CN1230546A - Extraction process of pheophorbide A from algae - Google Patents
Extraction process of pheophorbide A from algae Download PDFInfo
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- CN1230546A CN1230546A CN 98107247 CN98107247A CN1230546A CN 1230546 A CN1230546 A CN 1230546A CN 98107247 CN98107247 CN 98107247 CN 98107247 A CN98107247 A CN 98107247A CN 1230546 A CN1230546 A CN 1230546A
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Abstract
The present invention relates to the technology field of extracting chlorophyll. High purity pheohorbide A is extracted by using red alga, blue green alga and yellow green alga as raw material and through the processes of edible color extraction with low density solution of acid, alkali, salt and alcohol; extraction to refine chla, liquid phase process to prepare pheophorbide A and eliminating acid with adsorbent. Owing to that no chlorophyll B and C is contained in the raw material, the present invention can obtain high purity pheophorbide A and has economic utility greatly higher than that of conventional process.
Description
The invention belongs to and from algae, extract the chlorophyll technical field.
Pheophorbide A (Pheophorbide a, PhA)
Be the new laser chemistry method of a kind of usefulness treatment malignant tumour efficiency light sensitization medicament, the PhA vein is injected in patient's body, main retention can the kill tumor cell through the laser radiation tumor locus at tumor tissues after 24 hours, and does not damage normal cell.
At present the preparation of PhA is to adopt chromatographic technique to extract Chlorophyll A (Chla) from high green plants and pteridophyte in the world, and the phytol tail of sloughing Chla with acid or enzyme makes PhA then.All contain Chlorophyll B (Chlb) in these plants.The chemical structure of Chlb and Chla only differ from a group, and obtain highly purified Chla needs a large amount of reagent to carry out chromatography.Time-consuming, the waste fund, benefit is low.
The objective of the invention is with the algae that does not contain Chlb is raw material, earlier pigment is removed, and extraction obtains Chla and prepares PhA by liquid phase method more then, obtains water miscible PhA after the depickling.
The present invention extracts PhA from algae, substantially through three key steps:
1, extract pigment in the algae, salt, alkali, acid, the alcohol dissolving of the defibrination of algae with lower concentration, in high salt concentration solution, be settled out pigment then.
2, the Chlorophyll A of extraction ester solution, carotenoid, xenthophylls etc. adopt alcohol, ketone, ether, alkane, the extraction of benzene equal solvent, Chla can be separated through absorption, chromatography, extraction again.
3, the preparation of pheophorbide A
The diluted acid concentrated acid
(Pheophytinea) (Pheophorbide a) for Chlorophyll A---phoeophytin A---pheophorbide A (Chlorophlla)
Being described in detail as follows of extracting method of the present invention:
Choosing of raw material.With red algae, blue-green algae, yellowish green algaes such as laver, gelidium, Ceramium kondoi, carrageen is raw material.The new material of gathering will be extractd the algae root, uses flushing with clean water behind the dirts such as flush away silt, the empty moisture that removes.In, big shape frond will cut short and grind; Unicellular algae is used flushing with clean water after selecting the sieve of suitable mesh for use.Dry product is used flushing with clean water after wanting water to send out.
The extraction of food dye.The ground starting material are dipped into extraction agent concentration 12-24 hour after-filtration in the extracting solution of 2-60%.Collect filtrate and filter residue respectively, extraction agent uses acid, alkali, salt, alcohol such as methyl alcohol, ethanol, hydrochloric acid, sulfuric acid, potassium hydroxide, sodium hydroxide, sodium-chlor, sal epsom, ammonium sulfate.With salt, have precipitation to separate out in the filtrate, filter and collect throw out, food dye.Filter residue is dipped into 12-24 hour after-filtration in the extracting solution that extraction agent concentration is 50-99% again, collects filtrate.Add deionized water in the filtrate extraction agent concentration is reduced to 40-60%, add sorbent material again and stir after-filtration, collect filtrate and sorbent material respectively, evaporate to dryness filtrate can get food dye.Sorbent material adopts fossil meal, diatomite, absorbent cotton, Mierocrystalline cellulose, organic resin.
