CN103235067A - Method for enriching gallotannin with antioxidation activity from mangos - Google Patents
Method for enriching gallotannin with antioxidation activity from mangos Download PDFInfo
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- CN103235067A CN103235067A CN2013101424177A CN201310142417A CN103235067A CN 103235067 A CN103235067 A CN 103235067A CN 2013101424177 A CN2013101424177 A CN 2013101424177A CN 201310142417 A CN201310142417 A CN 201310142417A CN 103235067 A CN103235067 A CN 103235067A
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Abstract
The invention provides a method for enriching gallotannin with antioxidation activity from mangos. The gallotannin with antioxidation activity is prepared from the mango processing byproduct dried powder by a solid-phase extraction technique through the steps of crude extract preparation, crude extract purification and antioxidation activity analysis. Compared with the prior art, the invention has the advantages of safe and manageable production process and short production cycle; the whole purification process is accurately quantified by HPLC (high performance liquid chromatography), and thus, has high accuracy; the 1,1-diphenyl-2-trinitrophenylhydrazine antioxidation analysis on the obtained crude extract powder and Vc detects that the capacity for clearing the 1,1-diphenyl-2-trinitrophenylhydrazine is better than that of the Vc, which indicates that the gallotannin has very high antioxidation activity. The invention can be used for developing additives of functional foods, cosmetics and the like. The invention is simple to operate, has the advantages of simple equipment investment, high efficiency and stable and controllable conditions, and is applicable to large-scale production.
Description
Technical field
The invention belongs to the extraction and separation technology field of native compound, being specifically related to the mango processing byproduct is raw material, adopts the enrichment from mango of Solid-Phase Extraction method to have the method for the nutgall tannin of higher antioxidation activity.
Background technology
Mango be Anacardiaceae plant mango (
Mangifera indica) fruit, contain rich nutrient substances.Studies show that in recent years, in the mango because containing carotenoid, flavones and derivant thereof and phenolic acid class isoreactivity material and have anti-oxidant, anti-inflammatory, antibacterial, immunological regulation suppresses tumour and hypoglycemic isoreactivity, gets more and more people's extensive concerning.In the existing research, pay close attention to materials such as carotenoid and phenols mostly for the trophic function of mango, for the activity rating of mango nutgall tannin, yet there are no deep report.In recent years, the technology of preparation tannin has more report from the other plant material, but because the structure of tannin complexity, yet there are no the report of enrichment nutgall tannin.Mango is subjected to extensively liking of people as a kind of tropical fruit (tree) of delicious food, but account for the pericarp about fruit 30-65% and plant benevolence, still eating raw in the mango deep processing all becomes discarded object in the process, research enrichment nutgall tannin and estimate its antioxidation activity the accessory substance of mango process is turned waste into wealth from mango pericarp kind benevolence can also provide new approaches for the deep processing of mango.
Described five times of nutgall tannin structural formulas:
Summary of the invention
The object of the invention provide a kind of from mango the method for the nutgall tannin of enrichment tool antioxidation activity, realize by following steps:
(1) preparation of crude extract: the powder of mango processing byproduct, soaked ultrasonic 30 minutes the centrifuging and taking supernatant with 80% acetone (solid-to-liquid ratio 1:20), extract twice, filter, merge filtrate twice, evaporate to dryness and being dissolved in the distilled water on Rotary Evaporators obtains the water crude extract; The mango processing byproduct is selected dry mango pericarp for use or is planted benevolence;
(2) purifying of crude extract: adopt C18 solid-phase extraction column (Waters 12cc, 2 g), activate through methyl alcohol, behind the water balance, the water crude extract that step (1) is obtained joins on the solid-phase extraction column, fully after the absorption, wash with water earlier, remove water-solubility impurities such as saccharic acid, use methanol-eluted fractions then, the meoh eluate vacuum concentrates evaporate to dryness and makes powder;
(3) utilize the powder principal ingredient of high performance liquid chromatography (HPLC) and LC-MS (LC-MS) technology for detection authentication step (2) also quantitative, confirm that this powder is rich in the nutgall tannin.This powder through preliminary purification demonstrates higher antioxidation activity (table 1,2), can be directly used in other activity research and industrialization development and use, and need not again through further purifying.
Chromatographic condition: flowing comprises 2% acetic acid (A) and 0.5% acetic acid/chromatogram acetonitrile (1:1 mutually, v/v) (B), gradient elution program: 0-2 min:10% B, 2-8 min:10-30% B, 8-30 min:30-40% B, 30-45 min:40-50% B, 45-60 min:50-65% B, 60-70 min:65-90% B, 70-71 min:90-10% B, 71-75 min:10% B, flow velocity are 0.6 ml/min, and fixing is XBridge mutually
TM, Shield, RP18(5 μ m, 250*4.6 mm) and chromatographic column, 25 ℃ of column temperatures detect wavelength 280 nm, sampling volume 10 μ l.
