CN101248731B - Cultivation method of acanthopanax root tree plant and acanthopanax root jujube tea with reinforced immunity function - Google Patents
Cultivation method of acanthopanax root tree plant and acanthopanax root jujube tea with reinforced immunity function Download PDFInfo
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Abstract
The invention relates to 'a cultivation method of an acanthopanax tea plant and the acanthopanax root tea with the purpose of enhancing immunity', which belongs to the field of health protection tea. The cultivation method of the acanthopanax tea plant is characterized in that: (1) soybean cakes are used for fertilizing the root; (2) pistil leaves of the acanthopanax are sprayed with foliar fertilizer every day a week before picking, wherein, the foliar fertilizer are prepared by jujube extract and ginseng distilled water. The acanthopanax root tea prepared by the tea plant has no other ingredients, which can obviously enhance the immunity of the organism of people with low immunity; meanwhile, the tea also has the advantages of convenient drinking, no small pieces and moderate sweet and sour taste. Clinical trials verifies that the acanthopanax root tea has not only the efficacy of enhancing immunity, but also the advantages of balancing blood pressure, relieving physical fatigue, delaying senility, reducing the incidence rate of headache, strengthening memory power and even increasing working strength for some people; in addition, the tea has no side effect and adverse reaction.
Description
Technical field
The present invention relates to the health protection tea field, the health protection tea that particularly relates to a kind of wilsonii cultivation method and have the enhancing immunity function.
Background technology
The desirable immunologic function of human body should be to give full play to the effect favourable to body health, reduces or eliminates as much as possible the body adverse influence.In the middle of long term studies and practice, it is found that natural world exists the various materials that can regulate body's immunity, these materials are being transferred human body immune function, are improving body resistance against diseases, are being kept aspect such as self physiological equilibrium and brought into play positive effect.
China has long tea culture, and a lot of people are used as have tea as a kind of enjoyment of life.And the health protection tea that much has specific function that occurs on the market, be formulated by tealeaves and some health foods, in order to make its active ingredient better be dissolved in the water, these health foods generally all are ground into granule, therefore this health protection tea is contained in little paper bag or the non-woven bag more, when having tea bag tea is directly put into cup and soak and drink, but the quality of these Related products all exists the different defective of situation.Such as: prior art discloses a kind of health protection tea with enhancing immunity function, as the wilsonii green tea of announcing among the number of patent application 02109604.X, be to be that raw material is made by Wilsonii raw pulp extract and green tea, this tea possibility is leak-off pocket because particle is little, when drinking, to leave standstill a little while, and do not have conventional tea and be immersed in visual effect in the cup, therefore liking the people that samples tea for those, they dislike drinking bag tea.
Summary of the invention
At the defective in the above-mentioned field, the invention provides planting technique of a kind of wilsonii tea tree and the slender acanthopanax ginseng jujube tea that makes by this tea tree with enhancing immunity function, need not add all the other raw materials and auxiliary material, but by optimizing the planting technique of wilsonii tea tree, make the immunocompromised person when drinking this health protection tea, can obviously increase immunity of organisms, simultaneously this tealeaves drink very convenient, taste is moderate.
The cultivation method of wilsonii tea tree is characterized in that: use as root fertilizer with soybean cake (1); (2) wilsonii stamen leaf is plucked and is applied foliage fertilizer the last week, and described foliage fertilizer is formulated by Fructus Jujubae extract and ginseng distilled water.
The applied amount of described soybean cake is 80-100 kilogram/mu.
Described root fertilising is to imbed the radix Acanthopanacis senticosi tea usage tree root after soybean-cake flour is broken to the 30-60 order.
Described root fertilizing time is to freeze autumn before the feud.
Described Fructus Jujubae extract is the ethanol extract of date, and used concentration of alcohol is 60%.
The weight ratio of described Fructus Jujubae extract and ginseng distilled water is 1: 8-10.
The sprinkling amount of described foliage fertilizer is a 80-100Kg/ mu wilsonii tea tree, each spray with the blade face not drip be as the criterion.
The spraying time of described foliage fertilizer is to carry out after at 3 in afternoon.
Health protection tea with enhancing immunity function is a slender acanthopanax ginseng jujube tea, is formed by the stamen leaf frying of the wilsonii tea tree of said method cultivation.
The wilsonii tea tree is the mode that root fertilising and foliar application combine by special planting technique, root is used soybean cake by 100 kilograms every mu weight portion before freezing the feud annual autumn, dig at the radix Acanthopanacis senticosi tea usage tree root by strain and to apply after a pitting is pulverized an amount of soybean cake, then with grave lid, the powder of executing promptly can melt in soil and absorbed by plant root during early spring in the coming year; Pluck at wilsonii stamen leaf and to carry out foliar application the last week, the ethanol extract and the ginseng distilled water of date is mixed with foliage fertilizer is sprayed on the radix Acanthopanacis senticosi tea leaf, enter in the stamen leaf and the body that participates in the wilsonii plant circulates by the blade face pore.
Wilsonii stamen leaf is the leaf on the tender stem apex that the early spring, wilsonii tea tree New Development went out, and wilsonii stamen leaf is plucked, and frying tealeaves makes health protection tea of the present invention according to a conventional method.
