WO2004110465A1 - Extrait de faeces bombycis, et sa methode de preparation, et appareil pour diagnostiquer et pour traiter des tumeurs malignes - Google Patents

Extrait de faeces bombycis, et sa methode de preparation, et appareil pour diagnostiquer et pour traiter des tumeurs malignes Download PDF

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Publication number
WO2004110465A1
WO2004110465A1 PCT/CN2003/000465 CN0300465W WO2004110465A1 WO 2004110465 A1 WO2004110465 A1 WO 2004110465A1 CN 0300465 W CN0300465 W CN 0300465W WO 2004110465 A1 WO2004110465 A1 WO 2004110465A1
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Prior art keywords
fluorescence
light
extract
silkworm
optical fiber
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PCT/CN2003/000465
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English (en)
French (fr)
Inventor
Hongyuan Zhang
Zhifang Chen
Shujun Wang
Qinwen Zhong
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Beijing Zhongya Senlen Bio-Technology Development Ltd.
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Application filed by Beijing Zhongya Senlen Bio-Technology Development Ltd. filed Critical Beijing Zhongya Senlen Bio-Technology Development Ltd.
Priority to CNB038268248A priority Critical patent/CN100387240C/zh
Priority to AU2003242220A priority patent/AU2003242220A1/en
Priority to PCT/CN2003/000465 priority patent/WO2004110465A1/zh
Publication of WO2004110465A1 publication Critical patent/WO2004110465A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/41—Detecting, measuring or recording for evaluating the immune or lymphatic systems
    • A61B5/411—Detecting or monitoring allergy or intolerance reactions to an allergenic agent or substance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/0059—Measuring for diagnostic purposes; Identification of persons using light, e.g. diagnosis by transillumination, diascopy, fluorescence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N2021/6484—Optical fibres

Definitions

  • the invention relates to an extract of silkworm sand, a preparation method thereof, and use thereof in preparing a reagent or a medicine for diagnosis or treatment of malignant tumors, especially early malignant tumors, and a diagnosis or treatment of malignant tumors, especially early malignancy.
  • malignant tumor tissues have different characteristics of fluorescence from autofluorescence of normal tissues.
  • scientists have attempted to identify and diagnose malignant tumors by observing and recording the characteristic fluorescence of tumor tissues with fluorescence photogrammetry.
  • CN85100242 also discloses a spectral analysis method. This solution hopes to overcome the disadvantage of low sensitivity due to weak energy of the excitation light source.
  • the disclosed method improves the light source and transmission system.
  • the light source uses near-ultraviolet light of 3000-4000A to avoid exciting fluorescence.
  • the superposition of light pans can greatly increase the energy of the excitation light source and excite the autofluorescence of the tumor tissue to the greatest extent, which is convenient for visual observation and spectral diagnosis.
  • CN97106273 considers that the spectral analysis method is too complicated, and proposes a method for diagnosing cancer by the ratio of fluorescence values between normal and abnormal tissues.
  • the method and device based on visual observation or optical analysis for diagnosing malignant tumors can be misdiagnosed with naked eyes due to color distortion.
  • the device used for optical analysis has high cost and is difficult for operators.
  • the defect of high service level requirements restricts its use, especially the device has high requirements on the color separation system, circuit system and scanning system in the optical processing system, and it is easy to distort the signal (see ZL87105948 background technology section).
  • EP1610616, JP59-40869 propose to use the accumulation of the photosensitive substance hemaline derivative (HPD) in the tumor cells, and use fluorescent lamps and pump dye laser irradiation to obtain HPD-excited fluorescence to determine whether there is a tumor.
  • HPD photosensitive substance hemaline derivative
  • CN87103403 believes that the disadvantage of the above method is that it has low power and cannot illuminate deep layers.
  • This solution proposes a new medical pulse laser device, and points out a laser light source with a wavelength of 405 nm for diagnosis and a wavelength of 630 nm for treatment.
  • This solution also uses the photosensitizer HPD. And again pointed out that 405nm can effectively excite HPD-specific fluorescence, and the average power and peak output of the tissue are moderate.
  • HPD is very allergic, and the injection of HPD will not be visible for a few days, which is unacceptable in general examinations and tumor examinations.
  • HPD / PH1126 / NPE6 is selected as the photosensitizer, and a wavelength-controllable laser machine is used to generate excitation light and diagnose tumors by image analysis. .
  • Silkworm grit is a dried material of silkworm feces, which contains a large amount of chlorophyllate, protein and pectin, and is a cheap and readily available raw material for extracting chlorophyllate.
