CN107638433B - Tripterygium wilfordii hook F extract, preparation method thereof and application thereof in preparing eye drops - Google Patents
Tripterygium wilfordii hook F extract, preparation method thereof and application thereof in preparing eye drops Download PDFInfo
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Abstract
The invention discloses a tripterygium wilfordii extract, a preparation method thereof and application thereof in preparing eye drops. The tripterygium wilfordii extract is prepared by the following method: taking tripterygium wilfordii decoction pieces, extracting with 70-95% ethanol, filtering, combining filtrates, recovering ethanol under reduced pressure until no ethanol smell exists, continuously concentrating to obtain an extract with the relative density of 1.15-1.35, adsorbing with neutral alumina, drying under reduced pressure, and crushing to obtain a tripterygium wilfordii ethanol extract neutral alumina mixed sample; adding chloroform into the neutral alumina of the tripterygium wilfordii ethanol extract, extracting, filtering, collecting the filtrate, recovering chloroform, and drying to obtain the final product. The invention overcomes the self defect of high oral toxicity of the existing tripterygium wilfordii extract, has the advantages of safety, effectiveness, stability and the like when the tripterygium wilfordii extract is used for preparing eye drops, has excellent curative effect on xerophthalmia, and has good application prospect and market value.
Description
Technical Field
The invention belongs to the technical field of ophthalmic medicines and medicine preparation, and particularly relates to a tripterygium wilfordii extract, a preparation method thereof and application thereof in preparing eye drops.
Background
With the improvement of human living standard and technological level, especially the rapid development of electronic digital products, the average eye use frequency of people is increased rapidly, and environmental and individual factors increase the prevalence of xerophthalmia, which has seriously affected the life quality of some people. Due to the increase of the population suffering from the disease, the medicine for treating the dry eye disease is gradually the research and development hot spot of the medicine for ophthalmology.
In a normal human, the eye is maintained in a normal functional state by the presence of sufficient tear fluid on the surface of the eye, which in turn normalizes tear film components, coupled with a reasonable blink frequency. Dry eye is readily apparent when tear volume, tear film composition and blink frequency are altered. Many factors causing changes in tear volume and tear film components, such as inflammation of the eye, dysfunction of lacrimal glands, decreased blinking frequency (e.g. video terminal syndrome), surgery, immune factors, abnormal hormone metabolism, other drug interventions, etc., can cause changes in tear, tear film components and eye closure frequency, resulting in dry eye. The international dry eye working group in 2007 defines dry eye as: one type of tear and ocular surface disease is caused by a variety of factors, including ocular surface discomfort symptoms, vision changes and tear film instability and potential ocular surface damage, with increased tear osmolality and ocular surface inflammatory responses. Common clinical symptoms are: dry and astringent feeling, foreign body sensation, burning sensation, itching sensation, photophobia, red eyes, blurred vision, and fluctuation of vision. Severe dry eye can cause a significant loss of vision and thus affect their normal work and life, even leading to blindness.
At present, symptomatic treatment is mainly used for dry eye, the most important medicine is artificial tear replacement therapy, and other medicines mainly include anti-inflammatory medicines (such as nonsteroidal medicines, e.g., pranoprofen eye drops) and immunoregulatory medicines (such as cyclosporin a eye drops and corticosteroid eye drops). Other methods of dry eye treatment are surgical treatment, acupuncture and traditional Chinese medicine therapy. According to the development of dry eye treatment, the types of the medicines for treating dry eye are poor, the treatment means is single, and the action mechanism of the medicines is single, so that the method is an urgent field to be developed and enriched.
Tripterygium wilfordii hook is a perennial vine of Tripterygium wilfordii hook.f. of Celastraceae, is bitter in nature and taste, pungent and cool, has strong toxicity, is traditionally used as a medicine by peeling root xylem, and is also used as a medicine by peeling root and leaf. The modern chemical research result shows that the main chemical components of the compound have diterpenes, triterpenes, sesquiterpenes and alkaloids, and the modern pharmacological research shows that the compound has various pharmacological effects of immunosuppression, tumor resistance, rejection resistance, inflammation resistance, insecticide and the like, and is mainly applied to patients with immune-enhanced diseases such as rheumatic and rheumatoid arthritis, systemic lupus erythematosus and the like clinically at present. Although thunder god vine has various pharmacological actions and makes great contribution clinically, the oral preparation has great toxic and side effects and more toxic reactions, such as liver and kidney function damage, reproductive system damage, blood system adverse reaction and the like, and the application of thunder god vine is greatly limited. At present, no report related to the preparation of tripterygium wilfordii and active ingredients thereof into eye drops and the treatment of xerophthalmia is available.
Disclosure of Invention
The invention aims to: the problems of single treatment means of the existing xerophthalmia, great toxic and side effects of oral administration of the tripterygium wilfordii and the like are overcome, and the tripterygium wilfordii extract and the preparation method thereof are provided, and the tripterygium wilfordii extract is used for preparing eye drops and has the advantages of safety, effectiveness, stability and the like.
In order to achieve the above objects, it is considered that tripterygium wilfordii has various pharmacological actions such as immunosuppression, rejection resistance, and anti-inflammation, and clinical dry eye patients mostly suffer from dry eye caused by inflammation factors and immune factors, and clinical treatment schemes mainly involve anti-inflammation and immunoregulation methods, so if tripterygium wilfordii can be prepared into eye drops, it is presumed that tripterygium wilfordii will have a better therapeutic action on dry eye according to its pharmacological actions. In order to match the application, the invention provides a preparation method of the tripterygium wilfordii extract, which comprises the following steps:
(1) taking tripterygium wilfordii decoction pieces, extracting with 70-95% ethanol, filtering, combining filtrates, recovering ethanol under reduced pressure until no ethanol smell exists, continuously concentrating to obtain an extract with the relative density of 1.15-1.35, adsorbing with neutral alumina, drying under reduced pressure, and crushing to obtain a tripterygium wilfordii ethanol extract neutral alumina mixed sample;
(2) adding chloroform into neutral aluminum oxide sample of radix Tripterygii Wilfordii ethanol extract, extracting, filtering to obtain filtrate, recovering chloroform, and drying to obtain radix Tripterygii Wilfordii extract.