Experiment showed, that the health protection tea that makes by this method cultivation is high than its Radix Et Caulis Acanthopanacis Senticosi polysaccharide content of radix Acanthopanacis senticosi tea that the conventional cultivation method makes, and Radix Et Caulis Acanthopanacis Senticosi polysaccharide is the main component that really plays raise immunity.
Contain Radix Et Caulis Acanthopanacis Senticosi polysaccharide and eleutheroside A (eleutheroside A-cupreol glucoside), eleutheroside B (Syringin syringin), eleutheroside B1 (6 in the wilsonii, 8-escoparone-7-glucoside), eleutheroside L (galactite glycosides), eleutheroside D and E (being two kinds of not glucosides of the syringaresinol of isomorphism type) etc., what play the enhancing immunity effect is Radix Et Caulis Acanthopanacis Senticosi polysaccharide.We are in the influence of experimental study Radix Et Caulis Acanthopanacis Senticosi polysaccharide to normal mouse and immunosuppressed mice immune organ cell number and stereological parameter.The result shows that Radix Et Caulis Acanthopanacis Senticosi polysaccharide can strengthen cell number, spleen white pulp cumulative volume and the cortex of lymph node cumulative volume of mouse spleen and lymphonodi mesenterici, can obviously resist the immunosuppressive action of caused by cyclophosphamide.Conclusion: Radix Et Caulis Acanthopanacis Senticosi polysaccharide has the effect that strengthens immune function of mice.This also is that Radix Et Caulis Acanthopanacis Senticosi polysaccharide has the main effect to human body enhancing immunity function.
Date contains organic acid, triterpene glycoside, alkaloids, flavonoids, carbohydrate, vitamins and sitosterol, stigmasterol, desmosterol, also contains cAMP and cGMP.Organic acid has: betulinic acid, oleanolic acid, crataegolic acid, malic acid, tartaric acid, catechuic acid, oleic acid etc.The triterpene glycoside has: crataegolic acid-3-O-cis-right-coumaric acyl ester.Flavonoids has, 6,8-glucosulfone base-2 (R)-naringenin, rutin etc.Contained carbohydrate mainly is a glucose in the date, oligosaccharide, araban and the galacturonan etc. that also contain fructose, sucrose, be made up of glucose and fructose.In addition, also find to exist in the date a kind of acidic polysaccharose, called after zizyphus pectin A.Still contain multivitamins such as vitamin C, vitamin b3, thiamine, carotin, niacin in the date.In addition, 36 kinds of trace elements such as still resinous in the date, lymphatic temperament, Coumarins derivative, catechol, tannin, volatile oil, 13 seed amino acids and calcium, phosphorus, iron, selenium.
Date is the ripening fruits of Rhamnaceae plant wild jujube Zizipgus jujube Mill., also name red date (" Elementary Medicine "), dried jujube (" Mingyi Bielu ").It is sweet, warm in nature to distinguish the flavor of.Return spleen, stomach warp.Function: tonifying spleen and stomach, nourishing generate fluid, accent battalion defend, the antidote poison.
Cause that with cyclophosphamide mouse immune is low, the result shows that date polysaccharide can significantly promote generation and the activity of immunocompromised Turnover of Mouse Peritoneal Macrophages IL-Ia, promotes external splenocyte propagation.Conclusion: date polysaccharide has obvious immunological stimulation.
We test again that date polysaccharide produces splenocyte and secretion is white and be situated between (interleukin-2, IL-2) and serum soluble proleulzin acceptor (soluble interleukin-2 receptor, SIL-2R) influence of level and mechanism of action.With bloodletting and cyclophosphamide is research object with causing the immunocompromised mouse also, the date polysaccharide aqueous solution group that is divided into large, medium and small dosage (dosage be respectively 400,200,100mg/kg), the slow group of Chinese angelica tonifying blood oral liquid (6.6ml/kg, 3 times of original liquid dilutions) reach with volume physiological saline group, other establishes complete blank group, 6 every group.Modeling administration simultaneously, continuous 7d, generation and active and the serum SIL-R level of mensuration mouse boosting cell lL-2.The result shows, large, medium and small dosage date polysaccharide group and Chinese angelica tonifying blood oral liquid group all can significantly improve 1: 2 and 1: 4 immune generation and activity that resists mouse boosting cell lL-2 of dilution factor, compare with model group, difference has significance meaning (t=5.2～8.4, P＜0.01) with middle dosage date polysaccharide group mouse boosting cell lL-2 generation to be resisted in immunity and activity influence act as the strongest.Large, medium and small dosage date polysaccharide group and Chinese angelica tonifying blood oral liquid group all can significantly reduce serum SIL-2R level.Compare with model group, difference has significance meaning (t=14.1～16.3, P all＜0.01), and is the most remarkable to the influence of serum SIL-2R level with middle dosage date polysaccharide.Conclusion: date polysaccharide can promote immunity to resist mouse boosting cell generation and secretion IL-2, reduces serum SIL-2R level.