  • silkworm sand is degraded into a relatively stable chlorophyllic acid mixture by acid-base treatment, removing magnesium and phytol linked by ester bonds. Its aqueous solution can produce orange-red fluorescence of about 660nra under the excitation of red light, and it has selective uptake and photosensitivity for tumors (Hu Longqin et al. Application of chlorophyll derivatives in medicine. Foreign Medicine. , 1987, 8: 146).
  • the mixture is not stable enough, and its monomer compounds are difficult to obtain.
  • CN92103649. 3 discloses a method for preparing chlorophyllate from silkworm sand by using enzyme engineering technology. The method is mainly obtained by pretreating silkworm sand and hydrolyzing under the action of hydrolase, etc., and obtaining chlorophyllate through saponification and replacement.
  • CN93103432. 9 discloses a traditional Chinese medicine photosensitizer for photosensitivity therapy and a production process thereof, which contains 60-80% of chlorophyllic acid and its polymer.
  • the production process uses silkworm sand as raw material, mixed extraction with acetone, saponification with ethanol and NaOH, then acidification to remove magnesium, and finally mixed with silica gel G and adsorbed, and then separately with petroleum Ether and ethanol are eluted to remove impurities, and distilled water is dissolved and evaporated to obtain a finished product.
  • the photosensitizer has good diagnosis and treatment effect, convenient clinical application, simple and easy production process, and cheap and easy-to-obtain raw materials.
  • CN00121046. 7 discloses a process for preparing a natural green colorant chlorophyllin zinc.
  • the silkworm sand is leached with an organic solvent, the supernatant is concentrated, and then saponified.
  • the saponified solution is replaced with zinc acetate, and the oil-soluble chlorophyllin is obtained by post-treatment.
  • the optimal excitation wavelength of the silkworm sand extract in the prior art is about 405 nm.
  • an excitation light source capable of generating light at about 405 nm is required, which limits its use. Therefore, it has been developed to have short residence time in the body and tumor tissue.
  • the new photosensitizer with high internal retention concentration and appropriate excitation wavelength is very meaningful for the early diagnosis and timely treatment of cancer.
  • the main object of the present invention is to provide a silkworm sand extract, which is a photosensitizer for detecting fluorescence.
  • the silkworm sand extract has good uptake in tumor cells in the body, and the retention site is obvious, especially produced under the excitation of a specific light source. Strong characteristic fluorescence. By detecting the characteristic fluorescence and measuring the brightness, the mutation of the cell can be recognized, and therefore early canceration of the tissue and malignant tumor can be diagnosed.
  • Another object of the present invention is to provide a method for preparing the extract of the upper silkworm sand, which is simple and easy to perform, to obtain an effective photosensitive composition with detection specificity.
  • Still another object of the present invention is to provide the use of the silkworm sand extract for preparing a reagent or a medicine for diagnosing or treating early malignant tumors.
  • Another object of the present invention is to provide a device for diagnosing or treating early malignant tumors.
  • the device can generate excitation light that stimulates the silkworm sand extract of the present invention to generate characteristic fluorescence, and a detection system for receiving, detecting, and processing the characteristic fluorescence. Efficiently and accurately distinguish the early cancerous changes (precancerous lesions) of the tissue;
  • the irradiance of the excitation light source of the device is adjustable, making the test result highly reproducible.
  • the device has a simple structure, low production cost, operation and diagnosis. easily.
  • the object of the present invention can be achieved by:
  • An extract of silkworm grit which is the sodium or potassium salt of silkworm grit degradation product, its aqueous solution has the strongest fluorescence at a wavelength of 660nm ⁇ 15nm when excited by a near-violet light source of 365 ⁇ 15nm.
  • the preferred excitation wavelength is 365 ⁇ 10nm and the emission wavelength is 660nm ⁇ 10nm. More preferably, the excitation wavelength is 365 nm and the emission wavelength is 660 nm.
  • the silkworm sand extract of the present invention includes a sodium or potassium complex of a chlorin having a cyclopentanone structure having a chlorin as a mother nucleus, and the content thereof is about 30-58 wt ° /.
  • the preferred content is 40-50 wt ° /.
  • the effective components of the silkworm sand extract of the present invention are pheophytin a, pheophytin b, pyropheophytin &, rhodopsin-18, chlorin IV chlorin ⁇ dihydro A mixture of porphyrin ⁇ and other demagnesium green rings and their salts.
