CN108669531A - A kind of HERBA DENDROBII powder and its application - Google Patents
A kind of HERBA DENDROBII powder and its application Download PDFInfo
- Publication number
- CN108669531A CN108669531A CN201810271222.5A CN201810271222A CN108669531A CN 108669531 A CN108669531 A CN 108669531A CN 201810271222 A CN201810271222 A CN 201810271222A CN 108669531 A CN108669531 A CN 108669531A
- Authority
- CN
- China
- Prior art keywords
- herba dendrobii
- powder
- dendrobine
- plasma
- uplc
- Prior art date
- 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.)
- Pending
Links
- 239000000843 powder Substances 0.000 title claims abstract description 62
- 239000003814 drug Substances 0.000 claims description 23
- 229940079593 drug Drugs 0.000 claims description 15
- 230000001437 anti-cataract Effects 0.000 claims description 4
- 230000000259 anti-tumor effect Effects 0.000 claims description 4
- 239000003963 antioxidant agent Substances 0.000 claims description 4
- 230000003078 antioxidant effect Effects 0.000 claims description 4
- 235000006708 antioxidants Nutrition 0.000 claims description 4
- 230000006907 apoptotic process Effects 0.000 claims description 4
- 235000013305 food Nutrition 0.000 claims description 4
- 208000024827 Alzheimer disease Diseases 0.000 claims description 3
- 206010039966 Senile dementia Diseases 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- RYAHJFGVOCZDEI-UFFNCVEVSA-N Dendrobine Chemical compound C([C@H]1CC[C@@H]2[C@@]31C)N(C)[C@@H]3[C@H]1[C@@H](C(C)C)[C@@H]2C(=O)O1 RYAHJFGVOCZDEI-UFFNCVEVSA-N 0.000 abstract description 36
- RYAHJFGVOCZDEI-CZKZLRAZSA-N dendrobine Natural products O=C1O[C@@H]2[C@H](C(C)C)[C@H]1[C@H]1[C@@]3(C)[C@@H]2N(C)C[C@H]3CC1 RYAHJFGVOCZDEI-CZKZLRAZSA-N 0.000 abstract description 36
- 210000002381 plasma Anatomy 0.000 abstract description 36
- 241000700159 Rattus Species 0.000 abstract description 21
- 238000000034 method Methods 0.000 abstract description 20
- 230000000694 effects Effects 0.000 abstract description 19
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 abstract description 14
- 238000010521 absorption reaction Methods 0.000 abstract description 10
- 238000002474 experimental method Methods 0.000 abstract description 7
- 238000001727 in vivo Methods 0.000 abstract description 6
- 239000002245 particle Substances 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 4
- 229940126678 chinese medicines Drugs 0.000 abstract description 3
- 238000004704 ultra performance liquid chromatography Methods 0.000 abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 27
- 240000004638 Dendrobium nobile Species 0.000 description 10
- 239000011159 matrix material Substances 0.000 description 10
- 239000012224 working solution Substances 0.000 description 10
- 239000003513 alkali Substances 0.000 description 9
- 239000013558 reference substance Substances 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 238000011835 investigation Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 229930013930 alkaloid Natural products 0.000 description 5
- 150000003797 alkaloid derivatives Chemical class 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000004364 calculation method Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 238000003908 quality control method Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000002218 hypoglycaemic effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000003595 mist Substances 0.000 description 3
- 238000003305 oral gavage Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 230000000202 analgesic effect Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- KWGRBVOPPLSCSI-UHFFFAOYSA-N d-ephedrine Natural products CNC(C)C(O)C1=CC=CC=C1 KWGRBVOPPLSCSI-UHFFFAOYSA-N 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 238000003304 gavage Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000036470 plasma concentration Effects 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000003672 processing method Methods 0.000 description 2
- 229960003447 pseudoephedrine hydrochloride Drugs 0.000 description 2
- BALXUFOVQVENIU-KXNXZCPBSA-N pseudoephedrine hydrochloride Chemical compound [H+].[Cl-].CN[C@@H](C)[C@@H](O)C1=CC=CC=C1 BALXUFOVQVENIU-KXNXZCPBSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000004575 stone Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- 241000208140 Acer Species 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- MAEQVSBPCIMMJF-UHFFFAOYSA-N Nobilonin Natural products CC(C)C1C2C(=O)C3(C)C(CN(C)C)CCC3C1C(=O)O2 MAEQVSBPCIMMJF-UHFFFAOYSA-N 0.000 description 1
- MAEQVSBPCIMMJF-MSZYXCAISA-N Nobilonine Natural products O=C1[C@@]2(C)[C@@H](CN(C)C)CC[C@H]2[C@H]2[C@H](C(C)C)[C@H]1OC2=O MAEQVSBPCIMMJF-MSZYXCAISA-N 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000000151 deposition Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000004438 eyesight Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000009832 plasma treatment Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000007864 suspending Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