We test the date parenteral solution again to effect of immunologic function.Give mouse subcutaneous injection with 100% date parenteral solution, observe mouse thymus and spleen weight, serum lysozyme content, the acid a-aldehydic acid of T lymphocyte how lipase (ANAE) percentage and T lymphocyte transformation rate.The result shows that experimental mice thymus gland and spleen weight increase, and serum lysozyme content increases, and T lymphocyte ANAE is active to raise, and the T lymphocyte transformation rate increases.Conclusion: the date parenteral solution can make the mouse immune organ weight increase, and can strengthen mouse non-specific immune function and specific cellular immunity function.
People utilize fresh ginseng to be raw material in the genseng producing region, when the processing red ginseng, it is to steam ginseng that one procedure is arranged, in this procedure, produced a large amount of ginseng distilled waters, every processing 100kg genseng, can produce the ginseng distilled water about 150kg, these distilled water are generally all thrown away in vain, all do not carry out comprehensive utilization, we are through evidence, these ginseng distilled waters contain a large amount of ginsenosides, can make full use of it fully in the genseng process, the high-temperature sterilization of just having finished simultaneously naturally when steaming ginseng, and utilize the extraneous natural temperature (the average temperature is-5 ℃ to 5 ℃) during the red ginseng processing, this temperature helps these ginseng distilled waters and can put in container, keeping is to four of the coming year, the production operating period between May.
The present invention has optimized the cultivation method of wilsonii tea tree, by way with the such plant absorbing nutrient of root fertilising, with the soybean-cake flour landfill be processed in the wilsonii plant roots; Adopt the mixed solution that we is mixed with as the way of plant leaf surface being applied fertilizer, executes water again, reasonable time carries out foliage spray, carries out plant absorbing and photosynthesis before plucking wilsonii stamen leaf, and the immunologic function of wilsonii tealeaves is improved.
Health protection tea of the present invention, the suitable crowds such as physical labourer and brain worker, student, old man that give respectively take, thereby parts of body is adjusted to happy state, obviously alleviate physical fatigue and stress, tired that a variety of causes causes, thereby common disease reduces, energetic, be achieved the function of the enhancing immunity that the present invention has.
Soybean cake is the residue after the soybean oil refining, is commonly used for the big molecule fertilizer of animal feed and crop.Ginseng distilled water is the aqueous solution when steaming genseng, often is directly to emit, but more or less has some ginseng effective components to be dissolved in the water in steaming the process of genseng, and therefore above-mentioned two class raw materials all are twice laids, become the perfect fertilizer of wilsonii tea tree.Wilsonii early has application as tealeaves, but its bitter, common people are difficult to swallow, this flavor medicine-food two-purpose medicinal material of the preferred date of the present invention also is aided with ginseng distilled water, they are except powerful to enhancing immunity, utilize distinctive sweet, the taste that fragrance is regulated tea of its acid, sweet and genseng simultaneously, make health protection tea of the present invention become the mass product that the consumer takes like a shot.
Health protection tea of the present invention does not have all the other batchings, but make health protection tea of the present invention by the planting technique of optimizing the wilsonii tea tree, make the immunocompromised person when drinking this health protection tea, can obviously increase immunity of organisms, this tealeaves is drunk broken end very convenient, that do not have compound tealeaves to form simultaneously, and taste is sour-sweet moderate simultaneously.
The present invention has the instructions of taking of the health protection tea of enhancing immunity function:
(1) person that needs the enhancing immunity is as long as drink after brewing with 80 ℃ of hot boiling water.
(2) tea of the present invention can be made filter paper bag and make tea, conveniently to drink.
(3) this product brew the back cold and hot drink all can, effect is constant.
Many suitable crowds generally react clearheaded after trying out with tea of the present invention, sensation is light, and physical fatigue is eased, and immunity function improves, and major part has all been broken away from relevant for many years medicine.
Clinical verification, health protection tea of the present invention except effect with enhancing immunity, can also balancing blood pressure, alleviating physical fatigue, delay senility, reduce headache incidence, improve one's memory, the somebody also has the effect that increases working strength.Drink the without any side effects and bad reaction of this tea.
Embodiment
The present invention is described in further detail with experiment below in conjunction with embodiment.
Embodiment 1
1.1 root fertilising
Before freezing the feud annual autumn, also to use method as fertilising, weight portion by 80 kilograms every mu, taking by weighing soybean-cake flour, to be broken to 30-60 purpose powder standby, and establishment officer digs a pitting by strain at the wilsonii plant root the standby powder of an amount of soybean cake is applied, and covers with grave then.The powder of executing promptly can melt in soil and absorbed by plant root during early spring in the coming year.
1.2 foliar application
The ratio of sprinkling amount and wilsonii stamen leaf: every mu of plantation wilsonii plant is 4200 strains, every strain stamen leaf-making quantity is 0.05kg, the bright leaf-making quantity of every mu of wilsonii stamen leaf is 210kg, it is 80kg/ mu that Fructus Jujubae extract and ginseng distilled water (1: 10) are converted system aqueous solution sprinkling amount, is 21kg/ mu after fresh tea is given money as a gift.
1.2.1 weighting raw materials material: date 100Kg, the standard of above-mentioned raw materials is pressed under item of Pharmacopoeia of the People's Republic of China version in 2005.