Mass spectrum condition: ESI ion gun, negative ion mode (ESI
-); Dry gas (N
2) temperature: 325 ℃; Flow velocity: 5.0 l/min; Atomizer pressure: 45psi; Capillary voltage: 3500V.
The one-level mass spectrum is consistent with bibliographical information with the feature fragment that second order ms produces, and confirms that peak 1-5 is respectively five-nine times of nutgall tannin,, further each nutgall tannin component in the crude extract is carried out quantitatively as equivalent with five times of nutgall tannin.
The present invention adopts solid phase extraction techniques, is the nutgall tannin that the raw material enrichment has higher antioxidation activity with mango processing byproduct dried powder.The present invention has following advantage with respect to the technology in past: (1) production procedure safety is easy to operate; (2) with short production cycle; (4) whole purge process is carried out accurate quantification by HPLC, the accuracy height; (5) the crude extract powder that obtains and Vc do the anti-oxidant analysis of DPPH together, find its removing 1, the ability of 1-diphenyl-2-trinitrophenyl-hydrazine (DPPH) is better than Vc, illustrates to have very high antioxidation activity, can be used for the adjuvant of development function food, cosmetics etc.
Description of drawings
Fig. 1 is process chart of the present invention.
Fig. 2 A-F is the HPLC chromatogram that is rich in nutgall tannin mango crude extract.
Embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment 1
Referring to Fig. 1, enrichment nutgall tannin from mango kind benevolence: take by weighing 1.5 g Ma Qiesu, No. 1, platform farming, the real benevolence freeze drying powder of planting of pale reddish brown mango respectively, with 80% acetone (solid-to-liquid ratio 1:20) ultrasonic 30 minutes, the centrifuging and taking supernatant, repeat twice, merge supernatant, 45 ℃ of evaporates to dryness and be dissolved in the 25 ml water with deionized water on Rotary Evaporators.C18 solid-phase extraction column (Waters 12cc, 2 g) at first activate with 15 ml methyl alcohol, use 15 ml water balances again, then with sample on the 25 ml extracts, fully after the absorption, with 25 ml water wash, remove water-solubility impurities such as saccharic acid, use 25 ml methanol-eluted fractions then, with the impurity of leaving in the 15 ml chloroform wash-out pillars such as pigment, use 15 ml Mathanol regenerating pillars at last.Meoh eluate is the evaporate to dryness powdered in vacuum drying, yield is respectively 11.54%, 12.46%, 11.29% (table 1), identify that through mass spectrum the principal ingredient of this crude extract is five times to nine times nutgall tannin, HPLC detects collection of illustrative plates referring to Fig. 2 A, Fig. 2 B, Fig. 2 C, and HPLC quantitatively finds to be rich in the crude extract powder quantitative (table 1) of nutgall tannin.
Chromatographic condition: flowing comprises 2% acetic acid (A) and 0.5% acetic acid/chromatogram acetonitrile (1:1 mutually, v/v) (B), gradient elution program: 0-2 min:10% B, 2-8 min:10-30% B, 8-30 min:30-40% B, 30-45 min:40-50% B, 45-60 min:50-65% B, 60-70 min:65-90% B, 70-71 min:90-10% B, 71-75 min:10% B, flow velocity are 0.6 ml/min, and fixing is XBridge mutually
TM, Shield, RP18(5 μ m, 250*4.6 mm) and chromatographic column, 25 ℃ of column temperatures detect wavelength 280 nm, sampling volume 10 μ l.
Mass spectrum condition: ESI ion gun, negative ion mode (ESI
-); Dry gas (N
2) temperature: 325 ℃; Flow velocity: 5.0 l/min; Atomizer pressure: 45psi; Capillary voltage: 3500V.
Anti-oxidant analysis: Vc and the final concentration of mango kind benevolence crude extract in the DPPH reaction system are 5,2.5,1.25,0.625,0.3125,0.15625 μ g/ml.Get 2 μ l sample liquid, add the DPPH methanol solution 198 μ l of 25 μ g/ml, mixing is made blank with methyl alcohol, surveys absorbance under 517 nm, and reaction detected in 10 minutes under the room temperature.Identical experiment repeats 3 times.The free radical inhibiting rate is calculated as follows: inhibiting rate (%)=(A517 solvent-A517 sample)/A517 solvent * 100%.With half inhibiting rate (IC
50) the expression free radical scavenging activity.Suppress percent according to the free radical under each compound respective concentration, calculate IC with the Logit method
50Value is respectively 1.24 ± 0.19,1.15 ± 0.23,1.23 ± 0.02 μ g/ml(tables 1), the IC of Vc
50Be 1.42 ± 0.01 μ g/ml.
