by being filled with the chromatography column of D101 type macroporous adsorbent resin, wash away sugar and other water-soluble impurities with pure water after, re-use concentration of volume percent and be 10～100% aqueous ethanolic solution and carry out gradient elution.The collected volume percentage concentration is 60% elutriant, concentrated, the pH value of using sodium hydroxide solution to regulate concentrate eluant is 10, the ethyl acetate extraction of 3 times of volumes of use 2 times, it is 6 that the sodium hydroxide solution part is adjusted to the pH value with hydrochloric acid, and then with the ethyl acetate extraction of 3 times of volumes 2 times, wash with water to neutrality, then after reduction vaporization concentrates, lyophilize, through high-performance liquid chromatogram determination, calculate acquisition total flavones amount and count 1.0mg/g take sugarcane raw material (the moisture sugarcane toppers after the squeezing is as the basis).The collected volume percentage concentration is 30% elutriant, re-uses the ethyl acetate extraction 3 times of 2 times of volumes, and is concentrated, lyophilize, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.58mg/g take sugarcane raw material (moisture sugarcane toppers is as the basis), purity is 85%.
Embodiment 2
Use 60% methyl alcohol with the ultrasonic lixiviate of the solid-liquid ratio of 1: 6 (mass volume ratio) 4 times, each 1 hour the bagasse raw material 100g after squeezing.Then with the raw material extracting solution by being filled with the chromatography column of D301 type macroporous adsorbent resin, wash away sugar and other water-soluble impurities with pure water after, re-use concentration of volume percent and be 10～100% aqueous ethanolic solution and carry out gradient elution.The collected volume percentage concentration is 70% elutriant, concentrated, the pH value of using potassium hydroxide solution to regulate concentrate eluant is 9, the n-butanol extraction of 1 times of volume of use 2 times, it is 5 that the potassium hydroxide solution part is adjusted to the pH value with acetic acid, and then with the n-butanol extraction of 2 times of volumes 3 times, wash with water to neutrality, then after reduction vaporization concentrates, lyophilize, through high-performance liquid chromatogram determination, calculate acquisition total flavones amount and count 0.28mg/g take sugarcane raw material (the moisture bagasse after the squeezing is as the basis).The collected volume percentage concentration is 40% elutriant, re-uses the ethyl acetate extraction 1 time of 3 times of volumes, and is concentrated, lyophilize, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.054mg/g take sugarcane raw material (moisture bagasse is as the basis), purity is 75%.
Embodiment 3
Sugarcane toppers raw material 100g after the chopping used 80% ethanol extract each 1 hour 3 times with the solid-liquid ratio percolation of 1: 8 (mass volume ratio).Then with the raw material extracting solution by being filled with the chromatography column of AB-8 type macroporous adsorbent resin, wash away sugar and other water-soluble impurities with pure water after, re-use concentration of volume percent and be 10～100% aqueous ethanolic solution and carry out gradient elution.The collected volume percentage concentration is 80% elutriant, concentrated, the pH value of using sodium carbonate solution to regulate concentrate eluant is 9, the n-butanol extraction of 2 times of volumes of use 3 times, it is 4 that the sodium carbonate solution part is adjusted to the pH value with formic acid, and then with the ethyl acetate extraction of 1 times of volume 4 times, wash with water to neutrality, then after reduction vaporization concentrates, lyophilize, through high-performance liquid chromatogram determination, calculate acquisition total flavones amount and count 1.1mg/g take sugarcane raw material (the moisture sugarcane toppers after the squeezing is as the basis).The collected volume percentage concentration is 30% elutriant, re-uses the ethyl acetate extraction 2 times of 1 times of volume, and is concentrated, lyophilize, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.59mg/g take sugarcane raw material (moisture sugarcane toppers is as the basis), purity is 80%.