1.2.2 date with 60% alcohol reflux 3 hours, is extracted twice, reclaim ethanol and concentrate, concentrate and adopt vacuum concentration.Vacuum concentration claims concentrating under reduced pressure again, and evaporation not only can be saved a large amount of energy at a lower temperature; Simultaneously,, avoided the destruction and the loss of thermally labile component, preserved the nutrient component and the fragrance of raw material better because material is not subjected to temperatures involved.It is dry under 50-60 ℃ to concentrate the back, makes dry extract 28kg;
1.2.3 its dry extract is broken into 200 order powder, join in the ginseng distilled water according to 1: 10 ratio then, make ginseng distilled water aqueous solution 308kg.
1.2.4 be chosen in the season of winning wilsonii stamen leaf, whenever the week before winning, with the aqueous solution I that makes, in the sprayer that the up-to-standard specification got ready of packing into adapts to, the technical staff of tissue through training, to the plant leaf surface of the wilsonii stamen leaf that is about to win, implement to divide metering spray three times by the aqueous solution of every mu of 80kg.Carry out after 3 o'clock every afternoons, (the spray back need fill spray as the rainy day) implements a week continuously.
1.2.5 select weather fine morning, the technical staff of tissue through training carries out the work of winning of wilsonii stamen leaf.Win the fresh stamen leaf that gets off, be placed in the airing net dish that stainless steel makes, can not in heapsly stack, prevent heating, go mouldy.
1.2.6 frying tealeaves according to a conventional method, cake of press or pack are health protection tea of the present invention.
Embodiment 2
2.1 root fertilising
Before freezing the feud annual autumn, also to use method as fertilising, weight portion by 100 kilograms every mu, taking by weighing soybean-cake flour, to be broken to 30-60 purpose powder standby, and establishment officer digs a pitting by strain at the wilsonii plant root the standby powder of an amount of soybean cake is applied, and covers with grave then.The powder of executing promptly can melt in soil and absorbed by plant root during early spring in the coming year.
2.2 foliar application
The ratio of sprinkling amount and wilsonii stamen leaf: every mu of plantation wilsonii plant is 4200 strains, every strain stamen leaf-making quantity is 0.05kg, the bright leaf-making quantity of every mu of wilsonii stamen leaf is 210kg, it is 100kg/ mu that Fructus Jujubae extract and ginseng distilled water (1: 8) are converted system aqueous solution sprinkling amount, is 21kg/ mu after fresh tea is given money as a gift.
2.2.1 weighting raw materials material: date 100Kg, the standard of above-mentioned raw materials is pressed under item of Pharmacopoeia of the People's Republic of China version in 2005.
2.2.2 date with 60% alcohol reflux 3 hours, is extracted twice, reclaim ethanol and concentrate, concentrate and adopt vacuum concentration.Vacuum concentration claims concentrating under reduced pressure again, and evaporation not only can be saved a large amount of energy at a lower temperature; Simultaneously,, avoided the destruction and the loss of thermally labile component, preserved the nutrient component and the fragrance of raw material better because material is not subjected to temperatures involved.It is dry under 50-60 ℃ to concentrate the back, makes dry extract 28kg;
2.2.3 its dry extract is broken into 200 order powder, join in the ginseng distilled water according to 1: 8 ratio then, make ginseng distilled water aqueous solution 252kg.
2.2.4 be chosen in the season of winning wilsonii stamen leaf, whenever the week before winning, with the aqueous solution I that makes, in the sprayer that the up-to-standard specification got ready of packing into adapts to, the technical staff of tissue through training, to the plant leaf surface of the wilsonii stamen leaf that is about to win, implement to divide metering spray three times by the aqueous solution of every mu of 100kg.Carry out after 3 o'clock every afternoons, (the spray back need fill spray as the rainy day) implements a week continuously.
2.2.5 select weather fine morning, the technical staff of tissue through training carries out the work of winning of wilsonii stamen leaf.Win the fresh stamen leaf that gets off, be placed in the airing net dish that stainless steel makes, can not in heapsly stack, prevent heating, go mouldy.
2.2.6 frying tealeaves according to a conventional method, cake of press or pack are health protection tea of the present invention.
Experimental example 1
This health protection tea is the enhancing immunity function assessment test standard of slender acanthopanax ginseng jujube tea, carries out according to the Ministry of Public Health " health food check and assessment technique standard " (a 2003) year version, and result of the test is as follows:
1. materials and methods
1.1 test material: the slender acanthopanax ginseng jujube tea that the embodiment of the invention 1 makes; Adult's recommended amounts 2g/d every day.By adult 60kg weighing-machine, i.e. 33mg/ (kg body weight).
1.2 experimental animal: a cleaning level BALB/c mouse, male, 200,18～22g, available from Animal Experimental Study center, Hubei Province, the quality certification number is the moving word of doctor 2006-0812 number.
1.3 dosage is selected: three dosage groups are established in test: 10 times of human intaking amounts [0.33g/ (kg body weight)] group, 20 times of human intaking amounts [0.67g/ (kg body weight)] group, 30 times of human intaking amounts [1.00g/ (kg body weight)] group and control group.The mouse stomach volume is the 0.2ml/10g body weight.