Enrichment nutgall tannin from the mango pericarp: take by weighing 1.5 g Ma Qiesu, No. 1, platform farming, the real pericarp freeze drying of pale reddish brown mango powder respectively, with 80% acetone (solid-to-liquid ratio 1:20) ultrasonic 30 minutes, the centrifuging and taking supernatant, repeat twice, merge supernatant, 45 degree evaporates to dryness and be dissolved in the 25 ml water with deionized water on Rotary Evaporators.C18 solid-phase extraction column (Waters 12cc, 2 g) at first activate with 15 ml methyl alcohol, use 15 ml water balances again, then with sample on the 25 ml extracts, fully after the absorption, with 25 ml water wash, remove water-solubility impurities such as saccharic acid, use 25 ml methanol-eluted fractions then, with the impurity of leaving in the 15 ml chloroform wash-out pillars such as pigment, use 15 ml Mathanol regenerating pillars at last.Meoh eluate is the evaporate to dryness powdered in vacuum drying, yield is respectively 7.04%, 13.14%, 8.85%(table 2), through mass spectrum identify the principal ingredient of this crude extract be five times to nine times of nutgall tannin (table 3), HPLC detects collection of illustrative plates referring to Fig. 2 D, Fig. 2 E, Fig. 2 F, and HPLC quantitatively finds to be rich in the crude extract powder nutgall tannin (table 2).
Chromatographic condition: flowing comprises 2% acetic acid (A) and 0.5% acetic acid/chromatogram acetonitrile (1:1 mutually, v/v) (B), gradient elution program: 0-2 min:10% B, 2-8 min:10-30% B, 8-30 min:30-40% B, 30-45 min:40-50% B, 45-60 min:50-65% B, 60-70 min:65-90% B, 70-71 min:90-10% B, 71-75 min:10% B, flow velocity are 0.6 ml/min, and fixing is XBridge mutually
TM, Shield, RP18(5 μ m, 250*4.6 mm) and chromatographic column, 25 ℃ of column temperatures detect wavelength 280 nm, sampling volume 10 μ l.
Mass spectrum condition: ESI ion gun, negative ion mode (ESI
-); Dry gas (N
2) temperature: 325 ℃; Flow velocity: 5.0 l/min; Atomizer pressure: 45psi; Capillary voltage: 3500V.
Anti-oxidant analysis: Vc and the final concentration of mango pericarp crude extract in the DPPH reaction system are 5,2.5,1.25,0.625,0.3125,0.15625 μ g/ml.Get 2 μ l sample liquid, add the DPPH methanol solution 198 μ l of 25 μ g/ml, mixing is made blank with methyl alcohol, surveys absorbance under 517 nm, and reaction detected in 10 minutes under the room temperature.Identical experiment repeats 3 times.The free radical inhibiting rate is calculated as follows: inhibiting rate (%)=(A517 solvent-A517 sample)/A517 solvent * 100%.With half inhibiting rate (IC
50) the expression free radical scavenging activity.Suppress percent according to the free radical under each compound respective concentration, calculate IC with the Logit method
50Value is respectively 1.28 ± 0.07,1.22 ± 0.13,1.32 ± 0.22 μ g/ml(tables 2), the IC of Vc
50Be 1.42 ± 0.01 μ g/ml.
