CN102775276A - Preparation method of plant polyprenol with bacteriostatic and antioxidant activity and hydrogenated derivative thereof - Google Patents
Preparation method of plant polyprenol with bacteriostatic and antioxidant activity and hydrogenated derivative thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of plant polyprenol with bacteriostatic and antioxidant activity and a hydrogenated derivative thereof, which comprises the following steps of: taking gingko, pine needles and folium mori as raw materials, extracting a polyprenol lipid ointment through a non-polar solvent, including with a filler in urea alcohol solution, crystallizing, preparing a polyprenol type lipid urea inclusion compound and purifying polyprenol lipid through column chromatography; adding into 5-40 percent by weight of caustic soda solution for saponification reaction and preparing more than 95 percent by weight of polyprenol; adding a catalyst in methylene dichloride-methanol solution and ensuring that the mass ratio of the polyprenol to the catalyst is 80-200:1, the reaction pressure is 8-20 MPa, the reaction time is 18-48 hours, and the reaction temperature is 10-50 DEG C; and after reaction, filtering and concentrating under vacuum, wherein a concentrate is a polyprenol hydrogenated derivative. The polyprenol and the hydrogenated derivative thereof prepared by the method play roles in restraining staphylococcus aureus (Sa.), escherichia coli (Ec.), bacillus subtilis and salmonella and clearing superoxide free radicals and hydroxyl free radicals.
Description
Technical field
The present invention relates to a kind of have antibacterial and plant polyprenol and hydroderivating thing preparation method thereof anti-oxidant activity, belong to biomedicine field.
Technical background
Polyprenol all extensively exists in naked son, angiosperm; It also is present in bacterium, fungi and the mammiferous internal organs; The dolichol structural similitude of its structure and biologically active; Therefore be described as best synthetic intermediate, thus from plant extract birch type polyprenol and human body the dolichol structural similitude, so extraction separation plant polyprenol and synthesize dolichol and become a kind of new drug development theory.
Polyprenol is with C
5Isopentene group is the lipoid cpd of structural unit, on structure, can divide: betulol type ω-(trans)
2-(cis)
n-OH, the pure type ω of luxuriant and rich with fragrance card-(trans)
3-(cis)
n-OH and eggplant Buddhist nun alcohol type ω-(trans)
n-OH, Wang Chengzhang etc. isolate 7 kinds of betulol type polyprenols from Ginkgo Leaf, structural unit is 15～22.Like Fig. 1.
The form of polyprenol lipoid many alcohol with acetic ester and free state in plant materials exists, and manyly in animal body exists with SULPHOSUCCINIC ACID ESTER and pure forms.There are some researches show; In birch, be that 30～40 the unsaturated alcohol and the form of acetic ester exist with carbon chain lengths; Polyprenol in the lily magnolia leaf is the unsaturated alcohol of 45～65 carbon; Being that 20 and 45 saturated alcohol exists with carbon chain lengths in the phytol, is that 75～175 unsaturated alcohol exists with carbon chain lengths in the Herba Potentillae Discoloris, and the polyprenol in the yeast is that 30～40 saturated acetic ester exists with carbon chain lengths; Polyprenol carbon chain lengths in the pig liver is 55 saturated alcohol and SULPHOSUCCINIC ACID ESTER, contains carbon chain lengths in the leaf of fungi infestation and be 55 saturated alcohol.Report ginkgo polyprenol, Hyoscyamus niger L polyprenol, pine needle polyprenol, mulberry leaf polyprenol, CedarLeaves polyprenol etc. are arranged at present.
Plant polyprenol and hydroderivating thing thereof are the biosynthetic crucial carriers of human body gp, and tool is with effects such as antitumor, auxiliary chemicotherapy and antiviral and hypoglycemic, mutation, reducing blood-fat and adjusting immunologic functions.Especially the S-dolichol contains unsaturated double-bond, has anti-oxidant, removing lipid radical effect, can be anti-ageing, improve inferior key health crowd's quality of life.Although dolichol and SULPHOSUCCINIC ACID ESTER thereof are the crucial carriers of biological metabolism, because dolichol content in animal body is too low, its extraction, separation and purifying are cumbersome; Yield is extremely low, and is high from the natural dolichol cost of pluck extraction separation, and the supply of naturally occurring dolichol is extremely rare; Only contain the 200mg dolichol like the 5kg pork liver; Up to the present, do not solve a large amount of preparation problems of high purity dolichol both at home and abroad, thereby limited the drug development of dolichol and SULPHOSUCCINIC ACID ESTER thereof.Therefore, how to synthesize dolichol and SULPHOSUCCINIC ACID ESTER thereof, and study their biological activity and metabolic mechanism thereof, become the direction of international research.
The domestic and international at present purification for polyprenol is used for silica gel column chromatography more and separates; For urea entrapping method purifies and separates polyprenol report not both at home and abroad, especially very few as midbody hydrogenation products and oxidation-resistance thereof and antibacterial bioactive research for polyprenol.Dolichol be polyprenol not in the end unit isopentene group be hydrogenated to saturated primary isoamyl alcohol, be a kind of in the polyprenol hydrogenation products.Extraction separation betulol type structure of the present invention (ω-(trans)
2-(cis)
n-OH, n=10～20) polyprenol, through isopentene group unit in the synthetic preparation polyprenol molecular structure by hydrogen saturated be the unitary hydroderivating thing of isoamyl alkyl, synthetic product of the present invention has comprised dolichol.