-
- 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)
- A61K36/898—Orchidaceae (Orchid family)
- A61K36/8984—Dendrobium
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- 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
- A61P27/12—Ophthalmic agents for cataracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Diabetes (AREA)
- Epidemiology (AREA)
- Ophthalmology & Optometry (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- Nutrition Science (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Obesity (AREA)
- Hospice & Palliative Care (AREA)
- Endocrinology (AREA)
- Emergency Medicine (AREA)
- Psychiatry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
This application discloses a kind of HERBA DENDROBII powder, the grain size of the HERBA DENDROBII powder is 150~180 μm.It selects dendrobine as detection object, the content of dendrobine in rat plasma is detected using ultra performance liquid chromatography mass spectrometry (UPLC MS) technology, quantitatively evaluate the absorbing state of the HERBA DENDROBII powder of different-grain diameter in vivo.In conjunction with pharmacokinetic methods of the HERBA DENDROBII in rat body, the content of dendrobine in the blood plasma measured by UPLC MS, experiment finds that SD Oral Administration in Rats administration HERBA DENDROBII fine powder (150~180 μm) 0.8g/kg is compared with HERBA DENDROBII Ultramicro-powder (45 μm) 0.8g/kg, absorptivity of the HERBA DENDROBII fine powder in rat body is high, good absorption effect, soak time are short.It follows that different with other Chinese medicines, the powder particle diameter of HERBA DENDROBII powder is not the smaller the better, and the assimilation effect of fine powder (150 μm~180 μm) in vivo is much better than Ultramicro-powder (45 μm).
Description
Technical field
The invention belongs to Chinese medicine preparing technical fields, and in particular to a kind of HERBA DENDROBII and its application.
Background technology
The Chinese medicine stem of noble dendrobium has the effects that nourishing Yin and clearing heat, reinforcing stomach reg fluid, moistens the lung and relieve the cough, improving eyesight salubrity, the traditional Chinese medicines in 2015 editions
Clearly stipulate that one of the Source of Drug Plants of the stem of noble dendrobium is HERBA DENDROBII (DendrobiumnobileLindl.) in allusion quotation, it is based on stone
The drug action of dry measure used in former times, market demand are growing, and it is existing for how preferably developing and using HERBA DENDROBII this medicine resource
In one of study of pharmacy emphasis.HERBA DENDROBII on existing market mainly carries out primary product sale with fresh item, maple bucket.Secondary production
In the processing of product, the powder particle diameter degree of HERBA DENDROBII dry product does not provide explicitly, and the powder of what specific grain size can be preferably
In absorption of human body and distribution, also there are no relevant reported literatures.
In the production and preparation process of Chinese medicine preparation, it is necessary to carry out Crushing of Ultrafine to raw medicinal material, can be only achieved preparation work
Skill requirement.In general, in the preparation containing micro-powder of Chinese herbs, micro mist is thinner, and quality is higher.Micro mist generally refers to grain size 1
The powder of~75 μ ms, micro mist average grain diameter are generally less than 75 μm (traditional Chinese medicine beats powder at 100 μm or more).In this fineness
Under, 90% or more plant cell is broken, and is conducive to the absorption of drug and the raising of curative effect, is conducive to the release and suction of drug
Receipts drug-eluting rate is directly proportional to surface area (S) of drug, and the drug of equivalent, particle is smaller, and S is bigger, is more conducive to drug
Dissolution and absorption.The powder very little of micron Chinese medicine, cell-wall breaking ratio is high, active ingredient just exposure before not entering into body
Out, into after in vivo, soluble component dissolves rapidly, because of powder very little solubility low ingredient, has larger
Adhesive force and be firmly adhered on mucous membrane, thus be conducive to the absorption of drug.However, for HERBA DENDROBII, if crush journey
Degree is higher, and the bioavilability of drug is higherThe present invention is ground from the pharmacokinetics of the HERBA DENDROBII powder of different-grain diameter
Study carefully and set out, it was demonstrated that the absorbing state of the powder of clear grain size determines the powder diameter of optimum utilization.