Claims (2)
1. the method for the nutgall tannin of enrichment tool antioxidation activity from mango, realize by following steps:
(1) preparation of crude extract: dry mango processing byproduct, soaked the centrifuging and taking supernatant ultrasonic 30 minutes with 80% acetone of solid-to-liquid ratio 1:20, extract twice, filter, merge filtrate twice, evaporate to dryness and being dissolved in the distilled water on Rotary Evaporators obtains the water crude extract;
(2) purifying of crude extract: adopt the C18 solid-phase extraction column, activate through methyl alcohol, behind the water balance, the water crude extract that step (1) is obtained joins on the solid-phase extraction column, fully after the absorption, wash with water earlier, remove water-solubility impurities such as saccharic acid, use methanol-eluted fractions then, the meoh eluate vacuum concentrates evaporate to dryness and makes powder;
(3) utilize the powder of high performance liquid chromatography and LC-MS technology for detection authentication step (2), confirm that this powder is rich in the nutgall tannin;
Chromatographic condition: flowing comprises A:2% acetic acid and B mutually: 0.5% acetic acid/chromatogram acetonitrile, 1:1, v/v; Gradient elution program: 0-2 min:10% B, 2-8 min:10-30% B, 8-30 min:30-40% B, 30-45 min:40-50% B, 45-60 min:50-65% B, 60-70 min:65-90% B, 70-71 min:90-10% B, 71-75 min:10% B, flow velocity are 0.6 ml/min, and fixing is XBridge mutually
TM, Shield, RP18,5 μ m, 250*4.6 mm chromatographic column, 25 ℃ of column temperatures detect wavelength 280 nm, sampling volume 10 μ l;
Mass spectrum condition: ESI ion gun, negative ion mode; Dry N
2Temperature degree: 325 ℃; Flow velocity: 5.0 l/min; Atomizer pressure: 45psi; Capillary voltage: 3500V.
According to claim 1 a kind of from mango the method for the nutgall tannin of enrichment tool antioxidation activity, it is characterized in that the mango processing byproduct of the described drying of step (1) is selected dry mango pericarp for use or planted the benevolence powder.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105920114A (en) * | 2016-04-18 | 2016-09-07 | 浙江大学 | Black tartary buckwheat rice extract with blood sugar decreasing function and preparation method thereof |
| CN110954379A (en) * | 2018-09-27 | 2020-04-03 | 深圳钜运生物科技有限公司 | Method for evaluating antioxidant capacity of fruits |
| CN114740100A (en) * | 2022-03-10 | 2022-07-12 | 西北农林科技大学 | LC-MS/MS detection method of gallotannin in plant |
Citations (3)
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|---|---|---|---|---|
| GB818034A (en) * | 1955-10-12 | 1959-08-12 | Svit Np | Process for the production of tanning extracts |
| JP2006045121A (en) * | 2004-08-04 | 2006-02-16 | Maruzen Pharmaceut Co Ltd | Antibacterial composition |
| CN102250159A (en) * | 2011-04-28 | 2011-11-23 | 贵阳单宁科技有限公司 | Method for extracting and preparing high-purity tannic acid from plant raw material containing tannin |
-
2013
- 2013-04-23 CN CN2013101424177A patent/CN103235067A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB818034A (en) * | 1955-10-12 | 1959-08-12 | Svit Np | Process for the production of tanning extracts |
| JP2006045121A (en) * | 2004-08-04 | 2006-02-16 | Maruzen Pharmaceut Co Ltd | Antibacterial composition |
| CN102250159A (en) * | 2011-04-28 | 2011-11-23 | 贵阳单宁科技有限公司 | Method for extracting and preparing high-purity tannic acid from plant raw material containing tannin |
Non-Patent Citations (3)
| Title |
|---|
| CHRISTINA ENGELS ET AL.: "Antimicrobial Activity of Gallotannins Isolated from Mango (Mangifera indica L.) Kernels", 《J. AGRIC. FOOD CHEM.》, vol. 57, no. 17, 5 August 2009 (2009-08-05), pages 7712 - 7718 * |
| CHRISTINA ENGELS ET AL.: "Fractionation of Gallotannins from Mango (Mangifera indica L.) Kernels by High-Speed Counter-Current Chromatography and Determination of Their Antibacterial Activity", 《J. AGRIC. FOOD CHEM.》, vol. 58, no. 2, 18 December 2009 (2009-12-18), pages 775 - 780 * |
| GIULIANA D. NORATTO ET AL.: "Anticarcinogenic Effects of Polyphenolics from Mango (Mangifera indica) Varieties", 《J. AGRIC. FOOD CHEM.》, vol. 58, no. 7, 5 March 2010 (2010-03-05), pages 4104 - 4112 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105920114A (en) * | 2016-04-18 | 2016-09-07 | 浙江大学 | Black tartary buckwheat rice extract with blood sugar decreasing function and preparation method thereof |
| CN110954379A (en) * | 2018-09-27 | 2020-04-03 | 深圳钜运生物科技有限公司 | Method for evaluating antioxidant capacity of fruits |
| CN114740100A (en) * | 2022-03-10 | 2022-07-12 | 西北农林科技大学 | LC-MS/MS detection method of gallotannin in plant |
| CN114740100B (en) * | 2022-03-10 | 2024-03-01 | 西北农林科技大学 | LC-MS/MS detection method for gallotannins in plants |
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Application publication date: 20130807 |