1.4 key instrument and reagent: 5mm diameter card punch, microscope, Microhemagglutination breadboard, centrifuge, microplate reader, 96 well culture plates, 5%CO
2Incubator, 200 eye mesh screens, injection prepared Chinese ink, concanavalin A (ConA), MTT, DNFB, acetone, sesame oil, hair remover, SPBS, physiological saline, chicken red blood cell, methyl alcohol, Giemsa dye liquor, YAC-1 cell, Hanks liquid (pH7.2～7.4), RPMI 1640 complete culture solutions, lithium lactate, p-Iodonitrotetrazolium violet (INT), azophenlyene dimethyl ester sulphate (PMS), NAD, Tris-HCI buffer solution (pH8.2) 1%NP40, platform are expected orchid, 1mol/L hydrochloric acid etc.
1.5 experimental technique
1.5.1 carbon is cleaned up experiment and internal organs/weight ratio pH-value determination pH: test latter stage, after mouse is steady, inject the india ink (100mL/kg) of dilution from mouse tail vein, inject behind the prepared Chinese ink 2,10min by body weight, get blood 20uL from the angular vein clump respectively, existing side by side, soon it is added to 2mL 0.1%NaCO
3In the solution.With 721 spectrophotometers at 600nm place photometry density value (OD), with NaCO
3Solution is done blank.Mouse is put to death, dissect animal, take out liver, spleen and thymus gland, blot behind the bloodstain steady with filter paper.
1.5.2 delayed allergy (DTH): after giving mouse continuous irrigation stomach 30d with test material, every mouse skin of abdomen is with hair remover processings of losing hair or feathers, and the about 3cm * 3cm of scope then evenly smears sensitization with DNFB solution 50uL; Behind the 5d, evenly be applied in the mouse right ear two sides with DNFB solution 10u L again, attack; Behind the 24h, mouse is put to death in the cervical vertebra dislocation, and two ears about cutting take off the auricle of diameter 5mm and weigh with card punch.
1.5.3 HD50 value (HC
50) mensuration and antibody-producting cell detect (Jerne improve slide method): to behind the mouse continuous irrigation stomach 30d, the cell suspension 0.2mL of every lumbar injection 2% (volume fraction) SRBC carries out immunity with test material.Behind the 5d, get mouse blood and spleen and be HD50 value (HC respectively
50) mensuration and antibody-producting cell detect.
1.5.3.1 HD50 value (HC
50) mensuration: mouse is extractd eyeball get blood in centrifuge tube, place about 1h, serum is separated out, the centrifugal 10min of 2000r/min collects serum.With physiological saline dilute serum (300 times), serum 1mL after the dilution is put in vitro, add 10% (volume fraction) SRBC 0.5mL successively, complement 1mL (diluting by 1: 10) with physiological saline, other establishes the not control tube of increase serum (with physiologic saline for substitute), put be incubated 15～30min in 37 ℃ of waters bath with thermostatic control after, the ice bath cessation reaction.The centrifugal 10min of 2000r/min, get supernatant 1mL, add Dou Shi reagent 3mL in vitro, get 10% (volume fraction) SRBC 0.25ml simultaneously and add Dou Shi reagent to 4mL, in another in vitro, abundant mixing is behind the placement 10min, sentence control tube in 540nm and make blank, measure respectively and respectively manage optical density value.
1.5.3.2 antibody-producting cell detects (Jerne improve slide method): get the mouse spleen behind the SRBC immunity 5d, be placed in the little plate that fills Hanks liquid, grind spleen gently,, make single cell suspension through the filtration of 200 eye mesh screens.Centrifugal 10min (1000r/min) washes twice with Hanks liquid, with cell suspension in 5mL RPMI RPMI-1640, counting cells, and cell concentration is adjusted into 5 * 10
6Individual/mL.After the medium heating for dissolving of top layer, mix packing small test tube, every pipe 0.5mL with the Hanks liquid of equivalent PH7.2～7.4,2 times concentration, in pipe, add 50uL10%SRBC again, 20UL splenocyte county liquid, mixing is poured on the slide of brushing the agarose thin layer rapidly, do parallel plate, after solidifying in agar, slide buckled be placed on the horse, put into CO
2After hatching 1～1.5h in the incubator, counting hemolysis plaque number.
1.5.4 engulfing chicken red blood cell, Turnover of Mouse Peritoneal Macrophages tests (half intracorporal method): after giving mouse continuous irrigation stomach 30d with test material, and every mouse lumbar injection 2% chicken erythrocyte suspension 1mL.Behind the 30min, animal is put to death in the cervical vertebra dislocation, facing upward the position is fixed on the stencil plate, abdominal skin is cut off in the center, inject physiological saline 2mL through the abdominal cavity, rotate mouse plate 1min, sucking-off abdominal cavity washing lotion 1mL then, average mark drips on 2 slides, put into the enamel box that is lined with wet gauze, incubate completely in 37 ℃ of incubator incubation 30min., not paster cell is removed in rinsing in physiological saline. dry, with the fixing 20min of 1: 1 acetone first solution, 4% (volume fraction) Giemsa-phosphate buffer dyeing 3min uses distilled water rinsing airing again, in microscopically counting macrophage.
1.5.5 mouse spleen lymphocyte transformation experiment that ConA induces and NK cells in mice determination of activity (lactate dehydrogenase L DH determination method): after giving mouse continuous irrigation stomach 30d with test material, the cervical vertebra dislocation is put to death, the aseptic spleen of getting, place the little plate that fills an amount of aseptic Hanks liquid, gently spleen is torn up with tweezers, filter, make single cell suspension through 200 eye mesh screens, cell suspension is divided into two parts, the mouse spleen lymphocyte transformation experiment and the NK cells in mice determination of activity of inducing respectively at ConA.