  • the aqueous solution of silkworm sand extract of the present invention has a fluorescence value ratio of at least 8 to 12 times in a wavelength range of 660 nm to 15 nm under the excitation of a near-ultraviolet light source of 365 ⁇ 15 nm compared with a blank control; the preferred excitation wavelength is 365 Soil l Onm, emission wavelength 660nm ⁇ l Onm, at least 9-12 times the fluorescence value ratio; more preferably excitation wavelength is 365nffl, emission wavelength 660nm, at least 10-12 times the fluorescence value ratio.
  • the preparation method of silkworm sand extract of the present invention comprises: saponifying, post-treating and salting the silkworm or coarse silkworm sand extract, wherein the saponification is performed under heating in an alcohol and alkali aqueous solution, and the post-treatment is filtering, concentrating and using non-
  • the saponification solution is extracted with a polar organic solvent to discard the unsaponifiable matter, and the saponification solution is acidified with an acid; and then the salt is obtained to obtain a salt of the degradation product of silkworm sand.
  • the above crude silkworm sand extract is prepared by alkaline and / or acid degradation of silkworm sand using the methods of the prior art.
  • the non-saponifiable matter in the concentrated solution of the saponified liquid is extracted with a non-polar organic solvent in order to fully extract the ester-soluble and other non-saponifiable matter therein to obtain a completely water-soluble product.
  • the silkworm sand extract is a silkworm sand extract prepared by the following method: saponifying, extracting, acidifying, and alkalizing silkworm sand into a salt, wherein:
  • saponification is by adding 1-8 times alcohol and 5- 40wt ° / in silkworm sand.
  • the aqueous alkali solution is brought to a pH of 9-12 under heating and refluxing.
  • the alkali is NaOH or KOH, and the amount of NaOH or KOH is 3-30% by weight based on the weight of silkworm sand.
  • Saponification time is 4-10 hours.
  • the alcohol is methanol, ethanol, propanol or butanol, and ethanol is preferred.
  • the pH is preferably controlled at 9. 5-10. 5 and more preferably at pH 10.
  • the concentrated solution Before the non-polar organic solvent is used to extract the ester-soluble substances in the concentrated solution, the concentrated solution can also be first dissolved in water, and then the non-polar organic solvent can be used to extract the non-saponified substances or ester-soluble impurities in the dissolved solution. Layer, the saponification solution was removed, and the solvent was recovered and reused.
  • the amount of water used to dissolve the concentrated solution and the amount of solvent used during extraction are 15 times the concentration of the concentrated solution, the number of extractions is 1-4 times, or the extraction is colorless.
  • the non-polar organic solvent used for extraction is petroleum ether, ether, n-hexane, cyclohexane, carbon tetrachloride, chloroform, benzene or substituted benzene, and petroleum ether, ether, n-hexane, and cyclohexane are preferred; Most preferred is petroleum ether.
  • the lower saponification solution was acidified with an acid to a pH of 3-5, the supernatant was discarded, and the acidified saponification precipitated was collected.
  • a dilute solution of hydrochloric acid, sulfuric acid and nitric acid is used, and hydrochloric acid is preferred. And preferably acidified to a pH of 3.5-4.5.
  • the precipitated acidified saponification is reacted with potassium hydroxide, sodium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate to form a salt, and then filtered. The filtrate is concentrated or further dried to obtain silkworm.
  • the salt-forming reaction is preferably carried out in an alcohol and an aqueous solution of 1-30% by weight of a base or a salt.
  • the amount of alcohol used is that of silkworm sand.
  • the amount of alkali or salt is 1-25 wt% of silkworm sand.
  • the reaction is carried out at pH 9-12, and the preferred pH is 9-10.
  • the salt-forming reaction is generally completed by stirring at room temperature. If necessary, it can also be heated.
  • the ester-soluble impurities in the silkworm sand are extracted by a non-polar solvent after saponification, and then acidified, salt-formed, etc. are used to remove the magnesium, protein, and ester-soluble impurities in the silkworm sand.
  • Silkworm sand is fully degraded into pheophytin a, pheophytin b, pyropheophytin a, rhodopsin-18, chlorin P 6 , chlorin e 6 , chlorin the mixture pheophorbide ⁇ cycloalkyl-earth salt and the like, to obtain a high salt content of the chlorophyll extracts silkworm excrement, with the strongest fluorescence at a wavelength of 660nm ⁇ 15nm at 365 near the soil l 5 nm UV light excitation source.
  • the silkworm sand extract of the present invention can be completely dissolved in water, which is beneficial for specific aggregation and absorption in tumor tissues in vivo, and is easily digested and eliminated in normal tissues without any toxic and side effects.
  • the silkworm 'extract of the present invention can also be obtained by extracting before acidification, and adding alkali or salt to form a salt after acidification.
