CN110066265A - Method for extracting coenzyme Q10 and scopoletin from tobacco leaves - Google Patents

Abstract

The invention relates to a method for extracting coenzyme Q10 and scopoletin from tobacco leaves, which comprises the following steps: refluxing and extracting tobacco leaf raw materials by petroleum ether, filtering to obtain an extract I, and crystallizing the extract I by acetone to obtain a crystal I; fermenting the filter residue with bread yeast powder, autolyzing, reflux extracting with petroleum ether, filtering to obtain extract II, and crystallizing with acetone to obtain crystal II; recrystallizing the crystal I and the crystal II to obtain coenzyme Q10; extracting the residue II with ethanol, filtering, concentrating to obtain concentrated solution I, extracting with ethyl acetate, and performing silica gel column chromatography to obtain scopoletin. Coenzyme Q10 and scopoletin are synchronously extracted, so that the utilization rate of tobacco leaves is effectively improved, and the production cost is reduced. The yeast fermentation is utilized to convert a large amount of sugar substances in the tobacco leaves, so that the content of coenzyme Q10 is effectively improved, and the influence of the sugar substances on the extraction rate is avoided.

Description

A method of extracting Co-Q10 and Scopoletin from tobacco leaf

Technical field:

The invention belongs to natural products processing technique fields, relate to one kind and extract Co-Q10 and Anisodus luridus from tobacco leaf The method of element.

Background technique:

Nicotiana Solanaceae is annual or limited herbaceos perennial, base portion slightly lignifying.Inflorescence basidixed, it is coniform, it is more Flower；Capsule ovate or square round shape, length are approximately equal to persistent calyx.The average content of chemical component is respectively as follows: total reducing sugar in flue-cured tobacco 23.08%, reduced sugar 19.63%, total nitrogen 2%, nicotine 1.99%, protein 10.32%, schmuck value 2.45, total reducing sugar/nicotine Than 14.21, total nitrogen/nicotine is than 1.15.Also containing functional components such as Co-Q10, Salanesol, chlorogenic acids.Tobacco is warm-natured sweet in flavor, It is toxic, there is detumescence, removing toxic substances, desinsection and other effects, be mainly used for furuncle swelling toxin, favus of the scalp, tinea alba, the favus of the scalp, the diseases such as venomous snake bite, also The diseases such as a subcutaneous ulcer, carbuncle on the back, wind phlegm, crane knee (including bone tuberculosis, chronic purulent gonitis etc.) can be treated.

Co-Q10 also known as Ubidecarenone are a kind of fat-soluble quinones, are at room temperature orange-yellow crystal, and fusing point is 48 DEG C, Its odorless, tasteless, structure is similar to vitamin K, because of the side chain on its parent nucleus six --- and the degree of polymerization of polyisopreneyl is 10 and gain the name, be a kind of quinone cyclics.Be widely present in the tissues of animals and plants, microorganism it is intracellular, as end electricity The neccessary composition of sub- transmission system, plays an important role, first is that there is apparent lipoid peroxidization resistant；Second is that nutrients Matter plays an important role during being converted into energy in mitochondria.In addition, its pharmacological action is to congestive heart failure It is effective with muscular dystrophy caused by coronary heart disease, malnutrition etc., and be also costly substance as drug.Tradition both at home and abroad The method of production Co-Q10 be biological extraction method, extracted from animal viscera such as Pigs Hearts, cattle heart, specifically include as saponification method, Solvent extraction and adsorption chromatography etc..