Invention content
Smaller for the Chinese medicine grain size of the prior art, the better technology prejudice of assimilation effect, the present invention proposes one kind
HERBA DENDROBII fine powder more better than HERBA DENDROBII Ultramicro-powder assimilation effect.
The grain size of a kind of HERBA DENDROBII powder, the HERBA DENDROBII powder is 150~180 μm.
The working principle and beneficial effect of the present invention：HERBA DENDROBII chemical composition is various, wherein HERBA DENDROBII total alkaloid
It is main active material, there is hypoglycemic, antitumor, anti-oxidant, anti-senile dementia, inhibit Apoptosis, anti-cataract, solution
The modern pharmacologies effect such as heat, analgesic, removing toxic substances.HERBA DENDROBII total alkaloid is mainly sesquiterpenoids alkaloid, isolated at present
Dendrobine, stem of noble dendrobium ammonia alkali, nobilonine etc. amount to 14 kinds.Wherein, dendrobine (dendrobine, C16H25O2N) it is HERBA DENDROBII
Most important characteristic chemical constituent in total alkaloid, dendrobine account for 92% or more of HERBA DENDROBII total alkaloid content, chemical constitution
As follows.
Therefore, it selects dendrobine as detection object, uses ultra performance liquid chromatography-mass spectrometry (UPLC-MS) technology
The content that can quick, sensitive, accurately detect dendrobine in rat plasma, quantitatively evaluates the HERBA DENDROBII powder of different-grain diameter
The absorbing state of end in vivo.In conjunction with pharmacokinetic methods of the HERBA DENDROBII in rat body, measured by UPLC-MS
Blood plasma in dendrobine content, the absorption efficiency of the HERBA DENDROBII powder of different-grain diameter in vivo can be quantitatively determined, in turn
Determine the degree of grinding of HERBA DENDROBII.It is found through experiments that HERBA DENDROBII fine powder (150~180 μm) 0.8g/ is administered in SD Oral Administration in Rats
Kg investigates its Pharmacokinetic Characteristics by index of dendrobine, and stem of noble dendrobium alkali concentration average out to peak time is 11.71min in blood plasma,
Maximum plasma concentration average value is 212.17 μ g/L, mean half-life 351.95min, area under the drug-time curve AUC (0- ∞)
For 17792.49 μ gmin/L, same procedure is administered HERBA DENDROBII Ultramicro-powder (45 μm) 0.8g/kg, stem of noble dendrobium alkali concentration in blood plasma
Average peak time is 27.50min, and maximum plasma concentration average value is 42.58 μ g/L, mean half-life 333.55min, medicine
When area under the curve AUC (0- ∞) be 372.61 μ gmin/L.From the point of view of the above experimental result, compared to HERBA DENDROBII ultra micro
Powder, absorptivity of the HERBA DENDROBII fine powder in rat body is high, good absorption effect, and soak time is short.
It follows that different with other Chinese medicines, the powder particle diameter of HERBA DENDROBII powder is not the smaller the better, fine powder (150 μm~
180 μm) assimilation effect in vivo is much better than Ultramicro-powder (45 μm).
The application of HERBA DENDROBII powder, the HERBA DENDROBII powder is oral or injects.
It directly uses HERBA DENDROBII powder or is mixed with some other substance and solution and be used as oral agents and injection.
Preferably, the application of HERBA DENDROBII powder, the HERBA DENDROBII powder is hypoglycemic, antitumor, anti-oxidant, anti-ageing
Dementia inhibits the Apoptosis either food of anti-cataract or the application in drug.In HERBA DENDROBII powder it is main at
It is divided into dendrobine, dendrobine has hypoglycemic, antitumor, anti-oxidant, anti-senile dementia, inhibits Apoptosis, anti-cataract, solution
The modern pharmacologies effect such as heat, analgesic, removing toxic substances, using the HERBA DENDROBII fine powder of the present invention, therapeutic effect is more preferable.
Description of the drawings
Fig. 1 is the selection ion figure of dendrobine and internal standard compound in rat plasma；Wherein A：Blank plasma；B：Blood plasma sample
Product；1：Dendrobine；2：Internal standard.