Embodiment 4
The ethanol of the use of the bagasse raw material 100g after squeezing 70% is extracted each 1 hour 4 times with the solid-liquid ratio pickling process of 1: 9 (mass volume ratio).Then with the raw material extracting solution by being filled with the chromatography column of D100 type macroporous adsorbent resin, wash away sugar and other water-soluble impurities with pure water after, re-use concentration of volume percent and be 10～100% aqueous ethanolic solution and carry out gradient elution.The collected volume percentage concentration is 90% elutriant, concentrated, the pH value of using sodium hydrogen carbonate solution to regulate concentrate eluant is 8, the ethyl acetate extraction of 2 times of volumes of use 4 times, it is 3 that the sodium hydrogen carbonate solution part is adjusted to the pH value with phosphoric acid, and then with the n-butanol extraction of 1 times of volume 4 times, wash with water to neutrality, then after reduction vaporization concentrates, lyophilize, through high-performance liquid chromatogram determination, calculate acquisition total flavones amount and count 0.3mg/g take sugarcane raw material (the moisture bagasse after the squeezing is as the basis).The collected volume percentage concentration is 40% elutriant, re-uses the ethyl acetate extraction 3 times of 2 times of volumes, and is concentrated, lyophilize, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.058mg/g take sugarcane raw material (moisture bagasse is as the basis), purity is 70%.
Embodiment 5
Use 80% methyl alcohol with the ultrasonic lixiviate of the solid-liquid ratio of 1: 11 (mass volume ratio) 2 times, each 2 hours the sugarcane toppers raw material 100g after the chopping.Then with the raw material extracting solution by being filled with the chromatography column of D141 type macroporous adsorbent resin, wash away sugar and other water-soluble impurities with pure water after, re-use concentration of volume percent and be 10～100% aqueous ethanolic solution and carry out gradient elution.The collected volume percentage concentration is 100% elutriant, concentrated, the pH value of using sodium hydroxide solution to regulate concentrate eluant is 12, the n-butanol extraction of 3 times of volumes of use 4 times, it is 5 that the sodium hydroxide solution part is adjusted to the pH value with sulfuric acid, and then with the n-butanol extraction of 2 times of volumes 3 times, wash with water to neutrality, then after reduction vaporization concentrates, lyophilize, through high-performance liquid chromatogram determination, calculate acquisition total flavones amount and count 1.2mg/g take sugarcane raw material (the moisture sugarcane toppers after the squeezing is as the basis).The collected volume percentage concentration is 30% elutriant, re-uses the ethyl acetate extraction 1 time of 3 times of volumes, and is concentrated, lyophilize, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.63mg/g take sugarcane raw material (moisture sugarcane toppers is as the basis), purity is 75%.
Embodiment 6
Use 90% methyl alcohol with the ultrasonic lixiviate of the solid-liquid ratio of 1: 13 (mass volume ratio) 3 times, each 2 hours the bagasse raw material 100g after squeezing.Then with the raw material extracting solution by being filled with the chromatography column of D141 type macroporous adsorbent resin, wash away sugar and other water-soluble impurities with pure water after, re-use concentration of volume percent and be 10～100% aqueous ethanolic solution and carry out gradient elution.The collected volume percentage concentration is 60% elutriant, concentrated, the pH value of using potassium hydroxide solution to regulate concentrate eluant is 11, the n-butanol extraction of 1 times of volume of use 3 times, it is 6 that the potassium hydroxide solution part is adjusted to the pH value with sulfuric acid, and then with the ethyl acetate extraction of 3 times of volumes 2 times, wash with water to neutrality, then after reduction vaporization concentrates, lyophilize, through high-performance liquid chromatogram determination, calculate acquisition total flavones amount and count 0.32mg/g take sugarcane raw material (the moisture bagasse after the squeezing is as the basis).The collected volume percentage concentration is 20% elutriant, re-uses the ethyl acetate extraction 2 times of 1 times of volume, and is concentrated, lyophilize, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.055mg/g take sugarcane raw material (moisture bagasse is as the basis), purity is 65%.