Summary of the invention
Be to realize above-mentioned purpose, invented a kind of have antibacterial and plant polyprenol and hydroderivating thing preparation method thereof anti-oxidant activity, form by following steps:
The first step, the polyprenol lipoid extracts: will be rich in the fresh plant leaf of polyprenol lipoid, room temperature is dried in the shade; Be crushed to the following powder of 100 orders, add any solvent in anhydrous propanone or the C1～C3 alcohol, leaf and solvent are 1: 20 by mass ratio; Soaking at room temperature 10～72 hours, or 10 ℃～80 ℃ through microwave extraction or ultrasonic extraction 10～60 minutes, repeat to extract twice; United extraction liquid, vacuum reclaims organic solvent, and enriched material is a polyprenol class fat ointment;
In second step, the preparation of urea alcoholic solution: urea is put into anhydrous methanol, and heating in water bath to urea all dissolves under the nitrogen condition filling, and wherein the weightmeasurement ratio of urea and anhydrous methanol is 1: 1～10, and reflux temperature is 40～90 ℃;
The 3rd step; Polyprenol lipoid urea inclusion preparation: in the urea alcoholic solution, add polyprenol class fat ointment and filler, polyprenol class fat ointment: filler: the mass ratio 0.1～1: 1～3: 10～40 of urea alcoholic solution, 40～90 ℃ of stirring and refluxing; After being cooled to room temperature;-40～10 ℃ of crystallizations, suction filtration, crystallisate are polyprenol lipoid urea inclusion.
The 4th step; Polyprenol lipoid purifying: with polyprenol lipoid urea clathration crystallisate dress post, carry out wash-out with non-polar solvent, elution volume is column volume 1-4 times; TLC detects; Merge polyprenol lipoid elution fraction, concentrating back adding mass ratio is 1: 6～30 anhydrous propanones, the stirring at room dissolving; 40～200 order zeyssatite mix by mass ratio 1: 1～10 with quartz sand; Obtain mixed aid; Mixed aid and polyprenol lipoid mass ratio are to mix at 0.5～20: 100, stir 2～30 hours at-30 ℃～0 ℃, and 3000～20000 rev/mins of whizzers filter; Clear liquid reclaims organic solvent, and enriched material is a purifying polyprenol lipoid;
In the 5th step, the polyprenol preparation: the polyprenol lipoid joins in the 5%-40% sodium hydroxide solution, and polyprenol lipoid and sodium hydroxide solution mass ratio are 1: 10～50; 30 ℃～90 ℃ stirring reactions 0.5～3 hour, use normal hexane equal-volume extractive reaction liquid 2-5 time again, merge extraction phase; With equal-volume water backwash 2～5 times, the organic phase concentrating under reduced pressure, enriched material adds 5-20 times of silica gel mixing; With 2-6 times of normal hexane soaking at room temperature 0.5-2 hour, filter, it is dense dried filtrate again; Enriched material is faint yellow polyprenol oily matter, and polyprenol content is greater than 95%.
The 6th step, the preparation of polyprenol hydroderivating thing
With polyprenol be dissolved in methylene chloride-methanol solution (volume ratio is 8～1: 1), polyprenol and methylene chloride-methanol solution quality volume ratio (g: ml) 1: 20～100; Add catalyzer, polyprenol is 80～200: 1 with the catalyst quality ratio, reaction pressure 8～20MPa, and the reaction times is 18～48h, 10 ℃～50 ℃ of temperature of reaction; After reaction finishes, filter, vacuum concentration, enriched material are polyprenol hydroderivating thing.
Polyprenol and hydroderivating thing thereof are inhibited for streptococcus aureus (Sa.), intestinal bacteria (Ec.), bacillus subtilis and Salmonellas among the present invention; The polyprenol antibacterial circle diameter is 6.82-8.55mm; Hydroderivating thing antibacterial circle diameter is 8.02～9.98mm, and the hydrogenated products bacteriostatic activity is higher than polyprenol.Hydrogenated products is to O
2 -The removing result show, the increase of hydrogenated products sample concentration, the clearance rate of superoxide radical rises gradually; In 0.2～0.4mg/mL scope, be mild ascendant trend, be ascendant trend rapidly in 0.4～1.0mg/mL scope, 1.0mg/mL increases concentration later on; The clearance rate ascensional range is less, and trend tends towards stability after 1.4mL, but in general; Increase the concentration of product, can increase the clearance rate of superoxide radical, strengthen its oxidation resistant activity.Hydrogenated products shows the removing result of OH, the increase of hydrogenated products sample concentration, and the rate of removing of hydroxyl radical free radical rises gradually; In 0.4～1.2mg/mL scope, be mild ascendant trend, be ascendant trend rapidly in 1.2～2.0mg/mL scope, 2.0mg/mL increases concentration later on; The clearance rate ascendant trend tends towards stability, but in general, increases the concentration of product; Can increase the clearance rate of hydroxyl radical free radical, strengthen its oxidation resistant activity.The hydrogenated products of the present invention's preparation all has scavenging(action) preferably to removing superoxide radical and hydroxyl radical free radical, and good oxidation-resistance is arranged.
Polyprenol derives from a kind of in Ginkgo Leaf, pine needle, the mulberry leaf among the present invention, and the structure of polyprenol is betulol type ω-(trans)
2-(cis)
n-OH, wherein the scope of n is 10～20.Through catalytic hydrogenation reaction, with isopentene group unit in the polyprenol molecular structure by hydrogen saturated be the isoamyl alkyl unit, be the dolichol structure of primary isoamyl alcohol comprising end unit not.The hydrogenation catalyst of in this patent, selecting is (S)-Ru (OAc)
2(BINAP), Rh/PVP-TiO
2, Ru (BINAP) (OAc)
2In any.Like Fig. 1.
C1 in the first step of the present invention～C3 alcohol be in methyl alcohol, ethanol, n-propyl alcohol, the Virahol any, preferred Virahol.In the preparation of urea alcoholic solution, urea is put into anhydrous methanol, heating in water bath to urea all dissolves under the nitrogen condition filling; Wherein the weightmeasurement ratio of urea and anhydrous methanol is 1: 1～10; Preferred 1: 1～4, reflux temperature is 40～90 ℃, preferred 40～500 ℃; Filler is a kind of in silica gel, gac, polyamide resin, attapulgite, aluminum oxide, the zeyssatite in the 3rd step of the present invention.