Fig. 2 is rat oral gavage HERBA DENDROBII fine powder (0.8g/kg) dendrobine mean blood plasma concentration-time graph (n=7) afterwards
Figure.
Specific implementation mode
The present invention is further explained with reference to specific embodiment, but specific embodiment is not to the present invention
It is limited in any way.Unless stated otherwise, reagent involved in embodiment, method are reagent and method commonly used in the art.
1 materials and methods
1.1 instrument
Thermo Scientific Q Exactive quadrupole rods-electrostatic field orbit trap high resolution mass spectrum system, Thermo
3000 ultra performance liquid chromatography systems of Ultrmate (Thermo Fisher companies of the U.S.), 1022 liquid nitrogen generators of Genius
(PEAK companies of Britain), Milli-Q ultrapure water systems (Millipore companies of the U.S.), XS205 electronic balances (Switzerland METTLER
TOLEDO companies), XW-80A whirlpools mixed instrument (its woods Bell's instrument manufacturing Co., Ltd of Haimen City), Microfuge 20R high
Fast centrifuge (Beckman Coulter Inc. of the U.S.), STW-FT24 ice machines (SANYO GS company).
1.2 experimental animal
SPF grades of male and healthy SD rats 14 (production quality certification number：SCXK (Chongqing) 2012-0005), by Chongqing third army
Medical university's Experimental Animal Center provides, weight 200-240g, 21-22 DEG C of raising temperature, humidity 56%-65%, light and shade alt time
12/12h.This experiment meets the ethical standard of experimental animal Ethics Committee of Zunyi Medical College.
1.3 drugs and reagent
HERBA DENDROBII medicinal material (lot number：ZCXT20160503 it) is purchased from Crack In Guizhou Chishui city letter day Chinese Medicine Industry and develops limited public affairs
Department.Appropriate dry pulverizing medicinal materials are taken, until all sieved by No. five, and at least 95% is sieved by No. six to get fine powder (150-180 μ
m)；All sieved to get Ultramicro-powder (45 μm) by 300 mesh.
Dendrobine (lot number：D990100), be purchased from Beijing Hua Maike biotechnologys Co., Ltd, mass fraction >=
99.0%.Pseudoephedrine hydrochloride (lot number：171237-201208), be purchased from National Institute for Food and Drugs Control, mass fraction >=
99.9%.Acetonitrile, methanol, formic acid are mass spectrum grade, are purchased from Sigma-Aldrich chemicals Co., Ltd.Other reagents are equal
It is pure to analyze, it is purchased from Chinese medicines group chemical reagents corporation.
1.4 method
1.4.1 the analysis condition of UPLC-MS
Chromatographic condition：Using Hypersil Gold C18 (150 × 2.1mm, 1.9 μm) chromatography post separation；Flow phase composition
For -0.1% aqueous formic acid (B) of acetonitrile (A), condition of gradient elution 0-0.5min, 5%A；0.5-15min, 5%-95%A；
15-17min, 95%A；Volume flow 0.3mL/min；40 DEG C of column temperature；2 μ L of sample size.
Mass Spectrometry Conditions：Using HESI Ionization modes；Atomization temperature is 300 DEG C, and ion transfer tube temperature is 350 DEG C；Sheath
Gas velocity is 35arb, and secondary air speed is 15arb；Spray voltage 3.5kV；M/z detection ranges 100-1500；Scan pattern is
Full Scan/ cations scan；Resolution ratio uses MS Full Scan 70000FWHM.
1.4.2 the preparation of reference substance and internal standard working solution
Precision weighs dendrobine reference substance about 15mg, is placed in 10mL volumetric flasks, adds acetonitrile to dissolve and is diluted to graduation mark,
Mixing saves backup to get reference substance storing solution in 4 DEG C.Wait for that used time precision absorption reference substance storing solution is appropriate, it is dilute with acetonitrile
It releases to get reference substance working solution.
Precision weighs pseudoephedrine hydrochloride reference substance (IS) about 10mg, is placed in 10mL volumetric flasks, add acetonitrile dissolve and it is dilute
It releases to graduation mark, mixing saves backup to get internal standard storing solution in 4 DEG C.Wait for that used time precision absorption internal standard storing solution is appropriate, with
Dilution in acetonitrile is to get internal standard working solution, for use.
