US20160000847A1 - Method of using cistanche tubulosa extract for preparing drugs or food to protect eye cells - Google Patents
Method of using cistanche tubulosa extract for preparing drugs or food to protect eye cells Download PDFInfo
- Publication number
- US20160000847A1 US20160000847A1 US14/790,017 US201514790017A US2016000847A1 US 20160000847 A1 US20160000847 A1 US 20160000847A1 US 201514790017 A US201514790017 A US 201514790017A US 2016000847 A1 US2016000847 A1 US 2016000847A1
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- Prior art keywords
- cells
- extract
- macular degeneration
- drugs
- food
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/06—Antiglaucoma agents or miotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
Definitions
- This invention relates to a use of Cistanche tubulosa 's extract, especially relating to a use of preparing drugs or food for protecting eye cells.
- the eyes are the windows of the soul. The world can be seen through the eyes in an ultra-high resolution. Everyday life has to rely on the eyes.
- Current common eye diseases include age-related macular degeneration (AMD), diabetic retinopathy, and proliferative vitreoretinopathy (PVR), wherein the AMD is considered to be a critical eye disease to cause blindness in adults or elderly.
- AMD age-related macular degeneration
- PVR proliferative vitreoretinopathy
- Free radical will accumulate oxidative stress damages on DNA, proteins, lipids, and intercellular macromolecular materials and cause aging.
- some eye diseases especially macular degeneration and retinopathy
- This invention provides a method of using extract of Cistanche tubulosa for preparing drugs or food for protecting eye cells.
- the extract of Cistanche tubulosa comprises echinacoside, acteoside, isoacteoside, tubuloside A, or a combination thereof.
- the drugs or food are used to prevent, slow down, or treat eye diseases.
- the eye diseases are macular degeneration, macular hole, retinopathy, or glaucoma.
- the macular degeneration is age-related macular degeneration.
- the age-related macular degeneration is dry macular degeneration or wet macular degeneration.
- the retinopathy is diabetic retinopathy, retinitis pigmentosa, retina disease, retinal artery and vein occlusion, proliferative vitreoretinopathy, or central serous retinopathy.
- a form of the drugs is capsule, tablet, powder, or liquid.
- Cistanche tubulosa of the instant disclosure is used to prepare drugs or food having a function of protecting eye cells.
- FIGS. 1-5 are diagrams showing cytotoxicity testing results according to the instant disclosure the instant disclosure.
- FIGS. 6-10 are diagrams showing testing results of protective effect of the embodiments in the instant disclosure to H 2 O 2 -induced cytotoxicity.
- FIGS. 11-15 are diagrams showing testing results of protective effect of the embodiments in the instant disclosure to t-BHP-induced cytotoxicity.
- FIGS. 16-20 are diagrams showing testing results of protective effect of the embodiments in the instant disclosure to NaN 3 -induced cytotoxicity.
- FIGS. 21-25 are diagrams showing testing results of protective effect of the embodiments in the instant disclosure to blue light-induced cytotoxicity.
- This invention provides a method of using extract of Cistanche tubulosa for preparing drugs or food to protect eye cells.
- Cistanche tubulosa is also called “desert ginseng ” and is a type of Cistanche .
- Cistanche tubulosa is a drug used to invigorates the kidney and as an aphrodisiac. It can be used to treat impotence, weakness, sore waist and knee, and constipation.
- Cistanche tubulosa has been an entry in “Chinese Pharmacopoeia” since the 2005 edition. Cistanche has effects of anti-oxidation, protecting nerve cell (such as inhibiting neuronal apoptosis), promoting nerve growth factor secretion, regulating brain neurotransmitters, improving learning and memory, and reducing the generation of brain amyloid.
- the extract of Cistanche tubulosa comprises echinacoside, acteoside, isoacteoside, tubuloside A, or a combination thereof. All of the echinacoside, acteoside, isoacteoside, and tubuloside A have a protecting effect to the eye cells.
- the method of preparing drugs or food for protecting eye cells from the extract of Cistanche tubulosa comprises providing extract of Cistanche tubulosa and preparing drugs or food for protecting eye cells from the extract of Cistanche tubulosa .