1.5.5.1 the mouse spleen lymphocyte transformation experiment that ConA induces: use Hanks liquid washed cell suspension 2 times, each centrifugal 10min (1000r/min), then with cell suspension in the complete culture solution of 1mL, platform is expected blue dyeing counting viable count (more than 95%), and adjusting cell concentration is 3 * 10
6Individual/mL.Divide two holes to add in 24 well culture plates every part of cell suspension, every hole 1mL, a hole adds 75uLConA liquid, and another hole is put and is cultivated 72h in the incubator in contrast.Cultivate and finish preceding 4h, every hole is inhaled and is removed supernatant 0.7mL, adds the RPMI RPMI-1640 that 0.7mL does not contain serum, adds MTT 50uL/ hole simultaneously, continues to cultivate 4h.After cultivating end, every hole adds 1mL acid isopropyl alcohol, and the piping and druming mixing dissolves purple crystal fully.Divide then to install to 96 well culture plates, 3 parallel holes are done in every hole, measure optical density value with microplate reader with the 570nm wavelength.
1.5.5.2 NK cells in mice determination of activity (lactate dehydrogenase L DH determination method): 24h washes target cell cultivations of going down to posterity 3 times with Hanks liquid before using before the test, is 4 * 10 with RPMI 1640 complete culture solutions adjustment cell concentration
5Individual/mL.Wash splenocyte suspension 2 times with Hanks liquid, each centrifugal 10min (1000r/min).After abandoning supernatant, add 0.5mL aqua sterilisa 20s, add 0.5mL2 times of Hanks liquid and 8mLHanks liquid after the splitting erythrocyte again, centrifugal 10min (1000r/min), resuspended with the 1mL perfect medium, with 1% glacial acetic acid dilution back counting, platform is expected blue dyeing counting viable count (should more than 95%), and adjusting cell concentration is 2 * 10
7Individual/mL.Get each 100uL of target cell and effector cell (imitating target), add in 96 well culture plates than 50: 1; Target cell nature release aperture adds target cell and each 100uL of 1%N P40, and above-mentioned every three multiple holes of all establishing are in 37 ℃, 5%CO
2Cultivate 4h in the incubator, then with culture plate centrifugal (1500r/min) 5min, every hole is drawn supernatant 100uL and is put in the foster plate in 96 holes, add LDH matrix liquid 100uL simultaneously, reaction 3min, every hole adds the HCI 30uL of 1mol/L, measures optical density value at microplate reader 490nm place.The experimental data statistics: experiment gained The data SAS software is analyzed.
2. result
2.1 slender acanthopanax ginseng jujube tea the results are shown in Table 1 to table 5 to the influence of mouse body weight
Table 1 carbon is cleaned up experiment and internal organs/body weight experiment front and back the weight of animals record
The weight of animals record before and after the table 2 DTH experiment
Table 3 HD50 value (HC
50) experiment and antibody-producting cell experiment front and back the weight of animals record
Table 4 is engulfed experiment front and back the weight of animals record
The weight of animals record before and after table 5 mouse spleen lymphocyte transformation experiment and the NK cells in mice activity experiment
Through variance analysis, respectively test between three dosage groups and the control group forward and backward, body weight and weightening finish there are no significant difference, P＞0.05
2.2 slender acanthopanax ginseng jujube tea the results are shown in Table 6 to the influence of mouse internal organs/weight ratio.
Table 6 slender acanthopanax ginseng jujube tea is to the influence of mouse internal organs/weight ratio
Annotate: * shows and control group comparison, P＜0.05
Spleen/body weight ratio classical prescription difference analysis, F=0.40, P＞0.05.Thymus gland/body weight ratio classical prescription difference analysis, F=3.20, P＜0.05.The Q check, 30 multiple dose groups of human body recommended amounts and control group relatively have significant difference, P＜0.05.
2.3 slender acanthopanax ginseng jujube tea sees Table 7 to the influence of mouse cell immunologic function [mouse spleen lymphocyte transformation experiment and mouse delayed allergy (DTH)].
Table 7 slender acanthopanax ginseng jujube tea is to the mouse cell Immune Effects
20
Annotate: * shows and control group comparison P＜0.05
The OD difference of lymphocyte proliferation assay is through variance analysis, F=2.86, P＜0.05.Q check, three dosage groups of human body recommended amounts and control group comparison, there is significant difference P＜0.05.Ear swells degree through variance analysis, F=3.42, P＜0.05.The Q check, 30 multiple dose groups of human body recommended amounts and control group compare, and there is significant difference P＜0.05.
2.4 slender acanthopanax ginseng jujube tea sees Table 8 to the influence of mouse humoral immune function (antibody-producting cell is measured and mouse HD50 pH-value determination pH).