1.4.3 preparation and the processing method of Standard plasma samples
It tests same day precision and draws 100 μ L of blank plasma, the 20 μ L of dendrobine reference substance working solution of various concentration, whirlpool is added
30s is revolved, sequentially add 20 μ L of internal standard working solution and acetonitrile 200 μ L, vortex 30s is respectively to get alkali mass concentration containing the stem of noble dendrobium
912.94, the Standard plasma samples of 456.47,91.294,45.647,9.1294ng/mL and 3.04ng/mL.13000rpm is centrifuged
10min takes 50 μ L of supernatant to carry out UPLC-MS analyses.Be wherein height with mass concentration 912.94,91.294,9.1294ng/mL,
In, low concentration Quality Control plasma sample, mass concentration 3.04ng/mL be lower limit of quantitation (LLOQ) Standard plasma samples, parallel system
It is 5 parts standby.
1.4.4 the processing method of plasma sample
Precision draw 100 μ L of plasma sample, be added acetonitrile 20 μ L, vortex 30s, sequentially add 20 μ L of internal standard working solution and
200 μ L of acetonitrile, vortex 30s, 13000rpm centrifugation 10min, take 50 μ L of supernatant to set in sample injection bottle, pending UPLC-MS analyses.
1.4.5 the research of Pharmacokinetics in Rat
12h is deprived of food but not water before 14 rat experiments, and free water during experiment is fed after testing 12h.By 0.8g/kg
Dosage gavage gives HERBA DENDROBII fine powder (0.5%CMC-Na suspendings), 0 after administration, 2,6,10,15,20,30,40,
60,90,120,180,240,480,720,1440min takes a blood sample about 0.2mL through rat orbital venous plexus, sets sodium heparinized centrifugation
Guan Zhong, 4500rpm centrifuge 15min, and separated plasma is to be measured.Using biostatistics software DAS2.1, after rat oral gavage is administered
When m- plasma drug concentration data carry out non-compartment model (statistics moments method) and calculate.
2 results
The specificity of 2.1 methods
The rat blank plasma of 14 separate sources is taken, it is accurate respectively to draw each 100 μ L of blank plasma, 200 μ L second are added
Nitrile, after vortex 30s, 13000rpm centrifuges 10min, respectively takes investigation uses of the 50 μ L of supernatant for method specificity.Take 14 differences
Blood plasma after the rat administration in source is handled to obtain plasma sample, be analyzed with UPLC-MS, as a result by 1.4.4 lower methods
See Fig. 1.Measurement result shows that ingredient dendrobine to be measured and interior target retention time are respectively 7.76,5.96min, blank plasma
No appearance at the corresponding time shows that the plasma treatment process and determination condition have specificity, the endogenous object in blood plasma
Matter does not interfere the Accurate Determining of dendrobine.
2.2 linear and lower limit of quantitation investigations
By Standard plasma samples made from 1.4.3 lower section methods, after 1.4.1 lower condition analysis, data are using weighting
(1/X2) least square method carries out regressing calculation, with the ratio between dendrobine and interior target peak area (Y) corresponding stem of noble dendrobium alkali mass concentration
(X) linear regression equation acquired, Y=-0.6758+0.1393X (r=0.9988).The result shows that in 912.94-
Within the scope of 9.1294ng/mL, the ratio of stem of noble dendrobium alkali mass concentration and peak area is in good linear relationship in blood plasma.The stem of noble dendrobium in blood plasma
The LLOQ of alkali is 3.04ng/mL, and S/N rate (S/N) is more than 10.