The present invention prepares at polyprenol lipoid urea inclusion; With polyprenol class fat ointment: filler: the mass ratio 0.1～1: 1～3: 10～40 of urea alcoholic solution, 40～90 ℃ of stirring and refluxing, preferred 40～60 ℃; Cool off impurity elimination again; Preferably-20～-10 ℃ crystallization, suction filtration, crystallisate are polyprenol lipoid urea inclusion.In polyprenol lipoid purifying,, carry out wash-out with non-polar solvent with polyprenol lipoid urea clathration crystallisate dress post; Preferred normal hexane; Elution volume is column volume 1-4 times, and TLC detects, and merges polyprenol lipoid elution fraction; Concentrating back adding mass ratio is 1: 6～30 anhydrous propanones, the stirring at room dissolving; 40～200 order zeyssatite, preferred 100～120 orders mix by mass ratio 1: 1～10 with quartz sand; Obtain mixed aid; Mixed aid and polyprenol lipoid mass ratio are to mix at 0.5～20: 100, stir 2～30 hours at-30 ℃～0 ℃, and 3000～20000 rev/mins of whizzers filter; Clear liquid reclaims organic solvent, and enriched material is a purifying polyprenol lipoid;
Adopt TLC to detect among the present invention and HPLC detection polyprenol and hydroderivating thing thereof, iodine vapor colour developing when TLC detects.It is chromatographic column adopting KromasilC18 ODS-1 (4.6mm * 150mm, 5 μ m) that HPLC analyzes chromatographic condition, and moving phase is Virahol: methyl alcohol=1: 1 (V/V), 25 ℃ of column temperatures, flow velocity 1mL/min, detector wavelength 210nm.Patent of the present invention adopts HPLC, IR, NMR and MS to characterize the structure of polyprenol and hydroderivating thing thereof.Like Fig. 2 is the HPLC contrast of hydrogenated products and polyprenol, and the transformation efficiency of hydrogenated products is 25%～65%.
Fig. 3 is the IR of polyprenol and hydroderivating thing thereof, and the ir spectra basically identical of hydrogenated products and polyprenol is through analyzing: at 3322cm
-1And 835cm
-1Flexible and flexural vibration peak, 2958cm for=C-H
-1And 2860cm
-1Be CH
3-with-CH
2-stretching vibration peak, 1660cm
-1Stretching vibration for C=C.1450cm
-1Be CH
3-flexural vibration, 1380cm
-1For-CH
2-flexural vibration, 1000cm
-1Be the C-O-H stretching vibration.Fig. 4-5 is polyprenol and hydroderivating thing thereof
1H-NMR.Polyprenol
1H-NMR spectrum reference standard δ CDCl
3=7.26 (unimodal), through analyze δ: 1.59 (trans-CH
3), 1.68 (cis-CH
3), 1.74 (α-CH
3-cis), 1.99-2.09 (CH
3-), 4.07 (α-CH
2OH), 5.12 (C=CH).The nucleus magnetic hydrogen spectrum δ of hydrogenated products is α unit-CH (CH at 1.25 and 1.29 multiplets
3)-CH
2On methylene radical hydrogen, δ is the methyne hydrogen-CH (CH on the α unit at 0.85 place's multiplet
3)-CH
2, the prompting asymmetric hydrogenation results from unsaturated double-bond place, polyprenol α unit.Fig. 6-7 is polyprenol and hydroderivating thing thereof
13C-NMR.Polyprenol
13C-NMR spectrum reference standard δ CDCl
3=77.00 (triplets), δ: 135.56 (α-cis-CH
3CH=), 134.84 and 134.77 (trans-CH
3CH=), 134.71 (cis-CH
3CH=), 134.42 (trans-C (CH
3)=CH), 123.67～124.53 (=CH), 58.51 (CH
2-O-C=O), 39.23 (CH
2-), 31.71 (CH
2-), 31.49 (cis-CH
2C (CH
3)=), 26.14 (trans-(CH
3)
2C=CH-), 25.90 (trans-CH
2CH=), 25.82 (cis-CH
2CH=), 25.18 (cis-CH
3C=), 22.86 (CH
3), 17.17 (trans-C (CH
3)=CH), 15.48 (trans-CH
3C=).The nuclear-magnetism carbon of hydrogenated products spectrum is through analyzing: δ corresponding methyne peak height on 29.71 becomes, δ weakens (α-cis-CH in the corresponding explanation polyprenol at 139.1 place's peak heights
3CH=) locate to take place hydrogenation, the prompting asymmetric hydrogenation results from unsaturated double-bond place, polyprenol α unit.
This patent is to the mensuration of polyprenol hydrogenation reaction transformation efficiency; Adopt the HPLC area normalization method; As can beappreciated from fig. 2 to pass the polyprenol peak better with hydroderivating thing peak separating effect along with appearance time, so our selection is done typical curve with the peak area at the 5th peak, the linear relationship of derived peak area and polyprenol; Demarcate the content of polyprenol, thereby derive the transformation efficiency of reaction.The typical curve of polyprenol is as shown in Figure 9, tries to achieve equation of linear regression y=7.23664x+3.8945, R
2=0.9992, good in 5.1～25.2 μ g scope internal linear relation.
This patent is optimized the polyprenol hydrogenation reaction, has mainly chosen reaction pressure, reaction times, temperature of reaction and catalyst consumption, polyprenol and methylene chloride-methanol solution quality volume ratio (g: ml) 1: 20～100; Add catalyzer, polyprenol is 80～200: 1 with the catalyst quality ratio, reaction pressure 8～20MPa, and the reaction times is 18～48h, 10 ℃～50 ℃ of temperature of reaction; After reaction finishes, filter, vacuum concentration, enriched material are polyprenol hydroderivating thing.
Beneficial effect of the present invention shows as:
First with polyprenol lipoid and filler and urea alcoholic solution through mixed dissolution and freezing and crystallizing; Preparation polyprenol lipoid urea inclusion; Effectively removed pigment, lipid acid and ester thereof, polarity contains the oxygen functional group, can effectively improve the purity of polyprenol lipoid, again through the anhydrous propanone dewax; Zeyssatite separates with quartz sand, obtains 80% above polyprenol lipoid.With traditional method relatively, the alkali lye that the polyprenol lipoid of this method preparation needs when saponification reaction reduces more than 50%, environmental friendliness not only, and saponification resultant separates through post and do not obtain 95% polyprenol.