- the method of preparing drugs or food for protecting eye cells from the extract of Cistanche tubulosa comprises extracting the extract from Cistanche tubulosa .
- the extracted components of Cistanche tubulosa is added with excipients and processed to form drugs or food.
- the extracted components of Cistanche tubulosa may be directly obtained and then added with excipients and processed to directly form drugs or food.
- the drugs and food prepared from extract of Cistanche tubulosa may comprise at least one of the four components described above, and optionally comprise other common excipients used in drugs or food.
- the drugs and food prepared from extract of Cistanche tubulosa may be in any forms, such as capsules, tablets, powder, or liquid.
- the drugs or food are used to prevent, slow down, or treat eye diseases.
- the eye diseases are macular degeneration, macular hole, retinopathy, or glaucoma.
- the macular degeneration is age-related macular degeneration.
- the age-related macular degeneration may be divided into dry macular degeneration and wet macular degeneration.
- the retinopathy is diabetic retinopathy, retinitis pigmentosa, retina disease, retinal artery and vein occlusion, proliferative vitreoretinopathy, or central serous retinopathy.
- the extract of Cistanche tubulosa is used to prepare food or drugs, wherein the components of the extract of Cistanche tubulosa comprise echinacoside, acteoside, isoacteoside, tubuloside A, or a combination thereof. These components can decrease the damage of the oxidative stress to the eye cells, and thus make the prepared drugs or food have protecting effects to the eye cells.
- the extract of Cistanche tubulosa may be further developed to drugs for slowing down the progress of eye diseases and especially may be used to treat the retinopathy diseases.
- Embodiment 1 of the instant disclosure is to prepare extract of Cistanche tubulosa .
- the method comprises the following steps.
- echinacoside was used in embodiment 2.
- Acteoside was used in embodiment 3.
- Isoacteoside was used in embodiment 4.
- the above materials were all purchased from ChromaDex, USA.
- Tubuloside A purchased from Tauto Biotech, Shanghai, China was used in embodiment 5.
- Retinal pigment epithelial was located between the retinal neuroepithelial layer and the choroid.
- RPE is in charge of many physiological functions, such as retinal barrier, phagocytosis, participating the visual cycle and metabolism, antioxidant function, and secreting growth factors, etc.
- RPE cells are susceptible to oxidative stress damages and then cell death causing retinopathy, visual dysfunction, and even loss of visual function. Therefore, RPE cells are usually used to study the cell model of eye-related diseases.
- age-related macular degeneration since the age-related macular degeneration is closely related to the oxidative stress of vascular endothelial growth factor (VEGF), deposition of lipofuscin (Delor ⁇ F C et al, 2001), chronic inflammation, and mutation of complement system, RPE cells are used in the study.
- VEGF vascular endothelial growth factor
- lipofuscin Desposition of lipofuscin
- CCM extra cellular matrix
- RPE cells purchased from American Type Culture Collection, abbreviated as ATCC
- ATCC American Type Culture Collection
- RPE cells were respectively placed in a 96-well plate. After placed in an incubator, the cells were cultured for 24 hours after treated by various concentrations of embodiments 1-5. Next, 1.0 mg/mL 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MITT) was added and reacted at 37° C. for 1 hour. Afterwards, 200 ⁇ L dimethyl sulfoxide (DMSO) was added into each well and shake in the dark at room temperature for 10 minutes after slight mixing.
- DMSO dimethyl sulfoxide
- an enzyme immunoassay analyzer (MRX microplate reader, USA) was used to detect the 550 nm absorbance of each well.
- the cells untreated by embodiments were used as a control group after culturing for 24 hours.
- One purpose of the example 1 is to test the cytotoxicity of the embodiments 1-5 to the RPE cells and the promotion of the embodiments 1-5 to the RPE cell growth.
- the mitochondrial enzyme of live cells can reduce the MTT to formazan of purple crystal dissolved by DMSO.
- the metabolic activity of cells is used to determine whether cells are alive or not.
- the measured dissolving absorbance is equal to the cell viability. Therefore, the more the cells alive, the higher the absorbance.
- the calculating formula of the cell viability was shown below.