The Chinese patent of Publication No. CN105566085A describes a kind of work that Co-Q10 is extracted from tobacco leaf in detail Skill includes the following steps: 1) pre-treatment: waste/hypo-tobacco leaf being passed through the tobacco leaf extract that extraction obtains, the pure of equivalent weight is added Water is put into pressure pan, is forced into 4kg/cm², keep its pressure uniform, and keep 5-10min, then restore normal in 1-3s Pressure, obtained material obtain the medicinal extract containing Salanesol using saponification process, crystallization, filtering；2) molecular distillation: by step 1) medicinal extract containing Salanesol obtained, is cleaned by molecular distillation, obtains light phase Salanesol and heavy phase Salanesol；3) column chromatography Separation: heavy phase Salanesol acetone solution that step 2) is obtained, filtering collect eluent by pillar layer separation, concentration, Salanesol after obtaining molecular distillation purification；4) methylphenol the preparation of methyl dimethoxy oxygroup benzoquinones: is etherified oxygen by bromo Change obtains methyl dimethoxy oxygroup benzoquinones；5) after the molecular distillation purification obtained light phase Salanesol that step 2) obtains, step 3) Salanesol and step 4) obtain methyl dimethoxy oxygroup benzoquinones mixing, reaction obtain Co-Q10.

A kind of method that the Chinese patent of Publication No. CN101705258A describes fermentation preparation of cozymase Q 10 in detail, The following steps are included: first carry out Agrobacterium tumefaciens high density aerobic fermentation 60~84 hours with 5~15% inoculum concentration, Fermentation medium group is divided into glucose 12~20%, ammonium sulfate 0.5~1.5%, potassium dihydrogen phosphate 0.1~0.5%, magnesium sulfate 0.01~0.05%, corn pulp 0.5~2.5%, diammonium hydrogen phosphate 0.2~1%, pH6~7.5, fermentation temperature is 28~31 DEG C； Then the fermentation liquid of acquisition is separated, obtains separating thallus；After finally the separating thallus is resuspended, is flowed back, extracted, obtained Co-Q10.

The Chinese patent of Publication No. CN101987815A describes a kind of purifying work for preparing high-purity Co-Q10 in detail Skill, comprising the following steps: (1) dissolve Co-Q10 crude extract with organic solvent A, and will with 2~3 times of column volume/hour flow velocitys The solution is pumped into the glass resin column for being filled with absorption resin, is reached after leakage wears a little, stopping sample introduction；(2) it is washed with organic solvent B The resin column in step (1) is washed, after eluting 5 times of column volumes, C2 times of column volume elution resin column of organic solvent is used instead, obtains coenzyme Q10 eluent；(3) the Co-Q10 eluent of step (2) is concentrated, is statically placed at 0 DEG C with organic solvent D dissolution and crystallizes 12h, Recrystallization 2 times；(4) crystal further obtained to step (3) carries out purifying purification, with finished product and chromatographic silica gel mass ratio for 1: 25 loadings, organic solvent E collect Co-Q10 eluent as eluant, eluent.

The Chinese patent of Publication No. CN101967499A describes a kind of production method of reduced coenzyme Q 10 in detail, It is characterized by: using all humans and animals cell line or cell strain as production reduced coenzyme Q 10 female parent, by The cell culture carried out in biological culture agent DMEM-1 culture solution obtains the cell liquid for containing a large amount of reduced coenzyme Q 10s, then Reduced coenzyme is obtained with the method for ethyl alcohol extraction concentration, rear crystallisation by cooling by the method for rotary-classify technology, or first Q10 final products.

The Chinese patent of Publication No. CN104086425A describe in detail it is a kind of extract simultaneously simultaneously separate tobacco chlorogenic acid, The method of Salanesol, nicotine, rutin sophorin, step include: Step 1: extracting simultaneously: tobacco material being crushed, temperature 40~50 DEG C, supersonic frequency 45KHz, under the conditions of solid-liquid ratio 10: 1, extracted with 80% ethyl alcohol it is primary, filter or centrifugation after, residue is distinguished again It is respectively extracted once with 80%, 95% ethyl alcohol；Extracting solution merges three times, obtains Tobacco Chlorogenic Acid, Salanesol, nicotine, rutin sophorin Crude extract, Tobacco Chlorogenic Acid, Salanesol, nicotine, rutin sophorin recovery rate up to 100%；Step 2: separation: separation eggplant Buddhist nun Alcohol；Separating chlorogenic acid, rutin sophorin；Separate nicotine.