2. adopting the asymmetric hydrogenation catalyzer first, is (S)-Ru (OAc) like catalyzer
2(BINAP), Rh/PVP-TiO
2, Ru (BINAP) (OAc)
2, extraction separation betulol type structure (ω-(trans)
2-(cis)
n-OH, n=10～20) polyprenol, through catalytic hydrogenation reaction, with isopentene group unit in the polyprenol molecular structure by hydrogen saturated be the isoamyl alkyl unit, be the dolichol structure of primary isoamyl alcohol comprising end unit not, reaction conversion ratio is the highest by 65%.
3. preparation has antibacterial and plant polyprenol and hydroderivating thing thereof anti-oxidant activity first; Inhibited for streptococcus aureus (Sa.), intestinal bacteria (Ec.), bacillus subtilis and Salmonellas, have scavenging(action) for removing superoxide radical and hydroxyl radical free radical.
Description of drawings:
The chemical structure of the polyprenol that Fig. 1 is three types
The HPLC of Fig. 2 polyprenol and hydrogenated products (A: polyprenol, B: hydrogenated products)
The IR of Fig. 3 polyprenol and hydrogenated products (A: polyprenol, B: hydrogenated products)
The nucleus magnetic hydrogen spectrum of Fig. 4 polyprenol (
1H-NMR)
The nucleus magnetic hydrogen spectrum of Fig. 5 hydrogenated products (
1H-NMR)
The nuclear-magnetism carbon spectrum of Fig. 6 polyprenol (
13C-NMR)
The nuclear-magnetism carbon spectrum of Fig. 7 hydrogenated products (
13C-NMR)
The typical curve of Fig. 8 polyprenol
The TLC spectrogram of each elution fraction of Fig. 9 sample
Figure 10 hydroderivating thing is to O
2 -The clearance rate of radical
Figure 11 hydroderivating thing is to the OH free radical scavenging activity
Embodiment
Following examples are more of the present invention giving an example, and should not regarded as qualification of the present invention.
A kind of have antibacterial and plant polyprenol and hydroderivating thing preparation method thereof anti-oxidant activity, is made up of following steps:
The first step, polyprenol lipoid are extracted: will be rich in the fresh plant leaf of polyprenol lipoid, a kind of as in Ginkgo Leaf, pine needle, the mulberry leaf; Preferred Ginkgo Leaf, room temperature is dried in the shade, and is crushed to the following powder of 100 orders; Add any solvent in anhydrous propanone or the C1～C3 alcohol, preferred acetone; Leaf and solvent are to mix at 1: 20 by mass ratio, soaking at room temperature 10～72 hours, and preferred 24～48 hours; Or leaf and solvent by mass ratio be mix at 1: 20 the back at 10 ℃～80 ℃ through microwave extraction or ultrasonic extraction 10～60 minutes; Preferred UW extracts 10 ℃～40 ℃ of temperature, 10～30 minutes time; Repeat to extract twice; United extraction liquid, vacuum reclaims organic solvent, and enriched material is a polyprenol class fat ointment;
Second step; The preparation of urea alcoholic solution: urea is put into anhydrous methanol; Heating in water bath to urea all dissolves under the nitrogen condition filling, and wherein the weightmeasurement ratio of urea and anhydrous methanol was 1: 1～10, as 1: 3,1: 4,1: 5,1: 6,1: 8; Reflux temperature is 40～90 ℃, preferred 40～60 ℃;
The 3rd step; Polyprenol lipoid urea inclusion preparation: in the urea alcoholic solution, add polyprenol class fat ointment and filler; Filler is selected in silica gel, gac, polyamide resin, attapulgite, aluminum oxide, the zeyssatite a kind of, preferred silica gel, attapulgite, aluminum oxide.Polyprenol class fat ointment: filler: the mass ratio 0.1～1: 1～3: 10～40 of urea alcoholic solution, 40～90 ℃ of stirring and refluxing, be cooled to room temperature after;-40～10 ℃ of crystallizations; Preferably-20～-10 ℃ crystallization, suction filtration, crystallisate are polyprenol lipoid urea inclusion.
The 4th step, polyprenol lipoid purifying:, carry out wash-out with non-polar solvent with polyprenol lipoid urea clathration crystallisate dress post; Preferred normal hexane, sherwood oil; Elution volume is column volume 1-4 times, and TLC detects, and merges polyprenol lipoid elution fraction; Concentrating back adding mass ratio is 1: 6～30 anhydrous propanones, the dissolving of preferred 1: 6～15 stirring at room; 40～200 order zeyssatite mix by mass ratio 1: 1～10 with quartz sand, obtain mixed aid; Mixed aid is 0.5～20 with polyprenol lipoid mass ratio: 100 mix; Preferred 100～120 orders stirred 2～30 hours at-30 ℃～0 ℃, and 3000～20000 rev/mins of whizzers filter; Preferred 3000～10000 rev/mins of clear liquids reclaim organic solvent, and enriched material is a purifying polyprenol lipoid;
In the 5th step, the polyprenol preparation: the polyprenol lipoid joins in the 5%-40% sodium hydroxide solution, and polyprenol lipoid and sodium hydroxide solution mass ratio are 1: 10～50; 30 ℃～90 ℃ stirring reactions 0.5～3 hour, use normal hexane equal-volume extractive reaction liquid 2-5 time again, merge extraction phase; With equal-volume water backwash 2～5 times, the organic phase concentrating under reduced pressure, enriched material adds 5-20 times of silica gel mixing; With 2-6 times of normal hexane soaking at room temperature 0.5-2 hour, filter, it is dense dried filtrate again; Enriched material is faint yellow polyprenol oily matter, and polyprenol content is greater than 95%.