  • the silkworm wormwood extract liquid of the present invention is dried by the method of the prior art, and a dried silkworm wormworm extract can be obtained, which can be prepared into a solution with a certain concentration by adding distilled water, and according to the method of the prior art, supplemented with pharmacology Acceptable excipients are prepared in any dosage form that is pharmaceutically acceptable, such as powders, capsules, pills, tablets, oral liquid, injections, sprays or coatings.
  • pharmacology Acceptable excipients are prepared in any dosage form that is pharmaceutically acceptable, such as powders, capsules, pills, tablets, oral liquid, injections, sprays or coatings.
  • early tumor tissues are difficult to distinguish from normal tissues. This is why the auto-fluorescence misdiagnosis rate is high.
  • the prior art proposed the use of demagnesium chlorophyllic acid and chlorophyll three under the excitation of a wavelength-tunable laser.
  • Acid has higher sensitivity as a photosensitizer (CN94106907).
  • the near-ultraviolet light (such as generated by a mercury lamp) is used to excite the silkworm sand extract of the present invention, which has more significant specificity and high sensitivity. It also greatly simplifies the requirements of the detection device and the diagnostic level of the operator.
  • the silkworm sand extract of the present invention as a photosensitizer only stays in tumor cells or precancerous lesions for a certain period of time. Therefore, theoretically, whether fluorescence can be detected means that there is or is no tumor Cells, but in fact, normal cells and tumor cells can also fluoresce under the excitation of near-ultraviolet light. Under near-ultraviolet light excitation, the ratio of the fluorescence value of the injected photosensitizer to the fluorescence value generated by normal cells is greater. It means that the sensitivity is higher and the detection result is more accurate.
  • the photosensitizer of the present invention has at least 5-12 times or even higher fluorescence brightness value of normal cells under the excitation of near-ultraviolet light.
  • the silkworm sand extract of the present invention can also perform photodynamic therapy by killing cancer cells while detecting.
  • the extract of the present invention has higher fluorescence brightness under the same intensity of ultraviolet light irradiation for two reasons.
  • One is that the required excitation energy is lower, and the other is the specificity of the silkworm sand extract to tumors. And the affinity is stronger, so the concentration accumulated at the tumor site is higher.
  • the device includes: a light generating device with adjustable irradiance and outputting ultraviolet light, a light transmission device, an ultraviolet irradiance detection device, and a fluorescence detection device.
  • the light transmission device includes an input optical fiber and a fluorescence collection optical fiber And the input light detection optical fiber; the ultraviolet light emitted by the light generating device is transmitted to the detection end portion of the input optical fiber and the fluorescence detection device for the input optical fiber located in the target tissue through the input optical fiber and the input light detection optical fiber respectively;
  • the photodetector that detects the ultraviolet irradiance emitted from the light generating device, the input end of the fluorescence collection optical fiber and the detection end portion of the input optical fiber near the target tissue are coaxially arranged into a composite fiber, and the characteristics generated by the target tissue are Fluorescence is transmitted to the photomultiplier tube in the fluorescence detection device for detecting the fluorescence brightness through the output end of the fluorescence collection optical fiber.
  • the fluorescence signal output by the photomultiplier tube is processed by the electronic circuit to display the fluorescence brightness in a digital form on the fluorescence brightness display window , Or the fluorescence signal output by the photomultiplier tube is processed by the electronic circuit Output to a computer or a recorder for recording or data processing.
  • the ultraviolet light received by the photodetector of the ultraviolet irradiance is converted by a circuit, and the ultraviolet irradiance is displayed in a digital form on the ultraviolet display in the detector, or output to a recorder or computer for recording or data processing. .
  • the photodetector detects ultraviolet light in the wavelength range of 320-400nm.
  • a first filter device is disposed in the light generating device, or where the light generating device is connected to the input optical fiber or the input end portion of the input optical fiber. After color filtering by the filter, the narrow-band ultraviolet radiation of 365 ⁇ 15 nm is transmitted to the input end of the input optical fiber, and the narrow-band ultraviolet radiation of 365 ⁇ l Onra is preferably input.
  • the first filter device is two filters.
  • a second filter device is provided between the output end of the fluorescence collection optical fiber and the photomultiplier tube in the fluorescence detection device.
  • the second filter device is a filter that allows only red fluorescence with a wavelength of 600 to 750 legs to pass through, and its purpose is to filter out various stray light such as ultraviolet radiation, autofluorescence, and other scattering or superimposition on the detection accuracy, to avoid Detection results of ultraviolet radiation and autofluorescence interference.