The Chinese patent of Publication No. CN102060823A describes a kind of side that Scopoletin is extracted from saussurea involucrata in detail Method comprising the steps of: 1) ultrasonic alkali carries: take lanatechead saussurea herb with flower raw material, be crushed, alkali ultrasonic extraction 1-2 times of pH10-11 is added, filter Obtain extracting solution；2) acid reduction: said extracted is acidified to faintly acid, and precipitating is precipitated, filters out sediment and is washed with a small amount of benzene, takes solid Body；3) polyamide separate: by above-mentioned solid with 80-90% ethyl alcohol disperse, mix sample with polyamide, volatilize upper prop, spend from The ethanol solution stepwise elution of sub- water and different proportion collects height ethanol eluate, is concentrated and dried to obtain crude product；4) it recrystallizes: Add ethyl alcohol to dissolve by heating above-mentioned crude product, stand cooling, must crystallize, repeats 2-3 times, be drying to obtain product.

Show that Co-Q10 at present and Scopoletin isoreactivity physical market demand are very big from existing literature data, cost compared with Height, the problem of being primarily due to the prices of raw materials, but existing yield of tobacco and that most of the products are overstocked is superfluous, are not had and are extracted using tobacco leaf The method of Co-Q10 and Scopoletin.

Summary of the invention:

It is not high it is an object of the invention to be directed to superfluous tobacco leaf utilization rate at present, it proposes a kind of utilize and synchronizes extraction purification From tobacco leaf large-scale production Co-Q10 and Scopoletin, entire simple process convenient operation and control effectively improves the utilization of tobacco leaf Rate also may make two kinds of effective component receipts with higher while reducing Co-Q10 and Scopoletin production cost Rate, and can effectively ensure large-scale production Co-Q10 and Scopoletin.

The present invention provides the method that Co-Q10 and Scopoletin are extracted from tobacco leaf, comprises the following steps that

(1) raw tobacco material is weighed, petroleum ether, refluxing extraction 2h are added after crushing, ceramic membrane filter obtains filtrate I and filter residue I, Filtrate I evaporated under reduced pressure recycling petroleum ether obtains medicinal extract I, wherein the amount of petroleum ether, is 3~6: 1 by volume L/ tobacco leaf weight kg ratio；

(2) it takes medicinal extract I to melt in 50 DEG C of heating water baths, adds the acetone of 50 DEG C of preheatings, shaking to medicinal extract is dissolved, while hot It filters, is down to 0 DEG C with the rate of 1 DEG C/2min and keeps the temperature 4h, ceramic membrane filter obtains filtrate II and crystal I, wherein acetone Amount is 8~15: 1 by volume L/ medicinal extract I weight kg ratio；

(3) it takes filtrate II evaporated under reduced pressure to recycle acetone, obtains dried object, be sprayed directly on filter residue I after adding water stirring, then Saccharomyces cerevisiae powder is added and carries out 25~30 DEG C of fermentations 3~6 days, obtains fermentation material, wherein the weight kg/ tobacco leaf weight of Saccharomyces cerevisiae powder Kg ratio is 5~15:100；

(4) water for taking fermentation material sprinkling pH10~12, makes water content reach 25~35%, 50~60 DEG C of 4~10h of heat preservation, Petroleum ether refluxing extraction 2h is added, ceramic membrane filter obtains filtrate II I and filter residue II, and filtrate II I evaporated under reduced pressure recycles petroleum ether Medicinal extract II is obtained, wherein the amount of petroleum ether, is 3~6: 1 by volume L/ tobacco leaf weight kg ratio；

(5) it takes medicinal extract II to melt in 50 DEG C of heating water baths, adds the acetone of 50 DEG C of preheatings, shaking to medicinal extract is dissolved, taken advantage of Heat filters, and is down to 0 DEG C with the rate of 1 DEG C/2min and keeps the temperature 4h, ceramic membrane filter obtains filtrate IV and crystal II, wherein acetone Amount, by volume L/ medicinal extract II weight kg ratio be 8~15: 1；

(6) it takes crystal I and crystal II to melt in 50 DEG C of heating water baths, adds the acetone of 50 DEG C of preheatings, shaking is extremely Medicinal extract dissolution, filters while hot, is down to 0 DEG C with the rate of 1 DEG C/2min and keeps the temperature 4h, ceramic membrane filter obtains filtrate V and crystal III (Co-Q10), wherein the amount of acetone, is 5~8: 1 by volume L/ medicinal extract I weight kg ratio；