Embodiment 7
The ethanol of the use of the sugarcane toppers raw material 100g after the chopping 95% is extracted each 2 hours 2 times with the solid-liquid ratio pickling process of 1: 12 (mass volume ratio).Then with the raw material extracting solution by being filled with the chromatography column of D100 type macroporous adsorbent resin, wash away sugar and other water-soluble impurities with pure water after, re-use concentration of volume percent and be 10～100% aqueous ethanolic solution and carry out gradient elution.The collected volume percentage concentration is 70% elutriant, concentrated, the pH value of using sodium carbonate solution to regulate concentrate eluant is 8, the ethyl acetate extraction of 1 times of volume of use 2 times, it is 3 that the sodium carbonate solution part is adjusted to the pH value with phosphoric acid, and then with the n-butanol extraction of 2 times of volumes 3 times, wash with water to neutrality, then after reduction vaporization concentrates, lyophilize, through high-performance liquid chromatogram determination, calculate acquisition total flavones amount and count 1.3mg/g take sugarcane raw material (the moisture sugarcane toppers after the squeezing is as the basis).The collected volume percentage concentration is 10% elutriant, re-uses the ethyl acetate extraction 3 times of 2 times of volumes, and is concentrated, lyophilize, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.60mg/g take sugarcane raw material (moisture sugarcane toppers is as the basis), purity is 60%.
Embodiment 8
Bagasse raw material 100g after squeezing used 80% ethanol extract each 1 hour 3 times with the solid-liquid ratio percolation of 1: 10 (mass volume ratio).Then with the raw material extracting solution by being filled with the chromatography column of AB-8 type macroporous adsorbent resin, wash away sugar and other water-soluble impurities with pure water after, re-use concentration of volume percent and be 10～100% aqueous ethanolic solution and carry out gradient elution.The collected volume percentage concentration is 80% elutriant, concentrated, the pH value of using sodium hydrogen carbonate solution to regulate concentrate eluant is 9, the ethyl acetate extraction of 2 times of volumes of use 1 time, it is 4 that the sodium hydrogen carbonate solution part is adjusted to the pH value with formic acid, and then with the ethyl acetate extraction of 3 times of volumes 4 times, wash with water to neutrality, then after reduction vaporization concentrates, lyophilize, through high-performance liquid chromatogram determination, calculate acquisition total flavones amount and count 0.31mg/g take sugarcane raw material (the moisture bagasse after the squeezing is as the basis).The collected volume percentage concentration is 20% elutriant, re-uses the ethyl acetate extraction 1 time of 3 times of volumes, and is concentrated, lyophilize, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.056mg/g take sugarcane raw material (moisture bagasse is as the basis), purity is 70%.
Embodiment 9
Use 70% methyl alcohol with the ultrasonic lixiviate of the solid-liquid ratio of 1: 7 (mass volume ratio) 4 times, each 1 hour the sugarcane toppers raw material 100g after the chopping.Then with the raw material extracting solution by being filled with the chromatography column of D301 type macroporous adsorbent resin, wash away sugar and other water-soluble impurities with pure water after, re-use concentration of volume percent and be 10～100% aqueous ethanolic solution and carry out gradient elution.The collected volume percentage concentration is 90% elutriant, concentrated, the pH value of using sodium hydroxide solution to regulate concentrate eluant is 9, the ethyl acetate extraction of 3 times of volumes of use 3 times, it is 5 that the sodium hydroxide solution part is adjusted to the pH value with acetic acid, and then with the ethyl acetate extraction of 1 times of volume 2 times, wash with water to neutrality, then after reduction vaporization concentrates, lyophilize, through high-performance liquid chromatogram determination, calculate acquisition total flavones amount and count 1.0mg/g take sugarcane raw material (the moisture sugarcane toppers after the squeezing is as the basis).The collected volume percentage concentration is 30% elutriant, re-uses the ethyl acetate extraction 2 times of 1 times of volume, and is concentrated, lyophilize, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.61mg/g take sugarcane raw material (moisture sugarcane toppers is as the basis), purity is 80%.