  • the detection device preferably detects the fluorescence brightness at a wavelength of 610-700nm, and a more preferable wavelength is 650 ⁇ 15nm.
  • the response range of the photomultiplier tube in the fluorescence detection device of the device for diagnosing or treating early malignant tumors of the present invention is 300-S50nm, and the anode sensitivity is 100-300A / lm.
  • the light generating device outputs near-ultraviolet light whose irradiance is adjustable from greater than 0 to less than 1.999 mW / cm 2 . If the first filter device is arranged in the light generating device, the light generating device outputs near-ultraviolet light with a wavelength of 365 ⁇ 15 nm and an irradiance of greater than 0 to 1.999 mW / cm 2 .
  • the light generating device includes: a high-pressure mercury lamp emitting a light source, a parabolic mirror, a reflecting mirror, and a filter device arranged therein; the radiation light emitted by the high-pressure mercury lamp is condensed on the reflecting mirror through the parabolic mirror and passing through the reflecting mirror Reflect and focus to the filter device, and after filtering by the filter device, to the input end of the input optical fiber.
  • the high-pressure mercury lamp, parabolic mirror, and reflector are arranged in the chassis, and the filter device is arranged in the chassis, the input end of the input optical fiber, or a certain position in the input optical fiber.
  • a quartz light rod is provided on the side wall of the light output of the chassis.
  • One end of the quartz light rod receives ultraviolet light, and the other end is directly connected to the input end of the optical fiber or connected to the input end of the optical fiber through a filtering device. .
  • the ultraviolet light reflected by the mirror is focused on the quartz light rod and enters the input end of the input optical fiber through the first filter device.
  • the first filter device may also be disposed on the side of the quartz light rod that receives ultraviolet light.
  • the inner surface of the parabolic mirror is covered with a reflective film that reflects only ultraviolet light and passes all other long-wave light.
  • the light generating device is further provided with a controller for increasing and decreasing light, so that the output irradiance of the light generating device is greater than
  • the controller is similar to a controllable
  • the power switch realizes the increase or decrease of light by controlling the magnitude of the input current.
  • a window with an adjustable size is provided on the front of the quartz light rod to control the increase or decrease of light.
  • the amount of ultraviolet light is adjusted by controlling a wedge-shaped light bar on the convergence point of the parabolic reflector bowl light beam.
  • the light generating device is further provided with a cooling fan.
  • the input optical fiber and the input light detection optical fiber have a common optical input port.
  • the optical fibers are all quartz optical fibers, but the fluorescence collection fiber can also be changed to glass fibers.
  • the two fibers of the input optical fiber and the fluorescence collection optical fiber are arranged coaxially, It can also be determined according to the depth of the body to be detected in the body.
  • the brightness range of the red fluorescence detected by the fluorescence detection device is 0-999 cd / m 2 .
  • the fluorescence detection device further includes a fluorescent signal analog output port.
  • One end of the analog output port is connected to the output end of the A / D converter, and the other end is connected to a recorder or a computer for recording by the recorder. Or computer processing data.
  • the specific diagnosis process is: the silkworm sand extract of the present invention is injected into the human body by injection, oral or atomization in advance, and the composite fiber is used to replace the biopsy device of the endoscope and probe into the body or the surface, and the light generating device is used
  • a non-laser light source with a certain irradiance and a wavelength of 365 ⁇ 15 nm transmitted by the input optical fiber is irradiated to the suspicious site (target tissue), and the silkworm sand extract that has entered the abnormal tissue (target tissue) has a strong characteristic.
  • Fluorescence, the fluorescence fluorescein collects the optical fiber and transmits the fluorescence to the photomultiplier tube.
  • the brightness of the fluorescence is displayed on the fluorescence brightness display.
  • the occurrence of cancerous changes combined with endoscopic observation of the location and size of the tumor, realizes the diagnosis of early cancer.
  • the irradiance of ultraviolet light can be adjusted in the range of 0 to 1.999mW / cm 2 to display the ultraviolet brightness in the entire range.
  • the irradiance of ultraviolet light can also be fixed at a fixed value. A high reproducibility can be obtained, and it is easy to track the same patient.
  • the device of the invention can also be used alone.
  • the researchers of the present invention have unexpectedly discovered that silkworm sand extracts prepared according to the method of the present invention, which are different from the dichroic acid extracts of demagnesium chlorophyllic acid and chlorophyllic acid indicated in the prior art, are matched with the apparatus of the present invention and used
  • the ratio between the fluorescence value of the tissue in the tumor group and the fluorescence value in the tissue of the normal group was more than 8 times, further Right Compared with experiments, it was found that the ratio of the fluorescence value of various photosensitizers in the prior art to gastric cancer model mice and healthy mice is less than 3 times.