(7) take filter residue II that 60~80% ethanol solutions, refluxing extraction 2h are added, ceramic membrane filter obtains filtrate VI, filtrate VI It is concentrated under reduced pressure into the 1/10~1/20 of original volume, obtains concentrate I, wherein the amount of ethanol solution, by volume L/ tobacco leaf weight kg Than being 3~7: 1；

(8) filtrate IV and filtrate V is taken to merge, evaporated under reduced pressure recycles acetone, concentrate I is added, after being stirred, with two It is extracted again to the ethyl acetate of four times of volumes, is concentrated under reduced pressure into the 1/20~1/40 of original volume, obtain ethyl acetate extraction Liquid；

(9) acetic acid ethyl acetate extract is taken to be chromatographed with the silicagel column of 80~100 mesh, with three times to five times of column volume acetic acid Ethyl ester is eluted, and eluent is obtained, and is directly concentrated to dryness to obtain Scopoletin.

The aperture of above-mentioned ceramic membrane is between 0.2~0.3 micron.

The petroleum ether that the above-mentioned preferred boiling range of petroleum ether is 60~90 DEG C.

The temperature of above-mentioned reduced pressure is generally at 40~60 DEG C.

Above-mentioned Saccharomyces cerevisiae powder can directly from market, purchase be obtained, and can also voluntarily be cultivated.

Above-mentioned 60~80% ethanol solution is ethanol water, and 80% ethanol solution preparation method is that 80mL dehydrated alcohol adds Enter the mixing of 20mL water.

In a specific embodiment, wherein step (3) is described is added a certain amount of water, by volume L/ dried object weight Kg ratio is 2~6: 1.

Technical effect:

1, Co-Q10 and Scopoletin are extracted using synchronous, effectively increases the utilization rate of tobacco leaf, reduces and be produced into This.

2, using a large amount of glucides of yeast microbe conversion tobacco leaf, Co-Q10 content is effectively increased, carbohydrate is in turn avoided Influence of the substance to recovery rate.

3, when fermenting tobacco leaf, filtrate is crystallized by addition Co-Q10, can more be promoted supplemented by the substances conversion such as contained Salanesol Enzyme Q10 improves Co-Q10 yield, and prevents the loss of Scopoletin.

Specific embodiment:

Essentiality content of the invention is further illustrated with embodiment of the invention below, but this is not limited with this Invention.

Embodiment 1

Weigh 100kg raw tobacco material, after crushing be added 500L petroleum ether, refluxing extraction 2h, ceramic membrane filter obtain filtrate I and Filter residue I, filtrate I evaporated under reduced pressure recycling petroleum ether obtain 4.46kg medicinal extract I.4.46kg medicinal extract I is taken to melt in 50 DEG C of heating water baths, then The acetone 40L of 50 DEG C of preheatings is added, shaking to medicinal extract dissolves, filters while hot, be down to 0 DEG C with the rate of 1 DEG C/2min and keep the temperature 4h, ceramic membrane filter obtain filtrate II and 2.78kg crystal I.

It takes filtrate II evaporated under reduced pressure to recycle acetone, obtains dried object, add after water stirs and be sprayed directly on filter residue I, then plus Enter 8kg Saccharomyces cerevisiae powder and carry out 26 DEG C of fermentations 5 days, obtains fermentation material；The water for taking fermentation material sprinkling pH11, reaches water content 35%, 55 DEG C of heat preservation 6h add 400L petroleum ether refluxing extraction 2h, and ceramic membrane filter obtains filtrate II I and filter residue II, filtrate III evaporated under reduced pressure recycling petroleum ether obtains 3.68kg medicinal extract II；It takes 3.68kg medicinal extract II to melt in 50 DEG C of heating water baths, adds 50 DEG C preheating acetone 35L, shaking to medicinal extract dissolve, filter while hot, be down to 0 DEG C with the rate of 1 DEG C/2min and keep the temperature 4h, ceramics Film filters to get filtrate IV and 1.56kg crystal II.