Table 8 slender acanthopanax ginseng jujube tea is to the influence of mouse humoral immune function
Annotate: * and show and control group P＜0.05 relatively, * * shows with control group and compares P＜0.01
The hemolysis plaque number is through variance analysis, F=4.33, P＜0.05.The Q check, three dosage groups are compared with control group, and significant difference is arranged, P＜0.05; HC
50Through variance analysis, F=4.88, P＜0.01.The Q check, 30 multiple dose groups are compared with control group, and utmost point significant difference is arranged, P＜0.01.
2.5 slender acanthopanax ginseng jujube tea is engulfed the influence of chicken red blood cell ability to Turnover of Mouse Peritoneal Macrophages, sees Table 9.
Table 9 slender acanthopanax ginseng jujube tea is engulfed the influence of chicken red blood cell ability to Turnover of Mouse Peritoneal Macrophages
Annotate: * shows and control group comparison, P＜0.05
Turnover of Mouse Peritoneal Macrophages is engulfed the phagocytic rate of chicken red blood cell through variance analysis, F=1.94, P＞0.05.Phagocytic index is through variance analysis, F=3.16, P＜0.05.The Q check, 30 multiple dose groups are compared with control group, and there is significant difference P＜0.05.
2.6 slender acanthopanax ginseng jujube tea sees Table 10 to the influence of NK cells in mice activity.
Table 10 slender acanthopanax ginseng jujube tea is to the influence of NK cells in mice activity
Annotate: * shows and control group comparison, P＜0.05
Through variance analysis, F=4.14.P＜0.05.The Q check, 30 multiple dose groups are compared with control group, P＜0.05, there were significant differences.
3. conclusion
(1) three dosage groups of slender acanthopanax ginseng jujube tea there is no the weight of animals and body weight gain are had tangible influence.
(2) thymus gland/body weight ratio of 30 multiple dose groups of slender acanthopanax ginseng jujube tea human body recommended amounts and control group relatively have significant difference.
(3) test of mouse cell immunologic function shows, transforms in the realization in splenic lymphocyte, and three dosage group OD differences of slender acanthopanax ginseng jujube tea human body recommended amounts are significantly higher than control group, and the result is positive; The delayed allergy experiment shows that swollen degree of ear and control group compare, 30 multiple dose group significant differences, and the result is positive.
(4) the mouse humoral immune functional experiment shows, in the antibody-producting cell experiment, compares with control group, and three dosage groups of slender acanthopanax ginseng jujube tea splenocyte hemolysis plaque digital display work raises, and the result is positive; Mouse HD50 pH-value determination pH shows, the HC of 30 multiple dose groups
50Value and control group relatively have utmost point significant difference, and the result is positive.
(5) mouse monokaryon-macrophage function experimental result shows, cleans up in the experiment at mouse carbon, and the phagocytic index of slender acanthopanax ginseng jujube tea 30 multiple dose groups is significantly higher than control group, and the result is positive; Mouse macrophage is engulfed the chicken red blood cell test and is shown that the phagocytic index of 30 multiple dose groups is significantly higher than control group, and the result is positive
(6) NK cytoactive determination experiment shows, compares with control group, and slender acanthopanax ginseng jujube tea 30 multiple dose group NK cell activity significantly raise, and the result is positive.
According to the Ministry of Public Health " health food check and assessment technique standard " (2003) year version criterion, judge that by above result slender acanthopanax ginseng jujube tea set has the function of tangible enhancing immunity.
Experimental example 2: the clinical observation of enhancing immunity situation.
(1) suitable crowd's situation
The suitable crowd volunteer who participates in the experiment is totally 80 examples, wherein male 44 examples, and women 36 examples, the men and women was than 1.38: 1.Age 30-40 year 16 examples, 41-50 year 25 examples, 51-60 year 25 examples, 16 examples more than 60 years old.Wherein professional cadre 32 people, teacher 20 people, workman 13 people, student 5 people, other 10 people.
(2) diagnostic criteria
Take this product after one week, suitable crowd is under normal condition, and 89% the groups of people feel that needs enhancing immunity can obviously enter the good state of mind, continuous operation 8 hours.People's the state of mind is good, does not have obvious fatigue and uncomfortable phenomenon and common disease phenomenon.What the sensation health was clearly better accounts for 21%, after continuing to take, all enters normal condition generally speaking.
Drinking health protection tea of the present invention is an incremental process to the overall process of human body enhancing immunity, the Chinese herbal medicine effect, and haste brings no success.After date when drinking 1, the enhancing of body immunity is fluctuation status, continues to drink when crest.Body immunity tends towards stability gradually after drinking 1 period generally speaking, drinks about one month, and body immunity recovers normally substantially, holds on to drink the back and just do not had situation that body immunity is bad again.
Effectively: various need enhancing immunity crowd is after drinking health protection tea of the present invention, and body immunity is stabilized in the normal range (NR), does not generally need to stop drink, because this tea has multinomial health care simultaneously, can continue to take.We are by clinical verification, health protection tea of the present invention except effect with enhancing immunity, can also balancing blood pressure, reducing blood lipid, hypoglycemic, anti-oxidant, alleviating physical fatigue, delay senility, reduce headache incidence, improve one's memory, also have the effect that increases working strength.Without any side effects and the bad reaction of this health protection tea of long-term drinking.