The investigation of 2.3 matrix effects
Precision draws 30 parts of each 100 μ L of the different blank plasma in source, and 200 μ L acetonitriles, vortex 30s, 13000rpm is added
Centrifuge 10min.It is accurate respectively to draw 150 μ L, 10 μ L of reference substance working solution are added, the blood plasma of basic, normal, high stem of noble dendrobium alkali concentration is made
Matrix sample.10 μ L of internal standard working solution are added, vortex 30s, 13000rpm centrifuge 10min, and 50 μ L of supernatant is taken to carry out UPLC-MS
Analysis, dendrobine and internal standard peak area are denoted as A1 and B1 respectively.Precision draws 30 part of 100 μ L water, and 200 μ L acetonitriles are added, and is vortexed
30s, 13000rpm centrifuge 10min, take 150 μ L of supernatant respectively, and 10 μ L of reference substance working solution are added, basic, normal, high dendrobine is made
Concentration without matrix sample.Add 10 μ L of internal standard working solution, vortex 30s, 13000rpm centrifuge 10min, take 50 μ L of supernatant into
Row UPLC-MS analyses, dendrobine and internal standard peak area are denoted as A2 and B2 respectively.Dendrobine matrix effect (matrix effect,
ME it) is calculated by formula ME=A1/A2 × 100%, based on interior target matrix effect is carried out by formula ME=B1/B2 × 100%
It calculates.Result of calculation shows that the matrix effect of dendrobine is less than 6.0% in 95.5%-110.2%, RSD, interior target matrix effect
For 107.1%, RSD 5.3%, illustrates that matrix effect is not present to dendrobine and internal standard compound in blood plasma, do not influence blood plasma sample
Product accurate quantitative analysis.
The investigation of 2.4 extraction recoveries
High, medium and low concentration Quality Control plasma sample made from 1.4.3 lower section methods is subjected to UPLC-MS analyses, peak area point
Not compared with A1, B1 in matrix effect confirmatory experiment, dendrobine and interior target extraction recovery are calculated.Result of calculation shows stone
The average extraction recovery of dry measure used in former times alkali is 93.6%-109.4%, and RSD is less than 8.7%, and the interior target extraction recovery that is averaged is
97.8%, RSD 7.5%.
The investigation of 2.5 preci-sion and accuracies
High, medium and low concentration Quality Control plasma sample is made in 1.4.3 lower section methods and carries out UPLC-MS analyses, calculates precision
And accuracy.Result of calculation shows that the withinday precision RSD% of dendrobine and internal standard compound is respectively less than 9.1%, accurate in the daytime
Degree RSD% is respectively less than 10.3%, and accuracy in 92.5%-104.9%, meets the requirement of methodology.
The investigation of 2.6 stability
1.4.3 lower methods are prepared to the Quality Control plasma sample of high, medium and low concentration, are placed for 24 hours for 15 DEG C respectively, -70 DEG C of guarantors
It after depositing simultaneously multigelation 3 times, and -70 DEG C of preservation 15d, is detected with UPLC-MS, by retinue standard curve, calculates sample and measure
The deviation (RE%) of concentration and marker concentration, the stability of sample is investigated with this.The results show that dendrobine and internal standard compound exist
Under above-mentioned preservation condition, the relative deviation of measured value and ident value is respectively less than 11.6%, and sample is stablized, and can reach measurement and want
It asks.
The research of 2.7 HERBA DENDROBII fine powder Pharmacokinetics in Rat
Rat acquires plasma sample after gavage gives HERBA DENDROBII fine powder, in different time points, and treated, and sample is adopted
It is measured with the UPLC-MS detection methods of foundation, the results show that 2min dendrobines absorbed into serum after administration, is being administered
The time-concentration curve of dendrobine is shown in Fig. 2 under dosage.Pharmacokinetics ginseng is calculated using non-compartment model method (statistics moments method)
Number, pharmacokinetic parameter of the evaluation HERBA DENDROBII main component dendrobine in rat body, the results are shown in Table 1.
The pharmacokinetic parameters (n=7) of dendrobine after after 1 rat oral gavage HERBA DENDROBII fine powder of table, Ultramicro-powder
3 conclusions
Widely distributed after HERBA DENDROBII fine powder, Ultramicro-powder are oral, the apparent volume of distribution of Ultramicro-powder is far longer than fine powder, this
It is related to particle size.Seminar's primary-stage survey shows to find dendrobine in the heart, liver, spleen, lung, kidney, brain, intestinal tissue, and
It is distributed in liver and enteron aisle more, this is closely related with administering mode, the absorption of HERBA DENDROBII, metabolic way.Identical
Under experiment condition, in HERBA DENDROBII fine powder the blood concentration higher, infiltration rate of dendrobine faster, drug effect it is more preferable.
Above-described is only the embodiment of the present invention, and the common sense such as well known concrete structure and characteristic are not made herein in scheme
Excessive description, for those skilled in the art, without departing from the structure of the invention, can also make several changes
Shape and improvement, these should also be considered as protection scope of the present invention, these all do not interfere with the effect and patent that the present invention is implemented
Practicability.The scope of protection required by this application should be based on the content of the claims, the specific embodiment party in specification
The records such as formula can be used for explaining the content of claim.