The preparation of PhA:, collect elutriant (Chla) with 60-95% extracting solution drip washing sorbent material.Splash into the hydrochloric acid of several 1 volumetric molar concentrations in the elutriant, solution becomes is brown, and Chla distills after being reacted into phoeophytin A, till reducing extraction agent concentration and becoming turbid to the solution.Solution is placed separating funnel, add extraction agent and shake up static 1 hour of back.Extraction agent can be used alkane, aromatic hydrocarbons and derivatives thereof such as ether, sherwood oil, octane, trichloromethane, benzene, dimethylbenzene.Phoeophytin A abandons lower aqueous solution after coming together in the extraction agent, adds and leaves standstill after a little deionized water shakes up.In phoeophytin A extraction liquid, splash into sulfuric acid, make Chla take off chlorophyll alcohol tail and generate PhA.PhA is a water-soluble cpds, separates to enter among the lower aqueous solution from extraction agent.Isolate the PhA aqueous solution and add deionized water pH value is dropped to below 3, add sorbent material again and stir after-filtration, use deionized water drip washing again, the acid of wash-out surplus to PH be neutrality for extremely.Sorbent material is immersed wash-out goes out PhA in the extracting solution of 70-99%.Distillate the extraction agent in the PhA solution, obtain the PhA aqueous solution, carry out to carry out clinical use as injection after concentration and ionogen are regulated.Be convenient to preserve with getting black solid PhA behind the PhA aqueous solution evaporate to dryness.PhA and solution thereof all need keep in Dark Place.More than all recyclable extraction reagent in each still-process, reduce cost, avoid environmental pollution.
The drawing of accompanying drawing of the present invention is described as follows:
Fig. 1 is molecular structure and the electronic light spectrogram of the PhA that obtains of present method, also is Figure of abstract.
Solid line is an excitation spectrum among the figure, and dotted line is a fluorescence spectrum.
As seen from the figure, the PhA with method of the present invention obtains has two characteristic peaks at 418nm and 669nm, this Be consistent with standard sample, illustrate that the PhA that this method obtains is believable.
Do not contain chlorophyll B and C owing in the raw material among the present invention, thereby can obtain highly purified PhA. And we Method is easy, saves reagent, and benefit is more than ten times of conventional method.
Give further instruction below by example to the present invention.
Embodiment 1:
Taking by weighing 1 kilogram of laver plucking wash clean grinds the back to add 2 liters of concentration is 10% ethanolic soln.Soak and sieve with 100 mesh sieve after 24 hours, collect filtrate and filter residue.Measure 1 liter of filtrate and add 300 gram NaCl, stirring to leave standstill after 1 hour has red material to separate out, and its major ingredient is a phycoerythrin.Phycoerythrin is collected in filtration, and preserve dry back.Phycoerythrin can use as red food dye.
Add 1 liter of concentration in the filter residue and be 95% ethanolic soln, soak 12 hours after-filtration, collect filtrate.In filtrate, add 1 liter of deionized water and 200 gram diatomite, stir and leave standstill 1 hour after-filtration, collect sorbent material and filtrate.Evaporate to dryness filtrate gets yellow solid, can be as yellow food dye.
Toward the ethanolic soln of 1 liter 95% of diatomite adsorbant adding, the after-filtration that stirs is collected filtrate.Add several 1 mole of HCl in filtrate, solution is become brown by blue-greenish colour.Retort solution distillates till ethanol just become turbid to solution.Again solution is placed separating funnel, add octane, shake up to leave standstill and abandon lower floor's solution after 1 hour by 1: 1 (V/V).Add deionized water by 1: 0.1 (V/V) and shake up and leave standstill layering after 30 minutes, in the octane of upper strata, splash into the vitriol oil, have dark-brown solution to enter in the lower aqueous solution till no longer include brown solution and descend.Collect lower floor's brown solution, adding deionized water inward is 6 to end to PH.Take by weighing absorbent cotton 50 grams and soak, filter as sorbent material with it.Adsorbed the absorbent cotton of PhA with 95% ethanol drip washing, wash-out goes out PhA.Distillation PhA elutriant heats up in a steamer ethanol, gets the PhA aqueous solution, carries out just having made PhA injection injection after concentration and the ionogen adjustment.The evaporate to dryness elutriant can get black solid PhA powder and be convenient to preserve.