Embodiment 10
The methyl alcohol of the use of the bagasse raw material 100g after squeezing 95% is extracted each 2 hours 2 times with the solid-liquid ratio pickling process of 1: 5 (mass volume ratio).Then with the raw material extracting solution by being filled with the chromatography column of D101 type macroporous adsorbent resin, wash away sugar and other water-soluble impurities with pure water after, re-use concentration of volume percent and be 10～100% aqueous ethanolic solution and carry out gradient elution.The collected volume percentage concentration is 100% elutriant, concentrated, the pH value of using potassium hydroxide solution to regulate concentrate eluant is 10, the n-butanol extraction of 1 times of volume of use 2 times, it is 6 that the potassium hydroxide solution part is adjusted to the pH value with hydrochloric acid, and then with the ethyl acetate extraction of 2 times of volumes 3 times, wash with water to neutrality, then after reduction vaporization concentrates, lyophilize, through high-performance liquid chromatogram determination, calculate acquisition total flavones amount and count 0.29mg/g take sugarcane raw material (the moisture bagasse after the squeezing is as the basis).The collected volume percentage concentration is 40% elutriant, re-uses the ethyl acetate extraction 3 times of 2 times of volumes, and is concentrated, lyophilize, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.053mg/g take sugarcane raw material (moisture bagasse is as the basis), purity is 65%.
Embodiment 11
Sugarcane toppers raw material 100g after the chopping used 60% ethanol extract each 1 hour 4 times with the solid-liquid ratio percolation of 1: 14 (mass volume ratio).Then with the raw material extracting solution by being filled with the chromatography column of D301 type macroporous adsorbent resin, wash away sugar and other water-soluble impurities with pure water after, re-use concentration of volume percent and be 10～100% aqueous ethanolic solution and carry out gradient elution.The collected volume percentage concentration is 60% elutriant, concentrated, the pH value of using sodium hydroxide solution to regulate concentrate eluant is 8, the ethyl acetate extraction of 2 times of volumes of use 3 times, it is 5 that the sodium hydroxide solution part is adjusted to the pH value with acetic acid, and then with the ethyl acetate extraction of 2 times of volumes 2 times, wash with water to neutrality, then after reduction vaporization concentrates, lyophilize, through high-performance liquid chromatogram determination, calculate acquisition total flavones amount and count 1.2mg/g take sugarcane raw material (the moisture sugarcane toppers after the squeezing is as the basis).The collected volume percentage concentration is 20% elutriant, re-uses the ethyl acetate extraction 1 time of 3 times of volumes, and is concentrated, lyophilize, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.62mg/g take sugarcane raw material (moisture sugarcane toppers is as the basis), purity is 75%.
Embodiment 12
Bagasse raw material 100g after squeezing used 90% ethanol extract each 2 hours 3 times with the solid-liquid ratio percolation of 1: 5 (mass volume ratio).Then with the raw material extracting solution by being filled with the chromatography column of D101 type macroporous adsorbent resin, wash away sugar and other water-soluble impurities with pure water after, re-use concentration of volume percent and be 10～100% aqueous ethanolic solution and carry out gradient elution.The collected volume percentage concentration is 100% elutriant, concentrated, the pH value of using potassium hydroxide solution to regulate concentrate eluant is 12, the n-butanol extraction of 3 times of volumes of use 2 times, it is 4 that the potassium hydroxide solution part is adjusted to the pH value with hydrochloric acid, and then with the ethyl acetate extraction of 2 times of volumes 3 times, wash with water to neutrality, then after reduction vaporization concentrates, lyophilize, through high-performance liquid chromatogram determination, calculate acquisition total flavones amount and count 0.28mg/g take sugarcane raw material (the moisture bagasse after the squeezing is as the basis).The collected volume percentage concentration is 30% elutriant, re-uses the ethyl acetate extraction 2 times of 1 times of volume, and is concentrated, lyophilize, through high-performance liquid chromatogram determination, namely get the anthocyanidin amount and count 0.054mg/g take sugarcane raw material (moisture bagasse is as the basis), purity is 85%.