It takes 2.78kg crystal I and 1.56kg crystal II to melt in 50 DEG C of heating water baths, adds the third of 50 DEG C of preheatings Ketone 25L, shaking to medicinal extract dissolve, filter while hot, be down to 0 DEG C with the rate of 1 DEG C/2min and keep the temperature 4h, ceramic membrane filter must be filtered Liquid V and 1.84kg crystal III (Co-Q10).

Take filter residue II that 80% ethanol solution 600L, refluxing extraction 2h is added, ceramic membrane filter obtains filtrate VI, filtrate VI decompression It is concentrated to give 40L concentrate I；Filtrate IV and filtrate V is taken to merge, evaporated under reduced pressure recycles acetone, adds concentrate I, is stirred Afterwards, it is extracted with 120L ethyl acetate, 3L acetic acid ethyl acetate extract is concentrated under reduced pressure to obtain；Take acetic acid ethyl ester extract with 80~ The silicagel column of 100 mesh is chromatographed, and is eluted with five times of column volume ethyl acetate, obtains eluent, be directly concentrated to dryness Obtain 0.89kg Scopoletin.It is detected through HPLC, Co-Q10 content is 98.77% in gained sample, and Scopoletin content is 97.56%.

Embodiment 2

Weigh 200kg raw tobacco material, after crushing be added 900L petroleum ether, refluxing extraction 2h, ceramic membrane filter obtain filtrate I and Filter residue I, filtrate I evaporated under reduced pressure recycling petroleum ether obtain 9.23kg medicinal extract I, melt in 50 DEG C of heating water baths, add 50 DEG C of preheatings Acetone 100L, shaking to medicinal extract dissolve, filter while hot, be down to 0 DEG C with the rate of 1 DEG C/2min and keep the temperature 4h, ceramic membrane filter Obtain filtrate II and 6.13kg crystal I.

It takes filtrate II evaporated under reduced pressure to recycle acetone, obtains dried object, add after water stirs and be sprayed directly on filter residue I, then plus Enter 20kg Saccharomyces cerevisiae powder and carry out 26 DEG C to ferment 4 days, obtains fermentation material, then spray the water of pH10, water content is made to reach 25%, 60 DEG C 8h is kept the temperature, adds 900L petroleum ether refluxing extraction 2h, ceramic membrane filter obtains filtrate II I and filter residue II, filtrate II I evaporated under reduced pressure Recycling petroleum ether obtains 7.54kg medicinal extract II, melts in 50 DEG C of heating water baths, adds the acetone 80L of 50 DEG C of preheatings, and shaking is extremely soaked Cream dissolution, filters while hot, is down to 0 DEG C with the rate of 1 DEG C/2min and keeps the temperature 4h, and ceramic membrane filter obtains filtrate IV and 3.41kg knot Brilliant object II.

It takes 6.13kg crystal I and 3.41kg crystal II to melt in 50 DEG C of heating water baths, adds the third of 50 DEG C of preheatings Ketone 55L, shaking to medicinal extract dissolve, filter while hot, be down to 0 DEG C with the rate of 1 DEG C/2min and keep the temperature 4h, ceramic membrane filter must be filtered Liquid V and 3.83kg crystal III (Co-Q10).

Take filter residue II that 75% ethanol solution 1000L, refluxing extraction 2h is added, ceramic membrane filter obtains filtrate VI, and filtrate VI subtracts Pressure is concentrated to give 70L concentrate I；Filtrate IV and filtrate V is taken to merge, evaporated under reduced pressure recycles acetone, adds concentrate I, and stirring is mixed It after conjunction, is extracted with 200L ethyl acetate, 8L acetic acid ethyl acetate extract is concentrated under reduced pressure to obtain；Take acetic acid ethyl ester extract with 80~ The silicagel column of 100 mesh is chromatographed, and is eluted with four times of column volume ethyl acetate, obtains eluent, be directly concentrated to dryness Obtain 2.16kg Scopoletin.It is detected through HPLC, Co-Q10 content is 98.84% in gained sample, and Scopoletin content is 97.47%.