Embodiment 2:
Take by weighing 1 kilogram of the carrageen of plucking wash clean and grind the back to add 2 liters of concentration be 5% common salt aqueous solution, soak and sieve with 100 mesh sieve after 12 hours, collect filtrate and filter residue.Measure 1 liter of filtrate and add 300 gram NaCl, fully stirring to leave standstill after 1 hour has red material to separate out.Red material and drying are collected in filtration.
Add 1 liter of concentration in the filter residue and be 90% acetone soln, soak 12 hours after-filtration, collect filtrate.Add 1 liter of deionized water and 400 in the filtrate and digest stone flour, the static 1 hour after-filtration that stirs is collected filtrate and fossil meal.Evaporate to dryness filtrate gets the yellow solid powder.
In the fossil meal sorbent material, add 1 liter of dense acetone soln of 90% that becomes.Stir and leave standstill 1 hour after-filtration, collect filtrate.Addend drips 1 mole of HCl in filtrate, and solution becomes is brown, distill out that acetone treats that solution just become turbid till.With solution place separating funnel by 1: 1 (V/V) add benzene shake up leave standstill 1 hour after, discard lower floor's solution.Adding deionized water by 1: 0.1 (V/V) shakes up and leaves standstill layering after 1 hour.In the benzene of upper strata, splash into the vitriol oil, have the brownish black material to sink to enter in the lower aqueous solution till no longer produce.Collecting lower floor's liquid and adding deionized water to pH value is 6 to end, and adds and leaves standstill 1 hour after-filtration after 200 gram diatomite stir.With 90% acetone drip washing diatomite adsorbant, wash-out goes out PhA, and distills out acetone and get the PhA aqueous solution, carries out just having made the PhA injection after concentration and the ionogen adjustment.The evaporate to dryness elutriant gets black solid PhA and is convenient to preserve.
Embodiment 3:
The algae defibrination soaks with the KOH of 20% concentration, and adopting dimethylbenzene is organic extraction agent, and vitriol oil concentration adopts 80%, and other step is with embodiment 1.
Embodiment 4:
The algae defibrination soaks with the NaOH of 2% concentration, and organic extraction agent extracts the PhA sherwood oil with the acetone of 60% concentration, and the vitriol oil is 70% concentration, and other is with embodiment 1.
Embodiment 5:
The algae defibrination is with (the NH of 30% concentration
3)
2SO
4Soak, organic extraction agent is with the methyl alcohol of 80% concentration, and the extraction agent ether changes the hydrochloric acid that the vitriol oil is 99% concentration, and other is with embodiment 1.
Embodiment 6:
The algae defibrination soaks with the acetone of 20% concentration, and organic extraction agent is 90% with sherwood oil, sulfuric acid concentration, and other is with embodiment 1.
Claims (2)
1, a kind of method for preparing pheophorbide A (PhA) of from algae, extracting, adopting red algae, blue-green algae, yellowish green algae is raw material, it is characterized in that:
(1), acid, alkali, salt, alcohol or the ketone solution with lower concentration carries out the extraction of the food dye of algae defibrination;
(2), adopt alcohol, ketone, ether or alkane, aromatic hydrocarbons and derivative extracting process to extract refining chlorophyll A then;
(3), adopt again diluted acid liquid phase method de-magging, prepare PhA with the concentrated acid esterification again;
(4), employing diatomite, fossil meal, absorbent cotton, organic resin sorbent material carry out the depickling of PhA and make with extra care.