  • a near-ultraviolet radiation detection device is provided at an appropriate part of an optical fiber that transmits near-ultraviolet light, and the detection device actually obtains the irradiance of near-ultraviolet light in the optical fiber.
  • the second feature is the use of spectral analysis to diagnose malignant tumors.
  • the advantage of this detection device is that, in the process of gradually increasing the intensity of ultraviolet light irradiation, the fluorescence value excited by the photosensitizer can be directly detected correspondingly increased; in addition, the ultraviolet light irradiance can be accurately adjusted for different parts or organs to ensure that During the service life of the same light source, the UV irradiance can be adjusted to be consistent at all times.
  • the device of the present invention also overcomes the problem that the irradiance of the excitation light in the prior art cannot be adjusted. As the use time increases, the ultraviolet irradiance decreases, while other conditions in the device remain unchanged, and the detection result is not heavy. The shortcomings of probabilistic or prone to miss detection.
  • an alarm device is provided in the detection device, and a detection model is preset in the alarm device.
  • the alarm device can be diagnosed as cancerous through the peak sounder connected to it. In this way, the detection process can be simplified and the requirements for the operational level of the detection operator can be further reduced.
  • a second filter is provided between the output end of the fluorescence collection fiber and the photomultiplier tube in the detection device, and only allows red fluorescence with a wavelength of 600-750nm to pass through, and filters out ultraviolet radiation and autofluorescence, and excludes Because of the influence of various factors on the detection results, it can be determined that the detected fluorescence is the fluorescence from the photosensitizer in the target tissue.
  • the resolution of the device of the present invention for fluorescent brightness is 0.01cd / m 2 , and due to the strong selective affinity and adsorption of the photosensitizer of the present invention in mutant tissues, and normal metabolism in normal tissues, even for early cell mutations, the accuracy of detection can reach 98 ° / . .
  • the photosensitizer is preferably the silkworm sand extract of the present invention.
  • the dosage is from 0.1 to 100 ⁇ g / kg body weight.
  • the unabsorbed silkworms and extracts are naturally discharged from the body after 12 hours. Retention at the site of canceration for 12-24 hours.
  • the device of the present invention cooperates with an endoscope for naked eye resolution, and because of its higher fluorescence value, it is easier to distinguish and locate than the prior art.
  • the invention can be applied to the early diagnosis of various cancers, especially the detection and early diagnosis of cancerous changes in various organs that have been detected by endoscope, such as suspicious lung cancer, gastric cancer, bladder cancer, uterine cancer, prostate cancer, rectal cancer, skin Detection of cancer and other parts and diagnosis of precancerous lesions.
  • endoscope such as suspicious lung cancer, gastric cancer, bladder cancer, uterine cancer, prostate cancer, rectal cancer, skin Detection of cancer and other parts and diagnosis of precancerous lesions.
  • the silkworm sand extract of the present invention when used as a photosensitizer, because of its high sensitivity, it can retain even a slight tissue variation at an early stage, which can greatly reduce the diagnosis rate of early cancerous tissue.
  • the device of the present invention overcomes the defects in the prior art of using laser light sources and spectroscopic analysis equipment that are large, expensive, and inconvenient to use, as well as the defects of inaccurate detection of ultraviolet light sources and high operating requirements, and provides diagnosis or treatment of early malignant tumors.
  • the device is small in size, cheap in price, and simple in operation.
  • the absolute value of characteristic fluorescence is used to diagnose malignant tumors and early cancerous changes, and the accuracy is proved by animals. detailed description
  • FIG. 1 is a schematic diagram of a device for diagnosing or treating a tumor according to the present invention
  • FIG. 2 is a structural diagram of a device for diagnosing or treating a tumor of the present invention
  • FIG. 3 is a structural diagram of another embodiment of a device for diagnosing or treating a tumor of the present invention.
  • the above-mentioned silkworm sand extract powder is added with auxiliary materials such as starch, and is prepared into tablets with an active ingredient content of 5rag / tablet or granules of 5tng / bag according to the method of the prior art.
  • the precipitate is dissolved by adding 100ml of 10% NaOH solution and 1000ml of 95% ethanol.
  • the pH is controlled at about 9. Stir, heat and reduce pressure to recover ethanol, filter, and filtrate. Concentrated to give a creamy silkworm sand extract.
  • the paste-like silkworm sand extract was added with distilled water and prepared into an oral solution of 3-20 mg / ml by using the existing technology in formulation. The content was determined by fluorescence spectrophotometry.