In order to realize the purpose, the invention also provides the thunder god vine extract which is prepared by the preparation method of the thunder god vine extract.
In order to realize the purpose, the invention also provides the application of the tripterygium wilfordii extract in preparing tripterygium wilfordii extract eye drops. Correspondingly, the invention also provides the tripterygium wilfordii extract eye drops, which comprise the tripterygium wilfordii extract with effective concentration as an active ingredient and pharmaceutically acceptable auxiliary materials.
As a preferable technical scheme of the tripterygium wilfordii extract eye drops, the dosage of the tripterygium wilfordii extract in the eye drops is 0.25-4 mg/ml calculated by the extract; or the dosage of the tripterygium wilfordii extract is 0.025-0.4 wt% by mass concentration; or the dosage of the tripterygium wilfordii extract is 3.5-28 mg crude drug/ml based on the crude drug. It should be noted that, in practical application, the dosage of the tripterygium wilfordii extract is suitable for eye dropping, and the dosage of the tripterygium wilfordii extract is not limited to the above range.
As a preferable technical scheme of the tripterygium wilfordii extract eye drops, auxiliary materials meeting the national relevant technical requirements, such as pH regulators, osmotic pressure regulators, antioxidants, suspending thickening agents, auxiliary materials for improving bioavailability, solvents and the like can be added in the preparation process of the tripterygium wilfordii extract eye drops. The pH regulator includes but is not limited to borax, boric acid, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, acetic acid, sodium acetate and the like; such osmo-regulators include, but are not limited to, sodium chloride, boric acid, glucose, potassium chloride, borax, glycerol, and the like. The adjuvants for improving bioavailability include, but are not limited to, phospholipid, linoleic acid, polyunsaturated fatty acid, alpha-linolenic acid, etc. The antioxidant includes, but is not limited to, sodium sulfite, sodium metabisulfite, sodium thiosulfate, vitamin C, thiourea and the like. The suspending thickening agent includes but is not limited to methylcellulose, sodium carboxymethylcellulose, hypromellose, polyvinyl alcohol, polyvinylpyrrolidone, sodium alginate, etc. Such solvents include, but are not limited to, water for injection, ethanol, glycerol, propylene glycol, and the like. Such bacteriostatic agents include, but are not limited to, parabens, chlorobutanol, thimerosal, phenylethyl alcohol, phenylmercuric nitrate, and the like.
As a preferred technical scheme of the tripterygium wilfordii extract eye drops, the preparation method can be a conventional method in the field, and can also be the following method: mixing radix Tripterygii Wilfordii extract with adjuvant, adding injectable water to desired amount, filtering with G2 sintered funnel to obtain filtrate, sterilizing with flowing steam at 100 deg.C for 30min, and packaging.
Experiments prove that the tripterygium wilfordii extract eye drops have better curative effect on xerophthalmia caused by anterior ocular inflammation such as keratitis and conjunctivitis and immune imbalance, and the curative effect on the xerophthalmia caused by inflammatory factors is better than that of triptolide.
Compared with the prior art, the invention has the following beneficial effects:
the invention overcomes the self defect of high oral toxicity of the tripterygium wilfordii extract, provides the tripterygium wilfordii extract and the preparation method thereof, and uses the tripterygium wilfordii extract to prepare eye drops, in particular to prepare eye drops for treating xerophthalmia. Compared with the existing eye drops, the tripterygium wilfordii extract eye drops have the characteristics of wide adaptation diseases, good curative effect, small adverse reaction and the like, have obvious improvement effect on inflammation and pain at the front of eyes, have similar effect on non-steroidal anti-inflammatory analgesic eye drops, but have the effect on immune-dyscrasia type xerophthalmia which is not possessed by the non-steroidal anti-inflammatory analgesic eye drops. Compared with tacrolimus eye drops with immunosuppressive action, the tripterygium wilfordii extract eye drops have obvious improvement action on immune xerophthalmia, have similar action to tacrolimus eye drops, but have anti-inflammatory and analgesic actions which are not possessed by tacrolimus eye drops.
Detailed Description
In order to make the objects, technical solutions and advantageous technical effects of the present invention clearer, the present invention is further described in detail with reference to the following embodiments. It should be understood that the embodiments described in this specification are only for the purpose of illustrating the invention and are not to be construed as limiting the invention, and the parameters, proportions and the like of the embodiments may be suitably selected without materially affecting the results.
Example 1
1. Extracting radix Tripterygii Wilfordii decoction pieces with 8 times of 95% ethanol for 2 times, each for 1 hr, filtering, mixing filtrates, recovering ethanol under reduced pressure until ethanol smell disappears, concentrating to obtain soft extract with relative density of 1.25(60 deg.C), adsorbing with neutral alumina, drying under reduced pressure, and pulverizing to obtain radix Tripterygii Wilfordii ethanol extract neutral alumina mixed sample.
2. Mixing the neutral alumina of the alcohol extract of thunder god vine with the sample, adding 10 times of chloroform for extracting for 1 hour, filtering, taking the filtrate, recovering the chloroform, and drying to obtain the thunder god vine extract.
3. Taking 2.0G of tripterygium wilfordii extract, adding 0.6G of borax, 12G of boric acid, 0.3G of ethylparaben, 10.0G of methylcellulose and 10.0G of sodium alginate serving as pharmaceutical excipients of eye drops according to the conventional process in the field, adding water for injection to 1000ml, filtering by a G2 sintered funnel, taking the filtrate, sterilizing by flowing steam at 100 ℃ for 30 minutes, and subpackaging to obtain the tripterygium wilfordii extract.