The 6th step, the preparation of polyprenol hydroderivating thing
With polyprenol be dissolved in methylene chloride-methanol solution (volume ratio is 8～1: 1), polyprenol and methylene chloride-methanol solution quality volume ratio (g: ml) 1: 20～100; Add catalyzer, polyprenol is 80～200: 1 with the catalyst quality ratio, reaction pressure 8～20MPa, and the reaction times is 18～48h, 10 ℃～50 ℃ of temperature of reaction; After reaction finishes, filter, vacuum concentration, enriched material are polyprenol hydroderivating thing.
Adopting the asymmetric hydrogenation catalyzer in the present embodiment, is (S)-Ru (OAc) like catalyzer
2(BINAP), Rh/PVP-TiO
2, Ru (BINAP) (OAc)
2, the polyprenol hydrogenation reaction is optimized, mainly chosen reaction pressure, reaction times, temperature of reaction and catalyst consumption, the transformation efficiency of hydrogenated products is up to 65% 25%～65%.
Adopt TLC to detect in the present embodiment and HPLC detection polyprenol and hydroderivating thing thereof, iodine vapor colour developing when TLC detects.It is chromatographic column adopting KromasilC18 ODS-1 (4.6mm * 150mm, 5 μ m) that HPLC analyzes chromatographic condition, and moving phase is Virahol: methyl alcohol=1: 1 (V/V), 25 ℃ of column temperatures, flow velocity 1mL/min, detector wavelength 210nm.Adopt HPLC, IR, NMR and MS to characterize the structure of polyprenol and hydroderivating thing thereof.Result such as Fig. 2-9.
1. the selection in reaction times
30 ℃ of fixation reaction temperature, methylene chloride: methyl alcohol=2: 1 (V/V), catalyzer are (S)-Ru (OAc)
2(BINAP); The mass ratio of polyprenol and catalyzer is 100: 1; H2 pressure 10MPa; Reaction times is respectively 3h, 6h, 9h, 12h, 18h, 24h, 36h, gets supernatant in the reaction process and does the HPLC detection, and reaction conversion ratio is respectively 6.8%, 16%, 22%, 32%, 41%, 56% and 58%.Along with the increase in reaction times, productive rate progressively improves, and is basicly stable behind the 24h, so the preferred reaction time is 24h.
2. the selection of catalyst levels
20 ℃ of fixation reaction temperature, catalyzer be Ru (BINAP) (OAc)
2, methylene chloride: methyl alcohol=3: 1 (V/V), H
2Pressure 12MPa, the reaction times is 24h, the mass ratio of polyprenol and catalyzer was got respectively 50: 1,100: 1,200: 1,300: 1,500: 1,1000: 1; Get supernatant in the reaction process and do the HPLC detection; Reaction conversion ratio is respectively 48%, 46%, 40%, 36%, 24%, 15%, and along with the increase of catalyst levels, productive rate progressively improves; Transformation efficiency is stable when catalyst consumption reaches 100: 1, so preferred 100: 1 of catalyst consumption.
3. the selection of reaction pressure
40 ℃ of fixation reaction temperature, catalyzer are Rh/PVP-TiO
2, methylene chloride: methyl alcohol=5: 1 (V/V), the mass ratio of polyprenol and catalyzer are 100: 1, the reaction times is 24h.Reaction pressure is respectively 2MPa, 4MPa, 6MPa, 8MPa, 10MPa, 12MPa, 15MPa, 20MPa; Get supernatant in the reaction process and do the HPLC detection; Reaction conversion ratio is respectively 6%, 18%, 32%, 43%, 52%, 60%, 65%, 68%; Along with the increase transformation efficiency of reaction pressure raises gradually, transformation efficiency raises and slows down when pressure reaches 12MPa, reaches the highest by 68% during 2010MPa.Consider production security, the preferred 10～15MPa. of reaction pressure
The purifying experiment of embodiment 3 polyprenols
1. get mulberry leaf 1kg, be crushed to 40 order powder, add anhydrous propanone 20kg, soaking at room temperature 50 hours; Filter, add the 15kg anhydrous propanone again, 40 ℃ of ultrasonic extraction 20 minutes; United extraction liquid, vacuum reclaims organic solvent, and enriched material is a polyprenol class fat ointment;
2. four kinds of different types of absorption carriers such as silica gel, monosodium glutamate active carbon for decolorization, polyamide resin, attapulgite have been screened; Pure urea and urea and absorption carrier carry out single factor screening by 5 kinds of different mass proportionings such as mass ratio 10: 1,5: 1,5: 2,5: 3,1: 1 to the purifying of polyprenol class fat ointment, and the result is like table 1-2.
The purification result of the different proportionings of table 1 urea silica gel
By table 1 is carrier with silica gel; Sherwood oil is the different proportionings that eluent passes through urea and silica gel, and experimental result can find out that polyprenol yield under pure urea condition reaches 92.08%, and purity is merely 39.83%; Urea and silica gel mass ratio are that the Ginkgo Leaf polyprenol purity that obtains of 5: 1 purifying is the highest; Its purity is 72.34%, and along with its purity of increase of silica gel consumption diminishes again gradually, yield also descends thereupon.Take all factors into consideration the factor of its purity and yield, obtaining with silica gel is that the best purification condition of carrier is that urea silica gel mass ratio is 5: 1, and its purity is 72.34%.
The purification result of table 2 different carriers
By table 2; Four kinds of different adsorption carriers such as silica gel, monosodium glutamate active carbon for decolorization, attapulgite and polyamide resin have been selected; Mass ratio between urea and the carrier 5: 1; Find with silica gel to be that the product purity that obtains of carrier is the highest through the contrast experiment, the monosodium glutamate active carbon for decolorization takes second place, and polyamide resin and attapulgite improve not quite product purity; The product colour that polyamide resin, attapulgite purifying obtain shows red-brown more deeply, and silica gel takes second place, and the product colour that monosodium glutamate active carbon for decolorization purifying obtains is thin out to be deep yellow.Take all factors into consideration with silica gel is that carrier is best.