  • the filtrate was collected, heated to remove ethanol under reduced pressure, and 30% hydrochloric acid was added to adjust the pH to about 5.5, and the precipitate was precipitated, centrifuged, the precipitate was collected, the precipitate was washed with water, and dried to obtain 30 g of dark green silkworm silk extract powder.
  • the above-mentioned silkworm sand extract powder is added into auxiliary materials and prepared into a tablet or a spray according to the method of the prior art.
  • the salt forming step may be replaced by Na 2 C0 3 K 2 C0 3, NaHC0 3 or KHC0 3, to give the corresponding chlorophyll degradation silkworm excrement ring type K or Na salt.
  • Excitation conditions the excitation wavelength is 365 legs, and the bandwidth is 10 gall.
  • a standard curve was made with the fluorescence intensity at 65 Onm as the ordinate and the concentration as the abscissa, and the fluorescence intensity values were measured at different concentrations to make a standard curve.
  • This experimental example relates to the toxicity of silkworm sand extract of the present invention
  • Silkworm sand extract prepared by the method of Example 1.
  • mice 60 healthy mice were divided into two groups: male and female.
  • the mice in the administration group took 25g / kg of the silkworm sand extract of the invention orally once.
  • the control group did not take any medicine.
  • the weight and clinical manifestations of the fourth and tenth days before and after the administration of the above two groups were recorded.
  • the results showed that the maximum dose of silkworm sand extract 25 g / kg was orally administered to the mice at one time, no death occurred, and no clinical related to the administration was found. Performance, and no effect on body weight was found, suggesting that the human clinically proposed dose is a safe dose.
  • the input optical fiber and the input light detection optical fiber are collectively referred to as an excitation optical fiber and a fluorescence collecting optical fiber, that is, a collecting optical fiber.
  • the device used for photoelectric conversion is a photomultiplier tube.
  • the device for diagnosing or treating malignant tumors of the present invention includes: a light generating device 1, a light transmission device 2 and a detection device 3 that output adjustable irradiance ultraviolet light, and the detection device 3 includes an ultraviolet irradiance detection device and a fluorescence detection device .
  • the optical transmission device includes an input optical fiber 4, a fluorescence collection optical fiber 5 and an input light detection optical fiber 6; the input optical fiber 4 and the input light detection optical fiber 6 have a common light input end portion 21.
  • the ultraviolet light emitted from the light generating device 1 is transmitted to the detection end portion 8 and the detection device 3 of the input optical fiber located in the target tissue 7 via the input optical fiber 4 and the end of the input light detection optical fiber 6 respectively connected to it.
  • the photodetector 9 in the ultraviolet irradiance detection device that detects the ultraviolet irradiance emitted from the light generating device 1 the input end 10 of the fluorescence collection optical fiber 5 and the detection end of the input optical fiber close to the target tissue 7 8 parts are coaxially arranged as composite fibers 11.
  • the output end 12 of the fluorescence collection optical fiber 5 is connected to the photomultiplier tube 13 in the fluorescence detection device for detecting the fluorescence intensity in the detection device 3.
  • the fluorescence output from the photomultiplier tube 13 is The signal is displayed on the fluorescent brightness display window 15 after being processed by the electronic circuit 14.
  • the detection device 3 includes two independently-arranged ultraviolet irradiance detection devices 300 and a fluorescence detection device 301.
  • the ultraviolet irradiance detection device and the fluorescence detection device are disposed in the detection device 3 together.
  • a second filter device 45 is provided between the output end of the fluorescence collection optical fiber and the photomultiplier tube in the fluorescence detection device.
  • the second filter device 45 is a filter that allows only red fluorescence having a wavelength of 600-750 nm to pass through to prevent ultraviolet radiation from entering the photomultiplier tube to dilute the purity of the fluorescent signal.
  • the light generating device includes a high-pressure mercury lamp 16 emitting a light source, a parabolic mirror 17, a reflecting mirror 18, a quartz light rod 1, and a filter device 20.
  • the radiation light emitted by the high-pressure mercury lamp 16 is condensed by the parabolic mirror 17 and projected to the display.
  • a reflector 18 set at a 45-degree angle, after reflection to a quartz light rod 19, after filtering by a light filtering device 20, narrow-band ultraviolet radiation having a wavelength of 365 + 15 nm enters the input optical fiber 4 and the fluorescence detection light guide having a common input port. Input of fiber 6.