Claims (6)
1. comprehensive method of extracting anti-oxidation active substance anthocyanidin in the separation of sugarcane tip or the bagasse and not containing the total flavones of anthocyanidin, it is characterized in that the method is: first with sugarcane toppers or bagasse raw material pulverizing, to use concentration expressed in percentage by volume be 60～95% solvent with the solid-liquid ratio supersound extraction of 1: 5～1: 15 (mass volume ratio) 2～4 times, described solvent is ethanol-water solution or methanol-water solution, each lasting 1～3 hour, concentrating under reduced pressure obtained concentrated extracting solution after extracting solution merged; Then concentrated extracting solution is adsorbed by the chromatography column that is filled with macroporous adsorbent resin, with pure water wash away sugar and other water-soluble impurities after, re-use concentration of volume percent and be 10～100% ethanol-water solution and carry out gradient elution, collect the concentrated crude extract that obtains being rich in anthocyanidin of alcohol-water elutriant of 10～40% concentration sections, collect the concentrated crude extract that obtains being rich in total flavones of alcohol-water elutriant of 50～100% concentration sections; After above two kinds of crude extracts being adopted respectively solvent extration and the further enrichment of acid-alkali treatment method, purifying, concentrated and lyophilize, obtain respectively anthocyanidin and do not contain the total flavones of anthocyanidin.
2. method according to claim 1, the concrete grammar that it is characterized in that the vat liquor column chromatography is that concentrated extracting solution is adsorbed by the chromatography column that is filled with macroporous adsorbent resin, after washing away sugar and other water-soluble impurities with pure water, re-use concentration of volume percent and be 10～100% ethanol-water solution and carry out gradient elution; The collected volume percentage concentration is 10～40% elutriant, re-uses the ethyl acetate of 1～3 times of volume to this elutriant extraction 1～3 time; Raffinate gets anthocyanidin through concentrating under reduced pressure after the lyophilize.
3. method according to claim 1, the concrete grammar that it is characterized in that the vat liquor column chromatography is by being filled with the chromatography column of macroporous adsorbent resin with concentrated extracting solution, after washing away sugar and other water-soluble impurities with pure water, re-use concentration of volume percent and be 10～100% ethanol-water solution and carry out gradient elution; The collected volume percentage concentration is 50～100% elutriant, behind the concentrating under reduced pressure, the pH value to 8 of the aqueous solution adjusting concentrate eluant of use sodium hydroxide or potassium hydroxide or yellow soda ash or sodium bicarbonate～12, ethyl acetate or the propyl carbinol with 1～3 times of concentrate eluant volume extracts 1～4 time this concentrate eluant again; Collect the buck layer solution after the raffinate, with hydrochloric acid or acetic acid or sulfuric acid or formic acid or phosphate aqueous solution its pH value being adjusted to 3～6 becomes acidic aqueous solution, then uses the ethyl acetate of 1～3 times of volume or this acidic aqueous solution of n-butanol extraction 2～4 times; Merge repeatedly total flavones extraction liquid, wash with water again to neutrality, after concentrated, lyophilize do not contain the total flavones of anthocyanidin.
4. method according to claim 2 is characterized in that macroporous adsorbent resin used in the method is D101 type or D301 type or AB-8 type or D100 type or D141 type macroporous adsorbent resin.
5. method according to claim 2 is characterized in that productive rate take moisture sugarcane toppers or slag weighing scale anthocyanidin as 0.053～0.63mg/g, and purity is 60～85%.
6. method according to claim 3 is characterized in that not containing the productive rate of total flavones of anthocyanidin as 0.28～1.3mg/g take sugarcane toppers or slag weighing scale.
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| CN103149282A (en) * | 2013-01-16 | 2013-06-12 | 广西中医药大学 | Sugarcane top qualitative identification high performance liquid chromatography (HPLC) fingerprint spectrum detection method |
| CN104957602B (en) * | 2015-05-22 | 2018-06-19 | 长沙普源生物科技有限公司 | Sugarcane active component composition and functional health-care food and preparation method thereof |
| CN107043402B (en) * | 2017-01-19 | 2020-09-22 | 华南理工大学 | Preparation method of high-activity sugarcane anthocyanin |
| CN109705081A (en) * | 2019-01-20 | 2019-05-03 | 林燕 | A method of extracting anthocyanidin from sugarcane pericarp |
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