Example 2
1. Extracting radix Tripterygii Wilfordii decoction pieces with 95% ethanol 12 times for 1 time, each for 2 hr, filtering, recovering ethanol from the filtrate under reduced pressure until no ethanol smell exists, concentrating to obtain soft extract with relative density of 1.30(60 deg.C), adsorbing with neutral alumina, drying under reduced pressure, and pulverizing to obtain radix Tripterygii Wilfordii ethanol extract neutral alumina mixed sample.
2. Mixing the neutral alumina of the alcohol extract of Tripterygium wilfordii hook F with ethanol, adding 8 times of chloroform, extracting for 1 hr, filtering, collecting the filtrate, recovering chloroform, and drying to obtain the extract of Tripterygium wilfordii hook F.
3. Adding 10.0g of radix Tripterygii Wilfordii extract and 0.3g of ethylparaben into 80ml of propylene glycol to dissolve to obtain solution 1; dissolving 5.8g of disodium hydrogen phosphate, 8.7g of sodium dihydrogen phosphate and 5.0g of sodium sulfite in water for injection, adding 10.0g of polyvinylpyrrolidone, and swelling to obtain a solution 2; adding solution 1 into solution 2, stirring, adding water for injection to 1000ml, filtering with G2 sintered funnel, collecting filtrate, sterilizing with flowing steam at 100 deg.C for 30min, and packaging.
Experimental example 1 determination of effective concentration range of Tripterygium wilfordii hook F extract eye drops
1. Experimental Material
1.1 Coomassie Brilliant blue: nanjing is built into a bioengineering institute; batch number: 20121106, respectively; specification: 100T/96 samples.
1.2 complete Freund's adjuvant: specification: 10 mL/bottle, batch number: SLBC5083, stored at 2-8 deg.C, SIGMA Chemicals Inc.
1.3 fluorescein sodium injection, batch No.: 120901, specification 3 mL: 0.6 g/count, property: brown clear liquid, storage conditions: and (4) sealing and storing, wherein a manufacturer: mingxing pharmaceuticals, Inc., white cloud mountain, Guangzhou.
1.4 tacrolimus eye drops, product of Kawasaki factory, Qianshou pharmaceutical Co., Ltd; batch number: g011; specification: 0.1% (5ml:5mg (in tacrolimus)).
1.5 disodium hydrogen phosphate: batch number: 20120715, specification: 500 g/bottle, manufacturer: jiangsu Qiangsheng functional chemistry GmbH. Sodium dihydrogen phosphate: batch number: 20111201, specification: 500 g/bottle, manufacturer: taishan city Yuejie reagent plastics, Inc.
1.5 gentamicin sulfate injection: batch number: 1212022, specification: 2ml, 8 ten thousand units/bottle, sealing, storing in a cool and dark place (keeping out of the sun and not exceeding 20 ℃), and manufacturing factory: henan Ruhong pharmaceutical Co., Ltd.
1.6 positive control Beifushu (recombinant bovine basic fibroblast growth factor eye drops): specification: 21000IU/5 ml/bottle; batch number: 20121206, respectively; the validity period is as follows: 12 months and 4 days in 2013; the manufacturer: the Zhuhaisheng biopharmaceutical Co., Ltd,
1.8 tiger red: batch number: lot # ky201204, specification: 1 g/piece, storage conditions: normal temperature and light protection, and is a product of Qiyun biotechnology limited company in Guangzhou city.
1.9 sodium pentobarbital: china medicine (group) shanghai chemical reagent company (import split charging), batch number: 090205, specification: 25 g/bottle.
1.10BALB/c mice: 7-8 weeks old, 144 individuals, 18-22g, SPF grade, purchased from Experimental animals technology, Inc. of Wei Tong Li Hua, Beijing.
1.11 New Zealand rabbits (6-10 weeks old, 30 rabbits, 1.5-2.2kg, common grade), purchased from the center of medical laboratory animals in Guangdong province.
2. Main instrument
2.1 liquid transfer device: 2-20. mu.l and 10-100. mu.l, products of the company BRAND.
2.2 Spectrophotometer: type 722N, a product of shanghai cyanine scientific instruments ltd.
2.3 full-automatic tissue dehydrator: ASP300S, LEICA, Germany.
2.4 Paraffin embedding machine + Cold stage: model EG1150H paraffin embedding machine, model EG1150C cold plate, product of LEICA, Germany.
2.5 rotary slicer: RM2016, product of LEICA, Germany.
2.6 multifunctional dyeing machine: model ST5020, LEICA, Germany.
2.7 biological microscope: DM3000(ebp100-04-L fluorescence) type, LEICA, Germany.
2.8 pathological image analysis system: DFC450+ image acquisition software Leica Application Suite V4, LEICA, Germany.
2.9 medical purification workbench: the model is SW-CJ-2F (D), and the manufacturer is Suzhou scientific and technical equipment Co.
2.10 slit-lamp microscope and matching shooting system: model LYL-II, a product of east west science and technology limited.