- Cell viability(%) (light absorbance after cells treated by drugs for 24 hours/light absorbance of the control group after 24 hours) ⁇ 100%
- the results of the example 1 was expressed by the mean standard error of the number of experiments (mean ⁇ S.E.). The statistical analysis was performed by Student's t-test or one way ANOVA. If P ⁇ 0.05, it indicates a significant difference.
- FIGS. 1-5 are diagrams showing cytotoxicity testing results according to the instant disclosure respectively. From FIGS. 1-5 , IC 25 , IC 50 and IC 75 of embodiments 1-5 are listed, and the results are summarized in Table 1 below.
- the IC 50 of embodiments 1-5 is sorted by values, which shows (embodiment 5, embodiment 1)>embodiment 2>(embodiment 3, embodiment 4).
- the testing concentration used in the subsequent example 2 was determined by the cytotoxicity test result of example 1.
- cytotoxicity test result of example 1 three concentrations of embodiments 1-5 were used to test the damage of oxidative stress on the protective effect in example 2.
- the IC 50 of embodiments 1-5 were used as the highest concentrations in example 2 to reaffirm the action range of embodiments 1-5 to be toxic or to be a drug.
- the oxidative stress inducer used in example 2 includes hydrogen peroxide (H 2 O 2 , concentration 0.01-10 mM), tert-butyl hydroxyperoxide (t-BHP, concentration 0.01-10 mM), sodium azide (NaN 3 , concentration 0.01-100 mM), and blue light-induced damage.
- the blue light having a wavelength of 480 nm and an illuminance of 350 lux was emitted by a blue light-emitting-diode (LED).
- a blue light-emitting-diode LED
- 0.5 mg/mL of MTT was added and reacted at 37° C. for 2 hours.
- 200 ⁇ L DMSO was added to each well. After slight mixing, the plate was vibrated in the dark for 10 minutes. After completely dissolving the residual cells, the enzyme immunoassay analyzer was used to detect the absorbance of each well.
- the cells untreated by oxidative stress inducers and embodiments were used as the control group.
- the cell viabilities relative to the control group was calculated by the cell viability calculating formula described in the example 1.
- results of the example 2 were expressed by the mean standard error of the number of experiments (mean ⁇ S.E.). The statistical analysis was performed by Student's t-test or one way ANOVA. If P ⁇ 0.05, it indicates a significant difference.
- Free radical is a molecule having an unpaired electron, and the number thereof is an odd number.
- the free radical may be oxygen free radical, superoxide anion radical (O 2 ⁇ ), hydroxyl radical (OH ⁇ ), and an intermediate, i.e. hydrogen peroxide (H 2 O 2 ), for example.
- these free radicals are considered to be generated after local ischemia and reperfusion.
- These active oxygen species are toxic to cells by attacking fatty acids to form peroxides of lipids and thus is toxic to the cells and react with proteins to damage and oxidize the amino acids, oxidize the sulfhydryl groups, and break the polypeptide chains.
- t-BHP is an organic hydroperoxide, and can be metabolized by glutathione peroxidase and oxidized glutathione (GSSG) in liver cells. 2. The exhaustion of glutathione (GSH) and the oxidation of NADPH is related to the constant of calcium ions and causes the cell viability being reduced. 3. In addition, t-BHP can be converted to peroxide or alkoxyl radicals by cytochrome P450 enzyme and free iron-dependent reaction. 4. Theses radicals may induce peroxidation of lipids and form covalent bonds with cellular molecules, such as DNAs and proteins, to further reduce the GSH content.
- NaN 3 NaN 3 is usually used to induce chemically induced mode of ischemia in cells and in vivo experiments. The exact mechanism is unclear. 2. The above effect is considered to be caused by NaN 3 inhibiting the cytochrome c oxidase (or known as respiratory chain complex IV) and blocks the electron transport chain to form peroxides. Blue 1. Prolonged exposure to light may lead to age-related macular light degeneration. The oxidative stress induced by light may be the key factor of RPE cell degeneration. 2.
- Retina is protected from the UV injury by the cornea and crystalline lens absorbing UV having a wavelength below 400 nm, but retina may be damaged by visible light. 3.