3 purification by silica gel column chromatography
(1) wet method dress post
Take by weighing silica gel 250g, use the sherwood oil soaked overnight, fixedly (post specification Φ 3 * 25cm) goes into very thin absorbent cotton at the post bottom plug to silica gel column chromatography; Open piston, below chromatography column, connect beaker, topple over the good silica gel suspension of infusion, knock cylinder in the toppling process gently; Bubble is got rid of, when silica gel dress column volume reach chromatography column 2/3 the time stop to adorn post, treat that liquid slowly flows out; When the upper strata liquid level was equal with the silica gel face, closure piston was put silica sand in shop, silica gel face upper strata.
(2) appearance on the dry method
With sneaking into minor amount of silicon rubber powder end behind a small amount of petroleum ether dissolution, stir into pasty state, appearance is gone up in the oven dry back.
(3) wash-out
Use the sherwood oil of 800mL sherwood oil, 400mL successively: the sherwood oil of ETHYLE ACETATE=99: 1 (V/V), 400mL: the sherwood oil of ETHYLE ACETATE=98: 2 (V/V), 500mL: ETHYLE ACETATE=97: 3 (V/V) carries out wash-out.
(4) merge elution fraction
Each the component elutriant that obtains adopts the TLC qualitative detection, and developping agent is a sherwood oil: ETHYLE ACETATE (9: 1) through the iodine vapor colour developing, merges the recovery solvent and obtains the Ginkgo Leaf polyprenol.Like Fig. 9.
(5) HPLC quantitative analysis: adopt KromasilC18 ODS-1 (4.6mm * 150mm, 5 μ m), moving phase is Virahol: methyl alcohol=1: 1 (V/V), and 25 ℃ of column temperatures, flow velocity 1mL/min, detector wavelength 210nm, the time is 65min.Through sherwood oil: the purity of the contained polyprenol of elution fraction of ETHYLE ACETATE=97: 3 (V/V) wash-out gained has reached 90.22%.Result such as table 3.
Table 3 polyprenol HPLC purity detecting result
1. experimental strain, raw material
1.1 supply the examination bacterial classification
Intestinal bacteria (Ec.), streptococcus aureus (Sa.), black-koji mould, Salmonella, saccharomyces cerevisiae and bacillus subtilis are that the laboratory keeps bacterial classification.
1.2 testing sample
Polyprenol raw material and hydroderivating thing by own in prepared in laboratory
2 experimental techniques
2.1 the bacteriostatic activity of filter paper method working sample
(1) culture medium preparation
Heat of solution after the cooling, is regulated pH to 7.0～7.2, and this is a liquid nutrient medium, adds the 2.0g agar powder therein, and this is a solid medium, 121 ℃ of sterilization 20min.
(2) liquid nutrient medium inoculation and activated spawn
Open the ultraviolet sterilization lamp of clean work station, after sterilization half a hour, open the operating switch of clean bench, get a ring bacterial classification with the transfering loop after the sterilization and dissolve in the liquid nutrient medium; The Erlenmeyer flask wall that rubs gently lets and separates on bacterium colony and the transfering loop, takes out transfering loop; Seal Erlenmeyer flask, the shaking culture base is uniformly dispersed bacterial classification gently; Be put in the biochemical incubator, 37 ℃ of following activation 24h cultivate streptococcus aureus and intestinal bacteria respectively with this method.
(3) sterilization of the required instrument of experiment
The dull and stereotyped used petridish that falls is wrapped with kraft paper with the petridish that the 6mm filter paper is housed, and has filled in the test tube of plug, the box that the liquid-transfering gun head is housed is wrapped with kraft paper respectively, and the saline water for preparing, solid medium are sealed with gauze and kraft paper respectively.With it at 121 ℃, autoclaving 20min under the 100Pa.
(4) regulate bacterium colony concentration
The solid medium that will dissolve is poured into about 20mL in petridish, places on the clean bench, cools off from levelling.
Get 7 test tubes, dilute the good bacteria suspension of activation successively, No. 1 test tube is the bacteria suspension of stoste after saline water dilutes 10 times, and No. 2 test tubes are the bacteria suspension of 100 times of dilutions, and after the dilution, No. seven test tube is to dilute 107 times bacteria suspension successively.
Pipette the bacteria suspension applying solid substratum in 100 μ L1～No. 7 test tubes, leave standstill it is permeated in substratum, petridish is inverted in the back, in biochemical incubator, cultivates 24h for 37 ℃.
According to the bacterium colony computing rule: colony forming units cfu * extension rate=colony count, count denumerable bacterium colony plate, calculate every ware colony count, selecting the bacterium colony coating concentration is 10
5Cfu/mL.
(5) preparation of specimen
Respectively polyprenol and hydroderivating thing are mixed with 2mg/mL solution with normal hexane, the filter paper that will pass through sterilization is dipped in the sample solution, measures its bacteriostatic activity after half a hour.
(6) the filter paper method is measured the bacteriostatic activity that supplies test agent
Pipette 100 μ L bacteria suspensions with pipettor and coat liquid nutrient medium, treat that bacterium liquid permeates behind substratum after placement for some time, paste gently with the filter paper that is soaked with testing sample and obey in ware; Note not overexerting; It is sunk in the solid medium, and 3 filter paper equidistance are even, with two wares contrast parallel test; Be inverted the biochemical 24h of cultivation down for 37 ℃, measure sample is to the antibacterial circle diameter of Sa. and Ec..
2.2 the mensuration of sample MIC value
(1) by last method preparing culture medium, liquid nutrient medium inoculation and activated spawn; Regulate bacterium colony concentration and preparation specimen, sample solution is configured to 120mg/mL, in No. 1 plate, add sample solution and each 1mL of saline water; Therefrom take out 1mL behind the mixing in No. 2 wares; After add 1mL saline water, 2 times of dilutions of every ware, the back is poured the solid medium of 14mL in petridish.