3. Experimental methods
Selecting a plurality of BALB/c mice qualified for adaptability observation, wherein the number of the BALB/c mice is half of that of the female and the male, randomly selecting 10 animals as a negative control group, randomly selecting 16 animals for antigen preparation, and using the rest animals for model preparation. And after the adaptability observation is finished, animal selection is carried out according to the standard, and the model is made after the animal selection is finished. After the animal model is made, the mouse is raised under the normal raising condition for 5 weeks, the weight is weighed once a week in the period, after the model is made for 5 weeks, the 4 unmodeled animals and the 4 modeled animals are taken, the mouse is killed by using a cervical dislocation method, the binoculus conjunctiva of the mouse is taken as a pathological section to observe the gland destruction degree and the lymphocyte infiltration degree, and other animals detect the lacrimal secretion amount (binoculus) and the lacrimal rupture time (binoculus). The other animals are divided into 9 groups (excluding animals with light or heavy weight, specifically based on lacrimal secretion) according to lacrimal secretion amount, respectively, such as model control group, tacrolimus group, radix Tripterygii Wilfordii extract eye drop 0.00625%, 0.0125%, 0.025%, 0.05%, 0.1%, 0.2%, and 0.4% (respectively 0.0625mg/ml, 0.125mg/ml, 0.25mg/ml, 0.5mg/ml, 1.0mg/ml, 2.0mg/ml, and 4.0mg/ml, respectively, and crude drug amount is 0.875mg/ml, 1.75mg/ml, 3.5mg/ml, 7.0mg/ml, 14mg/ml, and 28mg/ml), each group contains not less than 10 animals, and each group contains both male and female. Grouping, and then, administering according to the set requirement, wherein 4 times (about 2h once) a day, continuously administering for 7 days, and when administering, a micro sample injector is used for sucking liquid medicine and then administering, the administration volume of each dose group of the tacrolimus and the tripterygium wilfordii extract is 5 mu l/eye/time, 4 times/day, and the administration interval is about 2 h; the negative control group and the model control group are given 5 mul/eye/time and 4 times/day of 0.9% sodium chloride injection with equal volume, and the dosing interval is 2 h. During the test period, the lacrimal fluid secretion amount (both eyes) and the tear film rupture time (both eyes) of the mice in the administration periods D4 and D7 were observed. Mice were euthanized by cervical dislocation the day after the last administration, and palpebral conjunctiva and fornix conjunctiva of each group of mice were taken and pathologically sectioned to observe the degree of recovery and lymphocyte infiltration after goblet cell destruction.
The preparation method of the antigen comprises the following steps: the same mouse is taken, euthanized by cervical dislocation, disinfected at the periphery of eyes by a conventional method, and the conjunctiva is completely taken out on a super clean workbench, washed by normal saline, put into normal saline containing 8 ten thousand/500 mL gentamicin, cut into fragments and ground into homogenate in a homogenizer. Keeping at 4 deg.C, crushing in ultrasonic crusher for 1h, and centrifuging in centrifuge (3000 r/min. times.15 min). Taking out supernatant, and determining protein content by Coomassie brilliant blue method. The protein concentration was then diluted to 100. mu.g/mL with 0.14M PBS. An equal volume of conjunctival antigen solution was added to complete Freund's adjuvant (FCA) and mixed well to form a water-in-oil solution.
The molding method comprises the following steps: the water-in-oil solution was injected into the subcutaneous tissue of the back, axillary lymph nodes of the extremities and foot pads of the mouse at multiple points in a total amount of 0.5 mL/mouse.
Note: (1) BALB/c mice lacrimal secretion assay BALB/c mice lacrimal secretion was measured using Schirmer I assay (Schirmer I test). The eye forceps are used for clamping the phenol red cotton thread, the phenol red cotton thread is placed at the outer canthus of a BALB/c mouse, and the phenol red cotton thread is taken out after 60 seconds to measure the wet length of the phenol red cotton thread. (2) Tear film Break Up Time (BUT): using a micro-liquid-adding device, 1. mu.l of cobalt blue light of a slit lamp microscope was added into the conjunctival sac under the BALB/c mouse to observe the cornea, and when a green film of the cornea appeared as a black area, it showed tear film rupture. The measurement was performed 3 times in succession, and the average value was obtained. And (3) repeatedly measuring for 3 times when the normal BUT is 10-15 seconds and the time less than 10 seconds is abnormal, and taking an average value.
4. Results of the experiment
The test results show (tables 1 to 3):
(1) on the first day of administration, compared with a negative control group, the length of the phenol red cotton thread and the tear film rupture time of the mice in the model control group are obviously shortened and have statistical difference (P < 0.01); compared with a model control group, the length of the phenol red cotton thread of the tacrolimus (0.1%) eye drop group is obviously increased, the tear film rupture time is obviously prolonged, and the statistical difference (P <0.01) exists, the length of the phenol red cotton thread of the thunder god vine extract eye drop group is also obviously increased by 0.5mg/mL, 1mg/mL, 2mg/mL and 4mg/mL, and the statistical difference (P <0.01) exists, and the tear film rupture time of the thunder god vine extract eye drop group is obviously prolonged by 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL and 4mg/mL, and the statistical difference (P <0.01) exists.
(2) On the fourth day of administration, compared with the negative control group, the length of the phenol red cotton thread and the tear film rupture time of the mice in the model control group are obviously shortened and have statistical difference (P < 0.01); compared with a model control group, the length of the phenol red cotton thread of the tacrolimus (0.1%) eye drop group is obviously increased, the tear film rupture time is obviously prolonged, the statistical difference (P <0.05 or 0.01) exists, the length of the phenol red cotton thread of the thunder god vine extract eye drop group is also obviously increased by 0.5mg/mL, 1mg/mL, 2mg/mL and 4mg/mL, the statistical difference (P <0.01) exists, the tear film rupture time of the thunder god vine extract eye drop group is obviously prolonged by 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL and 4mg/mL, and the statistical difference (P <0.01) exists.
(3) On the seventh day of administration, compared with the negative control group, the length of the phenol red cotton thread and the tear film rupture time of the mice in the model control group are obviously shortened and have statistical difference (P < 0.01); compared with a model control group, the length of the phenol red cotton thread of the tacrolimus (0.1%) eye drop group is obviously increased, the tear film rupture time is obviously prolonged, the statistical difference (P <0.01) exists, the length of the phenol red cotton thread of the thunder god vine extract eye drop group is also obviously increased by 0.5mg/mL, 1mg/mL, 2mg/mL and 4mg/mL, the statistical difference (P <0.05 or 0.01) exists, the tear film rupture time of the thunder god vine extract eye drop group is obviously prolonged by 0.0625mg/mL, 0.125mg/mL, 0.25mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL and 4mg/mL, and the statistical difference (P <0.01) exists.