- the blue region of spectrum (wavelength 400-500 nm) has relatively higher energy and may penetrate the tissue to the cells and the organelles. In particularly, it has been known that the blue light will damage the tissue of retina.
- Example 2 used embodiments 1-5 to respectively treat the RPE cells, and then used these oxidative stress inducers to treat the RPE cells.
- the protective effect of the embodiments 1-5 to the RPE cells was evaluated by the viability of the RPE cells.
- FIGS. 6-10 are diagrams showing testing results of protective effect of embodiments 1-5 to H 2 O 2 -induced cytotoxicity.
- the concentrations of embodiment 1 were 50, 100, and 200 ⁇ g/mL.
- the concentrations of embodiment 2 were 18.75, 37.5, and 75 ⁇ g/mL.
- the concentrations of embodiment 3 were 15.625, 31.25, and 62.5 ⁇ g/mL.
- the concentrations of embodiment 4 were 15.625, 31.25, and 62.5 ⁇ g/mL.
- the concentrations of embodiment 5 were 50, 100, and 200 ⁇ g/mL.
- the testing results showed that 50% of RPE cells were dead after treated by 0.1 mM H 2 O 2 , i.e. the viability was about 50%.
- the cells may be protected by the embodiment 1, embodiment 2, embodiment 3, and embodiment 5 from the damage made at this concentration.
- the embodiment 1 had the strongest protection effect, and the cell viability was increased from 50% to 100% when the embodiment 1 was at concentrations of 50 ⁇ g/mL and 100 ⁇ g/mL.
- the concentration of the embodiment 1 was 200 ⁇ g/mL, reasonable cytotoxicity response was observed since the concentration was at the IC 50 concentration.
- the test results shown in FIGS. 6-10 also proved that the most effective action concentration range of the embodiment 1 to H 2 O 2 was between 50 ⁇ g/mL and 100 ⁇ g/mL.
- FIGS. 11-15 are diagrams showing testing results of protective effect of the embodiments 1-5 to t-BHP-induced cytotoxicity.
- the concentrations of embodiment 1 were 50, 100, and 200 ⁇ g/mL.
- the concentrations of embodiment 2 were 18.75, 37.5, and 75 ⁇ g/mL.
- the concentrations of embodiment 3 were 15.625, 31.25, and 62.5 ⁇ g/mL.
- the concentrations of embodiment 4 were 15.625, 31.25, and 62.5 ⁇ g/mL.
- the concentrations of embodiment 5 were 50, 100, and 200 ⁇ g/mL.
- the testing results showed that 80% of RPE cells were dead after treated by 0.3 mM t-BHP, i.e. the viability was about 20%.
- the cells may be protected by all of the embodiments 1-5 from the damage made at this concentration.
- the embodiments 1, 2, and 3 had the strongest protection effect, and the cell viability was increased from 20% to above 80% when the embodiments 1 2, and 3 were at low and medium concentrations.
- embodiments 1 2, and 3 were at high concentrations, reasonable cytotoxicity responses were observed since the high concentrations were at the IC 50 concentrations.
- the test results shown in FIGS. 11-15 also proved that the most effective action concentration ranges of the embodiments 1, 2, and 3 to t-BHP was between low and medium concentrations.
- FIGS. 16-20 are diagrams showing testing results of protective effect of the embodiments 1-5 to NaN 3 -induced cytotoxicity.
- the concentrations of embodiment 1 were 75, 100, and 125 ⁇ g/mL.
- the concentrations of embodiment 2 were 25, 37.5, and 50 ⁇ g/mL.
- the concentrations of embodiment 3 were 15.625, 31.25, and 62.5 ⁇ g/mL.
- the concentrations of embodiment 4 were 15.625, 31.25, and 62.5 ⁇ g/mL.
- the concentrations of embodiment 5 were 75, 100, and 125 ⁇ g/mL.
- the testing results showed that 50% of RPE cells were dead after treated by 10 mM NaN 3 , i.e. the viability was about 50%.