Claims (3)
1. a kind of HERBA DENDROBII powder, it is characterised in that：The grain size of the HERBA DENDROBII powder is 150~180 μm.
2. the application of HERBA DENDROBII powder according to claim 1, it is characterised in that：The HERBA DENDROBII powder it is oral or
Injection.
3. the application of HERBA DENDROBII powder according to claim 2, it is characterised in that：The HERBA DENDROBII powder is in drop blood
Sugared, antitumor, anti-oxidant, anti-senile dementia inhibits the Apoptosis either food of anti-cataract or the application in drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810271222.5A CN108669531A (en) | 2018-03-29 | 2018-03-29 | A kind of HERBA DENDROBII powder and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810271222.5A CN108669531A (en) | 2018-03-29 | 2018-03-29 | A kind of HERBA DENDROBII powder and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108669531A true CN108669531A (en) | 2018-10-19 |
Family
ID=63800525
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810271222.5A Pending CN108669531A (en) | 2018-03-29 | 2018-03-29 | A kind of HERBA DENDROBII powder and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108669531A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109828053A (en) * | 2019-03-15 | 2019-05-31 | 遵义医科大学 | The raw HERBA DENDROBII of a kind of pair of stone and the method for setting raw HERBA DENDROBII progress chromatographic identification |
| CN109856264A (en) * | 2019-01-11 | 2019-06-07 | 遵义医学院 | The detection method of active constituent content in Blumea balsamifera medicinal material |
| CN111588728A (en) * | 2019-02-20 | 2020-08-28 | 安益药业股份有限公司 | Medicinal composition, dendrobe extract and application of medicinal composition |
| CN113413439A (en) * | 2021-07-12 | 2021-09-21 | 中山创天生物科技有限公司 | Traditional Chinese medicine formula for treating Alzheimer's disease and preparation method of compound preparation of traditional Chinese medicine formula |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103285242A (en) * | 2013-05-23 | 2013-09-11 | 重庆市中药研究院 | Dendrobium buccal tablets and preparation method for same |
| CN104623312A (en) * | 2015-01-27 | 2015-05-20 | 中国农业科学院农产品加工研究所 | Instant dendrobe powder as well as preparation method and application thereof |
| CN106266609A (en) * | 2016-08-23 | 2017-01-04 | 嘉兴思必佳生物科技有限公司 | A kind of Herba Dendrobii micropowder sheet and preparation method thereof |
| CN107019761A (en) * | 2016-01-29 | 2017-08-08 | 林东昊 | A kind of instant stem of noble dendrobium fine grained and preparation method thereof |
-
2018
- 2018-03-29 CN CN201810271222.5A patent/CN108669531A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103285242A (en) * | 2013-05-23 | 2013-09-11 | 重庆市中药研究院 | Dendrobium buccal tablets and preparation method for same |
| CN104623312A (en) * | 2015-01-27 | 2015-05-20 | 中国农业科学院农产品加工研究所 | Instant dendrobe powder as well as preparation method and application thereof |
| CN107019761A (en) * | 2016-01-29 | 2017-08-08 | 林东昊 | A kind of instant stem of noble dendrobium fine grained and preparation method thereof |
| CN106266609A (en) * | 2016-08-23 | 2017-01-04 | 嘉兴思必佳生物科技有限公司 | A kind of Herba Dendrobii micropowder sheet and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 周威，等: "金钗石斛的化学成分和药理作用研究现状", 《中国新药杂志》 * |
| 罗颖，等: "金钗石斛微粉细胞破壁率及其石斛碱含量测定", 《泸州医学院学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109856264A (en) * | 2019-01-11 | 2019-06-07 | 遵义医学院 | The detection method of active constituent content in Blumea balsamifera medicinal material |
| CN109856264B (en) * | 2019-01-11 | 2021-09-07 | 遵义医科大学 | Method for detecting content of effective components in blumea balsamifera medicinal material |
| CN111588728A (en) * | 2019-02-20 | 2020-08-28 | 安益药业股份有限公司 | Medicinal composition, dendrobe extract and application of medicinal composition |
| CN109828053A (en) * | 2019-03-15 | 2019-05-31 | 遵义医科大学 | The raw HERBA DENDROBII of a kind of pair of stone and the method for setting raw HERBA DENDROBII progress chromatographic identification |
| CN113413439A (en) * | 2021-07-12 | 2021-09-21 | 中山创天生物科技有限公司 | Traditional Chinese medicine formula for treating Alzheimer's disease and preparation method of compound preparation of traditional Chinese medicine formula |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108669531A (en) | A kind of HERBA DENDROBII powder and its application | |