Histopathological research results show that compared with a negative control group, the animals after the model building can see pathological changes of different degrees of conjunctiva, mainly manifested by different degrees of hyperplasia, increased cell layer number and disordered arrangement of epithelial cells at the fornix part of the conjunctiva, increased numbers of goblet cells of the epithelial layer, and vacuolated goblet cells of a small amount of animals. No obvious abnormality is found in the tissue structure of other parts of the conjunctiva.
Compared with the model group, the degree of conjunctival lesion and the number of cases are integrated, and the remission degree is decreased according to the principle that tacrolimus eye drops are 1mg/mL (C) and 1mg/mL (I) and 0.5mg/mL (H) and 2mg/mL (J) are respectively equal to (K) and 0.25mg/mL (G) and 4mg/mL (K) and 0.125mg/mL (F) and 0.0625mg/mL (E) respectively greater than model control group (B) respectively.
5. Conclusion of the experiment
Triptolide is one of known important components of radix Tripterygii Wilfordii extract, and is also the main component for controlling the amount of radix Tripterygii Wilfordii extract, and has molecular weight of 360.4 daltons. Triptolide has strong pharmacological action of immunosuppression and the like, and triptolide are mainly used for treating diseases of hyperfimmunity, such as rheumatic and rheumatoid arthritis, systemic lupus erythematosus and the like. The main components of the tacrolimus eye drops are tacrolimus, namely FK506 with the molecular weight of 822.05, and the tacrolimus eye drops are first-line medicines for clinical liver and kidney transplantation, and are mainly applied to spring conjunctivitis patients with unobvious antiallergic treatment effects. The invention mainly inspects the improvement effect of the tripterygium wilfordii extract eye drops with different concentrations on xerophthalmia through a mouse sicca syndrome model, and screens out proper drug concentration. The research results show that the tripterygium wilfordii extract eye drops can obviously increase the length of the phenol red cotton thread in 0.5, 1, 2 and 4mg/mL groups (0.05%, 0.1%, 0.2% and 0.4%) and tacrolimus groups (1mg/mL and 0.1%) on the first day, the fourth day and the seventh day of administration, and simultaneously, the corresponding dose groups can also prolong the tear film rupture time of mice, but the tripterygium wilfordii extract eye drops can only prolong the tear film rupture time of the mice on the first day, the fourth day and the seventh day of administration when the tripterygium wilfordii extract eye drops are in doses of 0.0625, 0.125 and 0.25mg/mL, but have no obvious influence on the length of the phenol red cotton thread. The histopathological research result shows that the tripterygium wilfordii extractive eye drops have more obvious improvement effect on the histology when the medicine concentration is 0.5 and 1mg/mL (0.05 percent and 0.1 percent), and have relatively weaker improvement effect at other concentrations.
By integrating the results of the tear secretion test, the tear film rupture time and the histopathology results, the tripterygium wilfordii extract eye drops can reach the plateau stage of drug effect when the concentration is 0.5-1.0%, and can reach better drug effect at the concentration, so that the possibility of obtaining larger drug effect result by continuously increasing the drug concentration is lower. According to the research results, the dosage of the tripterygium wilfordii extract eye drops is 0.25-4 mg/ml. (0.025%, 0.4%) and has a good effect of improving xerophthalmia, and the peak concentration of the drug effect is 0.5-1 mg/ml (0.05%, 0.1%).
TABLE 1 Effect of eye drops of Tripterygium wilfordii hook F extract on the length of the phenol red cotton thread and the tear film rupture time of model of mouse conjunctival sicca syndrome ((
Day1)
In comparison to the negative control group, # means P <0.05, # means P < 0.01; p <0.05, P <0.01, compared to the model control group.
TABLE 2 Effect of eye drops of Tripterygium wilfordii hook F extract on the length of the phenol red cotton thread and the tear film rupture time of model of mouse conjunctival sicca syndrome ((
Day4)
In comparison to the negative control group, # means P <0.05, # means P < 0.01; p <0.05, P <0.01, compared to the model control group.
TABLE 3 Effect of eye drops of Tripterygium wilfordii hook F extract on the length of the phenol red cotton thread and the tear film rupture time of model of mouse conjunctival sicca syndrome ((
Day7)
In comparison to the negative control group, # means P <0.05, # means P < 0.01; p <0.05, P <0.01, compared to the model control group.
Experimental example 2 therapeutic action of Tripterygium wilfordii hook F extract eye drops on rabbit alkali burn type xerophthalmia
1. Experimental methods
Selecting a new Zealand rabbit qualified for adaptive observation, firstly carrying out ophthalmic examination (a fluorescein sodium method), and eliminating animals with eye irritation symptoms and conjunctival injury. The remaining animals were molded (right eye of each animal), molding method: each new zealand rabbit was anaesthetised topically with 3% pentobarbital intravenous anaesthesia and 2% lidocaine as eye drops. Then 1 piece of filter paper with the size of about 10mm multiplied by 5mm is dipped with 1mol/L of NaOH solution and is placed on the palpebral conjunctiva about 2mm above the corneal limbus of the rabbit, and the conjunctival sac is repeatedly washed with 50mL of physiological saline immediately after 90 s. The first day after the operation, conjunctival fluorescein staining, conjunctival tiger red staining, phenol red cotton line testing, tear film rupture time and tear fern-like change testing are performed, and the test is divided into 5 groups by a first dividing method in a balanced and random manner according to the scores of the fluorescein staining and the tiger red staining, wherein the first dividing method comprises the following steps: the test method comprises the following steps of model group, tripterygium wilfordii extract eye drop low-dose group, tripterygium wilfordii extract eye drop high-dose group, positive control group Behcet group and triptolide eye drop group, wherein 6 animals in each group have both sexes, the left eye of each animal is used as negative control of self control, corresponding medicines are immediately administered after grouping, 4 times/day and 2h are provided at each administration interval, the eyelid is passively closed for at least 10s after each eye drop, the medicines are continuously administered for 10 days, the negative control eye and the model group are administered with physiological saline with the same volume, conjunctival congestion, corneal transparency and neovascularization conditions are observed under a slit lamp on days 1, 5 and 10 after model building, meanwhile, conjunctival fluorescein staining, conjunctival tiger red staining, phenol red cotton line test, tear film rupture time detection method and phenol red cotton line and tear film rupture time determination method are consistent with the method. The specific inspection method is as follows: (1) conjunctival fluorescein staining: dripping 1% fluorescein sodium 2 μ l into conjunctival sac, rinsing excessive fluorescein with 0.9% physiological saline after 5s, and observing conjunctival staining condition by cobalt blue diffusion under slit lamp microscope. Conjunctival fluorescent staining scoring criteria: the conjunctiva is divided into four quadrants, each quadrant being divided into four grades according to staining degree and area: 0 is no dyeing; 1, disperse spot dyeing; 2, dense point dyeing is carried out; 3 points are divided into flake dyeing and 12 points. (2) Dyeing conjunctival tiger red: dripping 1% tiger red 2 μ l into conjunctival sac, rinsing excessive tiger red with 0.9% physiological saline after 5s, and observing conjunctival staining condition with green light under slit lamp microscope. The conjunctival tiger red staining score standard was consistent with the conjunctival fluorescent staining standard. Taking the conjunctiva of the lesion area (modeling area) for histopathological examination.