(2) mensuration of the MIC value of each sample
The substratum that has mixed sample is fallen plate, from the levelling cooled and solidified, serves as to detect bacterial classification with Sa. and Ec., and spread plate is placed for some time and treated that bacteria suspension infiltrates, and is inverted the biochemical 24h of cultivation down for 37 ℃.Observe the minimum concentration plate of asepsis growth, being designated as this concentration is the MIC value of sample for this bacterial classification.
2.3 the investigation of sample antimicrobial spectrum
(1) culture medium preparation
Prepare streptococcus aureus, intestinal bacteria, Salmonellas and subtilis on request and use substratum; Black-koji mould according to the preparation of Cha Shi substratum, is 100mL water, 3.0g sucrose, 2.0g agar, 0.2g NaNO with substratum
3, 0.1g K
2HPO
4, 0.05g KCl, 0.05g MgSO
47H
2O, 0.001g FeSO
4Saccharomyces cerevisiae is used the YPD substratum, 100mL water, and 2.0g glucose, the 2.0g peptone, 2.0g agar, the 1.0g yeast powder is regulated pH 6.2～6.5.Above substratum all places 121 ℃ of following autoclaving 20min.
(2) the filter paper method is measured antimicrobial spectrum
With normal hexane polyprenol and hydroderivating thing are mixed with the solution of 2mg/mL, measure its antibacterial circle diameter for streptococcus aureus, intestinal bacteria, saccharomyces cerevisiae, black-koji mould, Salmonellas and bacillus subtilis.
2.4 antibacterial experiment result
The bacteriostatic activity result such as the table 4. of filter paper method working sample
The antibacterial circle diameter of table 4 sample and ethyl p-hydroxybenzoate relatively
The mensuration result such as the table 5 of sample MIC value
The MIC value result of table 5 sample
Mark: S is a streptococcus aureus, and E is intestinal bacteria
By table 5, for the MIC value of streptococcus aureus, polyprenol is 2mg/mL, and hydrogenated products is for being 0.125mg/mL, and for colibacillary MIC value, polyprenol is 4mg/mL, and hydrogenated products is 0.25mg/mL.The MIC value is a minimal inhibitory concentration, is worth more for a short time, and bacteriostatic activity is strong more, therefore, for fungistatic effect is: hydrogenated products＞polyprenol, its effect are worse than the MIC value of ethyl p-hydroxybenzoate.
The investigation result of sample antimicrobial spectrum is like table 6.
The antimicrobial spectrum result of table 6 sample
Antimicrobial spectrum result shows; Polyprenol and hydroderivating thing thereof are inhibited for streptococcus aureus (Sa.), intestinal bacteria (Ec.), bacillus subtilis and Salmonellas; The polyprenol antibacterial circle diameter is 6.82～8.55mm; Hydroderivating thing antibacterial circle diameter is 8.02～9.98mm, and the hydrogenated products bacteriostatic activity is higher than polyprenol.
1. to O
2 -Removing
(1) prepares the buffered soln of Tris-HCl of pyrogallol solution and the pH=8.2 of 7.5mmol/L respectively;
(2) in 1～No. 9 10mL color-comparison tube, add 5mL Tris-HCl buffered soln respectively;
(3) add successively that 0.3mL concentration is respectively 0.2,0.4,0.6,0.8,1.0,1.2,1.4, the sample solution of 1.6mg/mL in 1～No. 8 color-comparison tube, in No. 9 samples, add the 0.3mL deionized water and make reference;
(4) tube comparison tubes after will shaking up places 37 ℃ of water-bath constant temperature 20min;
(5) add 0.3mL pyrogallol solution in 1～No. 9 tube comparison tubes, under 37 ℃,, every behind the mixing at a distance from Abs value of 5s survey at 320nm place;
(6) 1～No. 8 Δ Abs/ Δ t of meter is Fx, and No. 9 Δ Abs/ Δ t is F0;
(7) clearance rate of calculating radical:
(8) with concentration being X-coordinate, is that ordinate zou is drawn anti-oxidant curve with the superoxide radical clearance rate.Like Figure 10.
The removing of 2 pairs of hydroxy radical qiaos (OH)
(1) preparation nitroso-R-salt strength of solution is 0.0016mol/L, CoSO
4Strength of solution is 0.0005mol/L, 1.8% H
2O
2Solution, working concentration dilution in time is 0.1%, preparation pH is 9.2 Na
2CO
3-NaHCO
3
(2) in 1～No. 8 color-comparison tube, add 2mL Na successively
2CO
3-NaHCO
3Buffered soln, 1mL CoSO
4Solution and 1mL nitroso-R-salt solution
(3) add successively in 1～No. 6 pipe that 1mL concentration is 0.4,0.8,1.2,1.6,2.0, the sample solution of 2.4mg/mL;
(4) in 1～No. 7 pipe, add 1mL 0.1%H
2O
2Solution;
(5) 1～No. 8 pipe is settled to behind the 10mL at 37 ℃ of following water bath with thermostatic control 45min with deionized water respectively;
(6) carry out spectral scan at 250～600nm place, about 410nm, survey Abs;
(7) calculate apparent anti-OH oxidation ratio:
(8) being X-coordinate with concentration, is ordinate zou with the hydroxyl radical free radical clearance rate, draws anti-oxidant curve.Like Figure 11.
The result shows: hydrogenated products is to O
2 -The removing result show, the increase of hydrogenated products sample concentration, the clearance rate of superoxide radical rises gradually; In 0.2～0.4mg/mL scope, be mild ascendant trend, be ascendant trend rapidly in 0.4～1.0mg/mL scope, 1.0mg/mL increases concentration later on; The clearance rate ascensional range is less, and trend tends towards stability after 1.4mL, but in general; Increase the concentration of product, can increase the clearance rate of superoxide radical, strengthen its oxidation resistant activity.Hydrogenated products shows the removing result of OH, the increase of hydrogenated products sample concentration, and the rate of removing of hydroxyl radical free radical rises gradually; In 0.4～1.2mg/mL scope, be mild ascendant trend, be ascendant trend rapidly in 1.2～2.0mg/mL scope, 2.0mg/mL increases concentration later on; The clearance rate ascendant trend tends towards stability, but in general, increases the concentration of product; Can increase the clearance rate of hydroxyl radical free radical, strengthen its oxidation resistant activity.The hydrogenated products of the present invention's preparation all has scavenging(action) preferably to removing superoxide radical and hydroxyl radical free radical, and good oxidation-resistance is arranged.