Experimental example 3: Radix Et Caulis Acanthopanacis Senticosi polysaccharide content comparative trial
Experiment material: tried thing (the slender acanthopanax ginseng jujube tea that experimental example 2 makes) and reference substance (sell, on August 1st, 2006 produced, the gloomy source board wilsonii tealeaves of gloomy source, Beijing slender acanthopanax drink Co., Ltd)
The Radix Et Caulis Acanthopanacis Senticosi polysaccharide extracting method adopts the Microwave Extraction method: get respectively and tried thing and reference substance 500g, after the fragmentation, respectively taking by weighing 100g puts in the 500ml bottle, this flask is put into MCL-3 type continuous microwave reactor, use 250mL benzinum (60～90 ℃), ether and 80% alcohol reflux to extract successively, adjust power 560W, 500W, 400W, 350W, 20 minutes reaction time.After residue volatilizes solvent, put into MCL-3 type continuous microwave reactor again, continue with water refluxing extraction 20 minutes, adjust power 630W, 600W, 560W, 450W, be evaporated to half volume, add 0.1% active carbon, decolouring, filter, filtrate adds 95% ethanol makes solution contain alcohol 80%, and standing over night filters, residue gets Radix Et Caulis Acanthopanacis Senticosi polysaccharide with ether, absolute ethyl alcohol cyclic washing.Weighing after 60 ℃ of oven dry.This product is pressed dry product and is calculated, and is tried to contain Radix Et Caulis Acanthopanacis Senticosi polysaccharide 0.075% in the thing, contains Radix Et Caulis Acanthopanacis Senticosi polysaccharide 0.025% in the tester.Contrast contained Radix Et Caulis Acanthopanacis Senticosi polysaccharide in the wilsonii of visible cultivation method of the present invention plantation thus and have more 2 times more than than the contained Radix Et Caulis Acanthopanacis Senticosi polysaccharide of wilsonii of conventional method plantation.Therefore the prepared slender acanthopanax ginseng of the present invention jujube tea is joined the function that the jujube tea set has better enhancing immunity with conventional slender acanthopanax.
Claims (8)
1. the cultivation method of wilsonii tea tree, it is characterized in that: use as root fertilizer with soybean cake (1); (2) wilsonii stamen leaf is plucked and is sprayed foliage fertilizer every day the last week, and described foliage fertilizer is formulated by Fructus Jujubae extract and ginseng distilled water, and described Fructus Jujubae extract is date 60% ethanol extract, and described ginseng distilled water is the aqueous solution when steaming genseng.
2. method according to claim 1, the applied amount of described soybean cake are 80-100 kilogram/mu.
3. method according to claim 1, described root fertilising is to imbed the radix Acanthopanacis senticosi tea usage tree root after soybean-cake flour is broken to the 30-60 order.
4. method according to claim 1, described root fertilizing time are to freeze autumn before the feud.
5. method according to claim 1, the weight ratio of described Fructus Jujubae extract and ginseng distilled water are 1: 8-10.
6. method according to claim 1, the sprinkling amount of described foliage fertilizer are 80-100Kg/ mu/sky, each spray with the blade face not drip be as the criterion.
7. method according to claim 1, the spraying time of described foliage fertilizer are to carry out after at 3 in afternoon.
8. the slender acanthopanax ginseng jujube tea that has the enhancing immunity function is formed by the stamen leaf frying of the wilsonii tea tree of the arbitrary method cultivation of claim 1-7.
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| CN101849592A (en) * | 2010-05-06 | 2010-10-06 | 安徽天品堂生态农业有限公司 | Polygonatum tea stable state processing method |
| CN102388737B (en) * | 2011-07-29 | 2013-07-17 | 成都中医药大学 | Acanthopanax giraldii Harms artificial cultivation method |
| CN102422937B (en) * | 2011-10-13 | 2013-04-24 | 陇东学院 | Preparation method of acanthopanax brachypus harms color-changing health care tea |
| CN103299745B (en) * | 2013-05-14 | 2015-07-15 | 天津泰达绿化集团有限公司 | Method for integrating punching and fertilization of border trees |
| CN105532350B (en) * | 2015-12-22 | 2018-09-18 | 桂林双象生物科技有限公司 | A kind of preparation method of hainan holly leaf ginkgo biloba extract compound tea |
| CN105613178A (en) * | 2016-01-15 | 2016-06-01 | 隋丽梅 | Method for cultivating acanthopanax sessiliflorus trees and acanthopanax tea with weight reducing function |
| CN107162805A (en) * | 2017-07-13 | 2017-09-15 | 烟台固特丽生物科技股份有限公司 | A kind of method that fresh flower extract prepares tea leaf quality modifying agent |
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| CN1164337A (en) * | 1996-05-03 | 1997-11-12 | 郝来勤 | Natural plant health-care tea capable of resisting low frenquency radiation of TV screen |
| CN1460419A (en) * | 2003-06-19 | 2003-12-10 | 柳忠润 | Acanthopanax root tea and its preparation method |
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| CN1164337A (en) * | 1996-05-03 | 1997-11-12 | 郝来勤 | Natural plant health-care tea capable of resisting low frenquency radiation of TV screen |
| CN1460419A (en) * | 2003-06-19 | 2003-12-10 | 柳忠润 | Acanthopanax root tea and its preparation method |
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| 杨春花等.刺五加的研究进展.人参研究 1.2004,(1),17-21. * |
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