2. Results of the experiment
The results of the ophthalmic examination tests showed (tables 4 to 6): d1 days after molding, compared with a negative control group, the wet length of the phenol red cotton thread of the model group and each drug group has no obvious difference and has no statistical significance (P >0.05), the tear film breaking time of the model group and each drug group is obviously shortened, compared with the negative control group, the model group has statistical significance (P <0.05 or 0.01), the fluorescence and tiger red staining score is obviously increased, and compared with the negative control group, the model group has statistical significance (P < 0.01); compared with the model group, the length of the phenol red cotton thread, the tear film rupture time, the fluorescence staining and the tiger red staining score of each drug group have no obvious difference, and have no statistical significance (P is more than 0.05). 5 days after the model is made, compared with a negative control group, the wetting length of the phenol red cotton thread of the model group and each medicine group is obviously shortened, the statistical significance is realized compared with the negative control group (P is less than 0.01), the tear film breaking time of the model group and each medicine group is obviously shortened, the statistical significance is realized compared with the negative control group (P is less than 0.01), the staining scores of the fluorescein sodium and the tiger red are obviously increased, and the statistical significance is realized compared with the negative control group (P is less than 0.01); compared with the model group, the length of the phenol red cotton thread, the tear film rupture time, the fluorescence staining and the tiger red staining score of each drug group have no obvious difference, and have no statistical significance (P is more than 0.05). At D10 days after molding, compared with the negative control group, the model group has the advantages that the wetting length of the phenol red cotton thread is reduced, the tear film breaking time is shortened, the staining scores of fluorescein and tiger red are obviously different from those of the negative control group, and the statistical significance is realized (P is less than 0.05 or 0.01). Compared with the model group, the low-dose group of the tripterygium wilfordii extract has no obvious difference in the wetting length of the phenol red cotton thread, the tear film breaking time, the fluorescein and the tiger red staining scores, and has no statistical difference (P > 0.05). The fluorescence and tiger red staining scores of the high-dose group and the triptolide group of the tripterygium wilfordii extracts are obviously reduced, the length of phenol red cotton threads is obviously increased, the tear film rupture time is obviously prolonged, and the statistical significance is realized compared with that of a model group (P is less than 0.05). The length of the benfusu phenol red cotton thread is obviously increased, the staining scores of fluorescein and tiger red are obviously reduced, and the benfusu phenol red cotton thread has statistical significance compared with the model group (P <0.05 or 0.01).
The results of the histopathological examination show that: the tissue structures of the left eye eyelid conjunctiva and the eyeball conjunctiva of each animal are basically normal, the epithelial layer goblet cells are rich and are arranged regularly, and other epithelial cells are not obviously damaged; the subepithelial tissue has no changes such as loose tissue arrangement, fibrous tissue hyperplasia, inflammatory cell infiltration and the like; no obvious expansion of blood vessels is observed; the eyelid conjunctiva of each model-making side animal can be damaged in different degrees, and the damage is mainly characterized in that goblet cells of the eyelid conjunctiva epithelial layer at the model-making part are obviously reduced or disappeared; other epithelial cells proliferate, increasing the number of cell layers; a small amount of or some fibrous tissue hyperplasia in the upper and lower skin with a small amount of inflammatory cell infiltration; blood vessels of fornix parts of animals expand, engorge and the like; compared with the model group, the high-dose group of the tripterygium wilfordii extract, the triptolide group and the positive control Behcet group experimental animal have the advantages that the symptom of the goblet cells of the palpebral conjunctiva epithelial layer of the animal is obviously reduced, the trend is slightly reduced, the difference among the groups is not obvious, and the reduction degree is not obvious; the positive control Befushu group has no obvious improvement on inflammatory reaction and vascular congestive; the degree of hyperplasia of eyelid conjunctival epithelial cells and proliferation change and lesion of a small amount of fibrous tissues is obvious in a positive control Befushu group, and the degree of other administration groups is slightly lower.
3. Conclusion of the experiment
The result shows that compared with the negative control eye, the model group animals after alkali burn injury have obviously shortened rupture time of right tear film and phenol red cotton thread wetting length, obviously increased fluorescein and tiger red staining scores, and local histopathology of molding injury shows that the eyelid conjunctiva of each model side animal can be damaged to different degrees, mainly showing that the epithelial layer goblet cells of the eyelid conjunctiva at the molding part are obviously reduced or disappeared. The results suggest that although the score of fluorescein and tiger red staining decreased gradually with the time after molding, the score was still significantly higher than that of the negative control group before the last treatment, and the tear film rupture time and the length of phenol red cotton thread wetting were also significantly shorter than that of the negative control group, indicating that the animal bulbar conjunctiva injury local self-recovery to some extent was obtained after the molding was stopped for 10 days, but the dry eye model still existed and the model was successfully replicated.