Claims (7)
1. one kind has antibacterial and plant polyprenol and hydroderivating thing preparation method thereof anti-oxidant activity, it is characterized in that being made up of following steps:
The first step, the polyprenol lipoid extracts: will be rich in the fresh plant leaf of polyprenol lipoid, room temperature is dried in the shade; Be crushed to the following powder of 100 orders, add any solvent in anhydrous propanone or the C1～C3 alcohol, leaf and solvent are 1: 20 by mass ratio; Soaking at room temperature 10～72 hours, or 10 ℃～80 ℃ through microwave extraction or ultrasonic extraction 10～60 minutes, repeat to extract twice; United extraction liquid, vacuum reclaims organic solvent, and enriched material is a polyprenol class fat ointment;
In second step, the preparation of urea alcoholic solution: urea is put into anhydrous methanol, and heating in water bath to urea all dissolves under the nitrogen condition filling, and wherein the weightmeasurement ratio of urea and anhydrous methanol is 1: 1～10, and reflux temperature is 40～90 ℃;
The 3rd step; Polyprenol lipoid urea inclusion preparation: in the urea alcoholic solution, add polyprenol class fat ointment and filler, polyprenol class fat ointment: filler: the mass ratio 0.1～1: 1～3: 10～40 of urea alcoholic solution, 40～90 ℃ of stirring and refluxing; After being cooled to room temperature;-40～10 ℃ of crystallizations, suction filtration, crystallisate are polyprenol lipoid urea inclusion;
The 4th step; Polyprenol lipoid purifying: with polyprenol lipoid urea clathration crystallisate dress post, carry out wash-out with non-polar solvent, elution volume is column volume 1-4 times; TLC detects; Merge polyprenol lipoid elution fraction, concentrating back adding mass ratio is 1: 6～30 anhydrous propanones, the stirring at room dissolving; 40～200 order zeyssatite mix by mass ratio 1: 1～10 with quartz sand; Obtain mixed aid; Mixed aid and polyprenol lipoid mass ratio are to mix at 0.5～20: 100, stir 2～30 hours at-30 ℃～0 ℃, and 3000～20000 rev/mins of whizzers filter; Clear liquid reclaims organic solvent, and enriched material is a purifying polyprenol lipoid;
In the 5th step, the polyprenol preparation: the polyprenol lipoid joins in the 5%-40% sodium hydroxide solution, and polyprenol lipoid and sodium hydroxide solution mass ratio are 1: 10～50; 30 ℃～90 ℃ stirring reactions 0.5～3 hour, use normal hexane equal-volume extractive reaction liquid 2-5 time again, merge extraction phase; With equal-volume water backwash 2～5 times, the organic phase concentrating under reduced pressure, enriched material adds 5-20 times of silica gel mixing; With 2-6 times of normal hexane soaking at room temperature 0.5-2 hour, filter, it is dense dried filtrate again; Enriched material is faint yellow polyprenol oily matter, and polyprenol content is greater than 95%;
The 6th step, the preparation of polyprenol hydroderivating thing
With polyprenol be dissolved in methylene chloride-methanol solution (volume ratio is 8～1: 1), polyprenol and methylene chloride-methanol solution quality volume ratio (g: ml) 1: 20～100; Add catalyzer, polyprenol is 80～200: 1 with the catalyst quality ratio, reaction pressure 8～20MPa, and the reaction times is 18～48h, 10 ℃～50 ℃ of temperature of reaction; After reaction finishes, filter, vacuum concentration, enriched material are polyprenol hydroderivating thing.
2. according to claim 1 said a kind of have antibacterial and plant polyprenol and hydroderivating thing preparation method thereof anti-oxidant activity; It is characterized in that polyprenol and hydroderivating thing thereof are inhibited for streptococcus aureus (Sa.), intestinal bacteria (Ec.), bacillus subtilis and Salmonellas, have scavenging(action) for removing superoxide radical and hydroxyl radical free radical.
3. according to claim 1 said a kind of have antibacterial and plant polyprenol and hydroderivating thing preparation method thereof anti-oxidant activity; It is characterized in that polyprenol derives from a kind of in Ginkgo Leaf, pine needle, the mulberry leaf, the structure of polyprenol is betulol type ω-(trans)
2-(cis)
n-OH, wherein the scope of n is 10～20.
4. according to claim 1 said a kind of have antibacterial and plant polyprenol and hydroderivating thing preparation method thereof anti-oxidant activity, it is characterized in that polyprenol hydroderivating thing is that isopentene group unit is the isoamyl alkyl unit by hydrogen is saturated in the polyprenol molecular structure.
5. according to claim 1 said a kind of have antibacterial and plant polyprenol and hydroderivating thing preparation method thereof anti-oxidant activity, it is characterized in that C1 in the first step～C3 alcohol in methyl alcohol, ethanol, n-propyl alcohol, the Virahol any.
6. according to claim 1 said a kind of have antibacterial and plant polyprenol and hydroderivating thing preparation method thereof anti-oxidant activity, it is characterized in that filler is a kind of in silica gel, gac, polyamide resin, attapulgite, aluminum oxide, the zeyssatite in the 3rd step.
7. according to claim 1 said a kind of have antibacterial and plant polyprenol and hydroderivating thing preparation method thereof anti-oxidant activity, it is characterized in that catalyzer is (S)-Ru (OAc) in the 6th step
2(BINAP), Rh/PVP-TiO
2, Ru (BINAP) (OAc)
2In any.
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