Claims (4)

1. extracting the method for Co-Q10 and Scopoletin from tobacco leaf, which is characterized in that comprise the following steps that

(1) raw tobacco material is weighed, petroleum ether, refluxing extraction 2h are added after crushing, ceramic membrane filter obtains filtrate I and filter residue I, filtrate I evaporated under reduced pressure recycling petroleum ether obtains medicinal extract I, wherein the amount of petroleum ether, is 3~6: 1 by volume L/ tobacco leaf weight kg ratio；

(2) it takes medicinal extract I to melt in 50 DEG C of heating water baths, adds the acetone of 50 DEG C of preheatings, shaking to medicinal extract is dissolved, taken out while hot Filter, is down to 0 DEG C with the rate of 1 DEG C/2min and keeps the temperature 4h, and ceramic membrane filter obtains filtrate II and crystal I, wherein the amount of acetone, It is 8~15: 1 by volume L/ medicinal extract I weight kg ratio；

(3) it takes filtrate II evaporated under reduced pressure to recycle acetone, obtains dried object, be sprayed directly on filter residue I after adding water stirring, add Saccharomyces cerevisiae powder carries out 25~30 DEG C and ferments 3~6 days, obtains fermentation material, wherein the weight kg/ tobacco leaf weight kg ratio of Saccharomyces cerevisiae powder It is 5~15: 100；

(4) water for taking fermentation material sprinkling pH10~12, makes water content reach 25~35%, 50~60 DEG C of 4~10h of heat preservation, then plus Enter petroleum ether refluxing extraction 2h, ceramic membrane filter obtains filtrate II I and filter residue II, and filtrate II I evaporated under reduced pressure recycling petroleum ether must soak Cream II, wherein the amount of petroleum ether, is 3~6: 1 by volume L/ tobacco leaf weight kg ratio；

(5) it takes medicinal extract II to melt in 50 DEG C of heating water baths, adds the acetone of 50 DEG C of preheatings, shaking to medicinal extract is dissolved, taken out while hot Filter, is down to 0 DEG C with the rate of 1 DEG C/2min and keeps the temperature 4h, and ceramic membrane filter obtains filtrate IV and crystal II, wherein the amount of acetone, It is 8~15: 1 by volume L/ medicinal extract II weight kg ratio；

(6) it takes crystal I and crystal II to melt in 50 DEG C of heating water baths, adds the acetone of 50 DEG C of preheatings, shaking to medicinal extract Dissolution, filters while hot, is down to 0 DEG C with the rate of 1 DEG C/2min and keeps the temperature 4h, and ceramic membrane filter obtains filtrate V and Co-Q10, wherein The amount of acetone is 5~8: 1 by volume L/ medicinal extract I weight kg ratio；

(7) take filter residue II that 60~80% ethanol solutions, refluxing extraction 2h are added, ceramic membrane filter obtains filtrate VI, filtrate VI decompression It is concentrated into the 1/10~1/20 of original volume, obtains concentrate I, wherein the amount of ethanol solution, is 3 by volume L/ tobacco leaf weight kg ratio ~7: 1；

(8) filtrate IV and filtrate V is taken to merge, evaporated under reduced pressure recycles acetone, add concentrate I, after being stirred, twice of use to The ethyl acetate of four times of volumes is extracted, and is concentrated under reduced pressure into the 1/20~1/40 of original volume, is obtained acetic acid ethyl acetate extract；

(9) acetic acid ethyl acetate extract is taken to be chromatographed with the silicagel column of 80~100 mesh, with three times to five times of column volume ethyl acetate It is eluted, obtains eluent, be directly concentrated to dryness to obtain Scopoletin.

2. the method according to claim 1, which is characterized in that the aperture of the ceramic membrane is between 0.2~0.3 micron.

3. the method according to claim 1, which is characterized in that the petroleum ether that the preferred boiling range of petroleum ether is 60~90 DEG C.

4. the method according to claim 1, which is characterized in that wherein step (3) is described is added a certain amount of water, dry by volume L/ Dry object weight kg ratio is 2~6: 1.