| Wang et al. | Pharmacokinetics screening for multi-components absorbed in the rat plasma after oral administration traditional Chinese medicine formula Yin-Chen-Hao-Tang by ultra performance liquid chromatography-electrospray ionization/quadrupole-time-of-flight mass spectrometry combined with pattern recognition methods | |
| CA2693577C (en) | Dry-powder medicament | |
| Li et al. | A strategy to improve the oral availability of baicalein: The baicalein-theophylline cocrystal | |
| Shao et al. | Identification of the active compounds and functional mechanisms of Jinshui Huanxian formula in pulmonary fibrosis by integrating serum pharmacochemistry with network pharmacology | |
| CN106124685B (en) | Quality detection method of fallopia multiflora (thunb.) harald-aloe constipation relieving capsules | |
| Guan et al. | Simultaneous determination of calycosin-7-O-β-D-glucoside, cinnamic acid, paeoniflorin and albiflorin in rat plasma by UHPLC-MS/MS and its application to a pharmacokinetic study of Huangqi Guizhi Wuwu Decoction | |
| Liu et al. | Quantitative determination and toxicity evaluation of aristolochic acid analogues in Asarum heterotropoides F. Schmidt (Xixin) and traditional Chinese patent medicines | |
| CN104914173B (en) | The assay method of Multiple components content in a kind of Chinese medicinal composition preparation | |
| Fan et al. | Comparative pharmacokinetics of ginsenoside Rg3 and ginsenoside Rh2 after oral administration of ginsenoside Rg3 in normal and walker 256 tumor-bearing rats | |
| Xiang et al. | Simultaneous determination of polysaccharides and 21 nucleosides and amino acids in different tissues of Salvia miltiorrhiza from different areas by UV–visible spectrophotometry and UHPLC with triple quadrupole MS/MS | |
| Chang et al. | Simultaneous determination of ten active components in traditional Chinese medicinal products containing both Gegen (Pueraria lobata) and Danshen (Salvia miltiorrhiza) by high‐performance liquid chromatography | |
| Li et al. | Establishment of modified biopharmaceutics classification system absorption model for oral Traditional Chinese Medicine (Sanye Tablet) | |
| CN101502549B (en) | Notoginsen triterpenes capsule as well as preparation method thereof and method for measuring content | |
| He et al. | Application of a liquid chromatography–tandem mass spectrometry method to the pharmacokinetics, tissue distribution and excretion in the study of anemoside B4, a novel antiviral agent candidate, in rats | |
| CN106214656A (en) | The preparation of a kind of compound tablet of glycyrrhizin and test method of quality control | |
| Song et al. | Pharmacokinetic–pharmacodynamic evaluation of the major component astragaloside IV on the immunomodulatory effects of Yu-ping-feng prescription | |
| CN104998071A (en) | Compound preparation ofherb of dense flower Bulbophyllum and preparation and detection method for said compound preparation | |
| CN110261514A (en) | The content assaying method of the dried venom of toads in pills for curing heart disease | |
| CN107551089A (en) | A kind of medicine for treating hyperlipidemia and preparation method thereof and detection method | |
| Zhao et al. | Untargeted metabolomics using liquid chromatography coupled with mass spectrometry for rapid discovery of metabolite biomarkers to reveal therapeutic effects of Psoralea corylifolia seeds against osteoporosis | |
| CN101987146A (en) | Preparation method of capsule for treating pharyngitis and infection of upper respiratory tract and detection method of capsule | |
| CN107389818B (en) | A kind of drug for treating Parkinson and preparation method thereof and detection method and purposes | |
| Yao et al. | Simultaneous quantitation and comparison of eight components in Jiao‐ai decoction and Si‐wu decoction by ultra high performance liquid chromatography with triple quadrupole tandem mass spectrometry | |
| CN107233511B (en) | A kind of dispelling wind detoxicating capsule and preparation method thereof and detection method and purposes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181019 |