- the cells may be protected by all of the embodiments 1-5 from the damage made at this concentration. Especially, the embodiments 1, 4, and 5 had the strongest protection effect, and the cell viability was increased to almost 100% when the embodiments 1, 4, and 5 were at medium and high concentrations. The embodiments 2 and 3 come next. The cell viability was increased from 50% to above 80%. Moreover, the high concentrations of the embodiments 1-5 under the damage of NaN 3 did not show serious toxicity. This result showed that the IC 50 might fall into the range of the drug effect but did not show dose-dependent relationship. The test results shown in FIGS. 16-20 proved that the most effective action concentration range of the embodiments 1-5 to NaN 3 was between low and medium concentrations.
- FIGS. 21-25 are diagrams showing testing results of protective effect of the embodiments 1-5 to blue light irradiation-induced cytotoxicity.
- the concentrations of embodiment 1 were 50, 100, and 200 ⁇ g/mL.
- the concentrations of embodiment 2 were 18.75, 37.5, and 75 ⁇ g/mL.
- the concentrations of embodiment 3 were 15.625, 31.25, and 62.5 ⁇ g/mL.
- the concentrations of embodiment 4 were 15.625, 31.25, and 62.5 ⁇ g/mL.
- the concentrations of embodiment 5 were 50, 100, and 200 ⁇ g/mL.
- the test results showed that 40% RPE cells were dead after treated by irradiating with blue light (480 nm, 30 lux) for 5 days, i.e.
- the viability was about 60%.
- the cells may be protected by all of the embodiments 1-5 from the damage made at this concentration.
- the embodiments 3 and 4 had the strongest protection effect, and the cell viability was increased from 60% to above 90%-100% when the embodiments 3 and 4 were at medium concentrations.
- embodiments 1-5 were at high concentrations, reasonable cytotoxicity responses were observed since the high concentrations were at the IC 50 concentrations.
- the test results shown in FIGS. 21-25 also proved that the most effective action concentration ranges of the embodiments 3 and 4 to blue light irradiation was between low and medium concentrations.
- the instant disclosure provides a method of using extract of Cistanche tubulosa for preparing drugs or food to protect eye cells.
- the extract of Cistanche tubulosa can decrease the damage of the oxidative stress to the eye cells. Therefore, the drugs and food prepared from the extract of Cistanche tubulosa have good protective effect to the eye cells and can be further developed to the drugs and food of preventing eye diseases or slow downing the progress of eye disease.
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US15/854,722 US10967029B2 (en) | 2014-07-03 | 2017-12-26 | Method of using Cistanche tubulosa extract to prevent, slow down, or treat an eye disease caused by oxidative stress |
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TWI830221B (zh) * | 2021-05-06 | 2024-01-21 | 杏國新藥股份有限公司 | 緩解乾眼症的方法 |
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2015
- 2015-07-01 TW TW104121352A patent/TWI650131B/zh active
- 2015-07-02 US US14/790,017 patent/US20160000847A1/en not_active Abandoned
- 2015-07-03 KR KR1020177002601A patent/KR20170039160A/ko active Search and Examination
- 2015-07-03 MA MA040369A patent/MA40369A/fr unknown
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KR20170039160A (ko) | 2017-04-10 |
EP3181143B1 (fr) | 2020-09-23 |
US20180117109A1 (en) | 2018-05-03 |
TWI650131B (zh) | 2019-02-11 |
MY192535A (en) | 2022-08-26 |
CN112870258A (zh) | 2021-06-01 |
US10967029B2 (en) | 2021-04-06 |
EP3181143A4 (fr) | 2018-05-16 |
EP3181143A1 (fr) | 2017-06-21 |
RU2017102407A (ru) | 2018-08-03 |
CN105232640A (zh) | 2016-01-13 |
JP2017531614A (ja) | 2017-10-26 |
RU2688939C2 (ru) | 2019-05-23 |
MA40369A (fr) | 2017-06-21 |
JP6573961B2 (ja) | 2019-09-11 |
TW201601740A (zh) | 2016-01-16 |
EP3181143B8 (fr) | 2020-11-18 |
RU2017102407A3 (fr) | 2018-08-03 |
WO2016000663A1 (fr) | 2016-01-07 |
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