The high-dose group administration of the thunder god vine extract can obviously reduce the fluorescein and tiger red staining score value of the eyes of a model animal on the 10 th day, the moistening length of a phenol red cotton line and the tear film breaking time also have obvious improvement effect, and the histopathology research result also shows that the thunder god vine extract has certain improvement effect on the reduction of goblet cells and also has obvious improvement effect on congestion and edema of a conjunctival injury part and a conjunctival fornix part. Analysis of the tripterygium wilfordii extract to reduce the eye fluorescein and tiger red staining score value of model animals and improve the congestion and edema of conjunctival injury parts and conjunctival fornix parts is related to the anti-inflammatory effect of the tripterygium wilfordii extract.
The positive control product Baifushu product mainly contains recombinant bovine basic fibroblast growth factor (DFGF) and polyvinyl alcohol (PVA liquid membrane component). Bovine basic fibroblast growth factor has repair and regeneration promoting effects on cells derived from mesoderm and ectoderm. The results of the invention show that the Befushu can prolong the wet length of the phenol red cotton thread after being administrated for 10 days, reduce the dyeing score of fluorescein and tiger red, has certain improvement effect on the reduction of cup cells at the damaged part, but has no obvious improvement effect on the hyperemia and edema at the damaged part and conjunctiva fornix part, analyzes that the increase of the wet length of the phenol red cotton thread is related to the repair of the damaged part, and the Befushu has no anti-inflammatory effect, so that the invention has no improvement effect on the hyperemia and edema at the damaged part and conjunctiva fornix part, and is very consistent with the action mechanism of the Befushu.
Triptolide is one of the main active ingredients of tripterygium wilfordii extracts, belongs to diterpenoid compounds, has the activity mainly related to immunosuppression, is also one of the main toxic ingredients of tripterygium wilfordii, and has relatively weak anti-inflammatory effect. The results of theoretically analyzing that all the medicines capable of reducing local inflammation and edema of the eyes, increasing tear secretion and enhancing the stability of the tear film can improve the clinical symptoms of model animals, show that the triptolide eye drops have slightly weaker improvement effect on the alkali burn dry eye model than that of triptolide eye drops with equal dosage, and are probably related to other compounds which contain not only equal amount of triptolide in the triptolide extract, but also diterpenes, triterpenes and alkaloids in the triptolide extract.
In conclusion, the tripterygium wilfordii extract eye drops have obvious improvement effect on alkali burn New Zealand rabbit xerophthalmia models when the dosage is 400 mug/eye/day (1mg/ml, 100 mug/eye/time, 4 times/day), the improvement effect is mainly shown in that the tear secretion of model animals is increased, the tear film breaking time is prolonged, the conjunctival congestion and edema degree of the xerophthalmia models are reduced, the reduction condition of alkali burn local goblet cells is improved, and the improvement effect on the alkali burn models is stronger than that of the tripterygium wilfordii extract eye drops.
TABLE 4 evaluation results of phenol red cotton thread, tear film rupture time and staining on the first day of molding
In comparison to the negative control group, # means P <0.05, # means P < 0.01; compared to the model group, denotes P <0.05, denotes P < 0.01.
TABLE 5 phenol red cotton thread, tear film break-up time, staining score results table for fifth day of molding
In comparison to the negative control group, # means P <0.05, # means P < 0.01; compared to the model group, denotes P <0.05, denotes P < 0.01.
TABLE 6 phenol red cotton thread, tear film break-up time, staining score results table for 10 days of molding
In comparison to the negative control group, # means P <0.05, # means P < 0.01; compared to the model group, denotes P <0.05, denotes P < 0.01.
Experimental example 3 therapeutic action of Tripterygium wilfordii hook F extract eye drops on fungal keratitis
Selecting a new Zealand rabbit which is qualified in adaptability observation, carrying out ophthalmic examination (fluorescein sodium method) first, and eliminating animals with eye irritation symptoms, corneal defects and conjunctival injuries. 4 animals were randomly selected as a negative control group. Then, the remaining animals were subjected to third eyelid excision, and after excision, ofloxacin eye ointment was applied, and after 3 days of observation, ofloxacin eye drops were preventively applied to the eye for 3 days, 4 times a day, 1-2 drops each time. And molding after preventive eye dropping is finished. In the molding process, except for the negative control group, the other New Zealand rabbits are subjected to 3% of pentobarbital vein anesthesia and 2% of lidocaine eye-drop surface anesthesia, 7.0mm of trephine is drilled at the central indentation of the cornea of the experimental eye, a 15 ℃ corneal puncture knife is used for scraping the whole corneal epithelium within the cutting range, and then a microsyringe is used for intracorneal injection with the concentration of 1.5 multiplied by 10810 μ l of CFU/mL Fusarium solani, which was then covered with a contact lens and the air bubbles in the space between the cornea and the contact lens were squeezed out. Coating the conjunctival sac with ofloxacin eye ointment, intermittently suturing 2 needles of an eyelid by 3-0 silk threads to close the eyelid, removing eyelid suture after 48h of operation, removing a corneal contact lens, scoring the cornea, and dividing the animals into 4 groups according to the score, namely: model group, low and high dosage group of tripterygium wilfordii extract eye drops, dosage group of natamycin eye drops and half of male and female. The corresponding drugs were administered immediately after the grouping, 4 times/day, with an interval of about 2 hours between each administration, the eyelids were passively closed for at least 10s after each eye drop, and the negative control group and the model group were administered with 0.9% sodium chloride solution. The administration was continued for 7 days. The infection of the cornea of each group of rabbits is observed daily after the operation, and the slit lamp microscope examination and the photography are carried out respectively on the 3 rd, 5 th and 7 th days after the inoculation of the strain, and meanwhile, the examination is carried out by grouping forward scraping. Wherein, the scoring standard of the rabbit eye infection degree is scored according to the table 1 and is carried out on models 3, 5,Corneal scores were performed for 7 days. The grouped forward doctor blade inspection operation steps are as follows: scraping corneal tissue at the edge of the corneal ulcer area by using a sharp blade, staining Giemsa to observe the germ form, and determining the model building condition. At the end of the test, the animals were sacrificed by gas embolization, after the eye balls were removed, the entire cornea was cut off by an ophthalmological scissors extending the limbus, and the specimens were subjected to pathological examination.