2,, it is characterized in that adopting following step to carry out according to the method for claim 1:
(1), to choose laver, gelidium, Ceramium kondoi, carrageen be raw material, extracts the algae root, dirts such as flush away silt, clear water is cleaned, the empty moisture that removes is cut and is ground or be screened into defibrination;
(2), the extraction of food dye: the raw material soaking that (1) is got ready is 12-24 hour after-filtration in the extracting solution of 2-60% in concentration, collect filtrate and filter residue respectively, extraction agent can use methyl alcohol, ethanol, hydrochloric acid, sulfuric acid, potassium hydroxide, sodium hydroxide, sodium-chlor, sal epsom, ammonium sulfate etc.;
(3), add sodium-chlor in the filtrate that obtains in (2), there is precipitation to separate out, filters and collect throw out, obtain food dye;
(4), the filter residue that (2) are obtained is dipped in organic extracting solution that concentration is 50-99% 12-24 hour again, filter then, collect filtrate, add deionized water in the filtrate extraction agent concentration is reduced to 40-60%, add sorbent material again and stir after-filtration, collect filtrate and sorbent material respectively, evaporate to dryness filtrate can get food dye, extraction agent can be alcohols acetone, ethers preferably ethanol, acetone, sherwood oil, and sorbent material can adopt fossil meal, diatomite, absorbent cotton, Mierocrystalline cellulose, organic resin etc.;
(5), the preparation of PhA: soak the sorbent material that obtains in (4) after-filtration that stirs with the ethanol extract of 60-95% concentration, collect filtrate (Chla), splash into the hydrochloric acid of several 1 volumetric molar concentrations in the filtrate, solution becomes is brown, Chla is reacted into phoeophytin A, distill then, to extract concentration be reduced to become turbid in the solution till; This solution is placed separating funnel, after the adding extraction agent shakes up, static 1 hour, abandon lower aqueous solution, add again and leave standstill after deionized water shakes up, splashing into sulfuric acid in extraction liquid has dark-brown solution to enter in the lower aqueous solution till no longer include brown solution and descend, and makes Chla take off the phytol tail and generates PhA, and PhA isolates from extraction agent and enters in the lower aqueous solution; Extraction agent can be used ether, sherwood oil, octane, trichloromethane, benzene, dimethylbenzene etc.;
(6), collect the brown PhA aqueous solution of lower floor that (5) obtain, add deionized water PH reduced to below 3, add sorbent material again and fully stir after-filtration, use deionized water drip washing again, be neutrality to PH till;
(7), sorbent material is immersed wash-out goes out PhA in the extracting solution of 70-99% concentration; Distill out the extraction agent in the PhA solution then, obtain the PhA aqueous solution;
(8), with the PhA aqueous solution evaporate to dryness that (7) obtain, obtain the PhA of black solid;
(9), recyclable extraction agent in above each still-process.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98107247 CN1230546A (en) | 1998-04-01 | 1998-04-01 | Extraction process of pheophorbide A from algae |
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| CN 98107247 CN1230546A (en) | 1998-04-01 | 1998-04-01 | Extraction process of pheophorbide A from algae |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002040488A1 (en) * | 2000-11-20 | 2002-05-23 | Korea Kumho Petrochemical Co., Ltd. | Novel photosensitizers of 9-hydroxypheophorbide-a derivatives used for photocynamic therapy |
| CN102206221A (en) * | 2011-04-14 | 2011-10-05 | 天津科技大学 | Method for extracting chlorophyll from dunaliella by using inorganic alkali |
| CN102807471A (en) * | 2011-05-30 | 2012-12-05 | 浙江科技学院 | Method for separation preparation of high purity natural phytol |
| CN103833763A (en) * | 2014-03-21 | 2014-06-04 | 中国海洋大学 | Method of quickly preparing stable enteromorpha chlorophyll |
| CN112521393A (en) * | 2019-09-18 | 2021-03-19 | 康俄(上海)医疗科技有限公司 | Preparation method of methyl pheophorbide a |
-
1998
- 1998-04-01 CN CN 98107247 patent/CN1230546A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002040488A1 (en) * | 2000-11-20 | 2002-05-23 | Korea Kumho Petrochemical Co., Ltd. | Novel photosensitizers of 9-hydroxypheophorbide-a derivatives used for photocynamic therapy |
| CN102206221A (en) * | 2011-04-14 | 2011-10-05 | 天津科技大学 | Method for extracting chlorophyll from dunaliella by using inorganic alkali |
| CN102807471A (en) * | 2011-05-30 | 2012-12-05 | 浙江科技学院 | Method for separation preparation of high purity natural phytol |
| CN103833763A (en) * | 2014-03-21 | 2014-06-04 | 中国海洋大学 | Method of quickly preparing stable enteromorpha chlorophyll |
| CN103833763B (en) * | 2014-03-21 | 2015-11-18 | 中国海洋大学 | The chlorophyllous method of Enteromorpha is stablized in a kind of preparation fast |
| CN112521393A (en) * | 2019-09-18 | 2021-03-19 | 康俄(上海)医疗科技有限公司 | Preparation method of methyl pheophorbide a |
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