TABLE 7 evaluation criteria for degree of infection in New Zealand Rabbit eyes
The experimental results are as follows:
the results of the ophthalmic observations show (tables 8-10): the model group and each drug group have obviously higher corneal opacity area score, corneal transparency score, corneal surface score and total score (corneal opacity area score + corneal transparency score + corneal surface score) and have statistical significance (P is less than 0.05 or 0.01) compared with the negative control group after Day1 and Day3 administration; compared with the model group, the indexes of each drug group are not obviously changed and have no statistical significance (P is more than 0.05). After Day5 administration, the corneal opacity area score, corneal transparency score, secretion score, and total score (corneal opacity area score + corneal transparency score + corneal surface score) were significantly increased in the model group and each drug group compared to the negative control group, and were statistically significant (P <0.05 or 0.01) compared to the negative control group. Compared with the model group, the cornea surface scores of the thunder god vine extract high-dose group and the positive control natamycin group are obviously reduced, and the statistic significance (P <0.01) is achieved compared with the model group, and the scores of other indexes are not obviously reduced and have no statistic significance (P >0.05) compared with the model group.
The histopathological research result shows that the cornea structure of each animal in the negative control group is not abnormal: the epithelial layer has complete structure, the epithelial cells are arranged regularly and have normal shape, and no hyperplasia or shedding is generated; the fibrous tissues of the stroma layer are arranged in parallel, no edema or inflammatory cell infiltration is seen, and no small blood vessels are seen in the limbal stroma. Compared with the negative control group, pathological changes of different degrees can be seen on the cornea of the bilateral eyeball of the animal after the model is made, and the pathological changes are mainly manifested by corneal epithelial cell hyperplasia, corneal stroma edema, stroma fibrous tissue arrangement disorder, stroma inflammation cell infiltration and corneal limbus neovascularization. The pathological changes are relieved in each administration group compared with the model group. Wherein, the action of the high-dose group of the tripterygium wilfordii extract on relieving the necrosis of fibrous tissues in the matrix is obviously higher than that of the natamycin group.
And (4) experimental conclusion:
the radix Tripterygii Wilfordii extract contains alkaloids, diterpenes, triterpenes and sesquiterpene compounds, wherein triptolide of diterpenes has strong immunosuppressive effect and is considered as one of the main active ingredients of radix Tripterygii Wilfordii and its preparation, and in addition, the radix Tripterygii Wilfordii extract eye drop also contains triterpenes, alkaloids and sesquiterpene compounds, and has anti-inflammatory effect. The results of the invention show that compared with the model group, the cornea surface score is obviously reduced on the 5 th day of administration of the thunder god vine extract high-dose group, and the histopathology also shows that after 5 days of administration, the corneal epithelial cell hyperplasia, corneal stroma edema, stroma fiber tissue arrangement disorder, stroma inflammation cell infiltration and corneal limbus neovascularization pathological changes of each administration group and the model group are all relieved. The natamycin can be used as an effective medicine for treating fungal keratitis, and can effectively prevent the occurrence of complications such as neovessels, iritis, pannus and the like. It is considered that the present model is a model of replication of fungi injected into the cornea, whose early phase is dominated by an acute inflammatory response, whereas natamycin has no significant anti-inflammatory effect and therefore no significant effect on the model.
In conclusion, the tripterygium wilfordii extract eye drops are judged to have obvious improvement effect on a fungal keratitis model when the dosage is 200 and 400 mu g/eye/day (1mg/ml, 50 mu l, 100 mu l/eye/time and 4 times/day), and the improvement effect is mainly shown to reduce the rabbit corneal surface score and inhibit the necrosis of fibrous tissues in corneal stroma and the proliferation of new blood vessels.
Table 8 results of the scoring of eye infections with fungal keratitis in new zealand rabbits for the eye drops of tripterygium wilfordii extracts (D1,
)
in comparison to the negative control group, # means P <0.05, # means P < 0.01; compared to the model group, denotes P <0.05, denotes P < 0.01.
Table 9 eye drops of tripterygium wilfordii extract scored eye irritation results for new zealand rabbit fungal keratitis (D3,
)
in comparison to the negative control group, # means P <0.05, # means P < 0.01; compared to the model group, denotes P <0.05, denotes P < 0.01.
Table 10 eye drops of tripterygium wilfordii extract scored eye irritation results for new zealand rabbit fungal keratitis (D5,
)
in comparison to the negative control group, # means P <0.05, # means P < 0.01; compared to the model group, denotes P <0.05, denotes P < 0.01.
Appropriate changes and modifications to the embodiments described above will become apparent to those skilled in the art from the disclosure and teachings of the foregoing description. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present invention should fall within the scope of the claims of the present invention. Furthermore, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
Claims (1)
1. The application of the tripterygium wilfordii extract in preparing eye drops for treating xerophthalmia; the preparation method of the tripterygium wilfordii extract comprises the following steps:
(1) taking tripterygium wilfordii decoction pieces, extracting with 70-95% ethanol, filtering, combining filtrates, recovering ethanol under reduced pressure until no ethanol smell exists, continuously concentrating to obtain an extract with the relative density of 1.15-1.35, adsorbing with neutral alumina, drying under reduced pressure, and crushing to obtain a tripterygium wilfordii ethanol extract neutral alumina mixed sample;
(2) adding chloroform into neutral aluminum oxide sample of radix Tripterygii Wilfordii ethanol extract, extracting, filtering to obtain filtrate, recovering chloroform, and drying to obtain radix Tripterygii Wilfordii extract.
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