  • the surface of the parabolic mirror 17 is covered with reflection only
  • the filter device 20 includes two filters, which are filtered by the filters and transmitted to the input optical fiber input end with a narrow-band ultraviolet radiation of 365 + 15 nm, preferably 365 ⁇ 10 nm.
  • the high-pressure mercury lamp 16 in the light generating device is electrically connected to an adjustable control switch (not shown), and the irradiance of the output near-ultraviolet light is adjusted from 0 to 0 by controlling the size of the input current. 1. 999mW / cm 2 .
  • a front portion of the quartz light rod 19 is provided similar to A light-adjustable window of the camera shutter to control the increase or decrease of light, as shown in FIG. 1, a light blocking plate 30 may be provided at the front of the quartz light rod 19, and the light blocking plate 30 is fan-shaped, semi-circular, or wedge-shaped.
  • the light bar drives the light blocking sheet 30 to rotate by the rotation of the knob 31 on the panel to control the irradiance of the output ultraviolet light.
  • a light-shielding plate can also be set in the optical path between the reflector 18 and the convergence point of the reflected light beam.
  • the light-shielding plate can be controlled by a knob to partially block the light to control the irradiance of the output ultraviolet light ( Figure Not shown).
  • the input optical fiber 4, the input light detection optical fiber 6, and the fluorescence collection optical fiber 5 are quartz optical fibers.
  • the detection end of the input optical fiber close to the target tissue and the input end of the fluorescence collection optical fiber to 0.5 m above it are coaxially arranged as a composite fiber 8, the optical fiber is placed in a hose, and the composite fiber 8 15nm ⁇ The diameter of the part is 2 ⁇ 0. 15nm.
  • the photomultiplier tube 13 of the detection device 3 preferably has a photomultiplier tube with a response range of 300-850nm, a multiplier 8-15 grade, an anode sensitivity of 100-300A / lm, a voltage of 1000V, and a dark current of ⁇ , which is a preferred implementation of the present invention.
  • the photomultiplier tube is a photomultiplier tube of model GBB24 produced by Beijing Nuclear Instrument Factory, and the brightness of the red fluorescence detected by the photomultiplier tube 13 ranges from 0 to 1999 cd / m 2 .
  • the fluorescent brightness is displayed on the fluorescent brightness display 15.
  • the resolution of the fluorescence brightness is 0.01 cd / m 2 .
  • the accuracy indicated by the fluorescent brightness display 15 is: tl O%. '
  • the fluorescence collection optical fiber 5 is a glass fiber.
  • the ultraviolet light intensity photodetector 9 receives the ultraviolet light emitted from the light generating device 1 through the input light detection optical fiber 6 and converts the ultraviolet light through a circuit (A / D converter 46) to display the irradiance of the ultraviolet light in digital form.
  • a circuit A / D converter 46
  • the ultraviolet irradiance at the lcfff exit end of the transmitted light beam that has penetrated into the human body branch can be adjusted as required from 0 to 1.999mW / cm 2 .
  • the accuracy indicated by the ultraviolet display 22 is ⁇ 10%.
  • the ultraviolet irradiance detection device in the further detection device 3 is also provided with an ultraviolet signal analog output port for recording by a recorder or processing data by a computer.
  • the photodetector 9 is a silicon photocell, and the light entering the silicon photocell is filtered by two filters, ZTO and QB21.
  • the photodetector 9 is a UV-A photodetector produced by the Photoelectric Instrument Factory of Beijing Normal University.
  • the detection device 3 includes a fluorescent signal analog output port 40.
  • One end of the analog output port is connected to the output end of the A / D converter 14, and the other end is connected to a recorder or a computer (Fig. (Not shown) for recording data with a recorder or computer processing.
  • the analog output port 40 and the fluorescent brightness display 15 may be set at the same time.
  • an alarm device 41 with an adjustable threshold is provided in the detection device, and a detection model is preset in a circuit 43 of the alarm device. When the brightness of the fluorescence exceeds the prescribed amount of the diagnostic model, the alarm device can be diagnosed as cancerous through the peak sounder 42 connected to it. In this way, the detection process can be simplified, and the requirements for the operational level of the detection operator can be further reduced.
  • the fluorescence brightness detection is divided into three ranges, and the range is selected by a button control on the panel of the detection device.
  • the ultraviolet irradiance detection device of the present invention is calibrated with an ultraviolet standard irradiator.
  • the calibration light source of the fluorescence brightness detection device is an A light source plus a 1.5 mm thick CB550 filter.

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Description

蚕砂提取物、 其制备方法、 用途及诊断或治疗恶性肿瘤的装置 发明领域
本发明涉及一种蚕砂的提取物、 其制备方法、 及其在制备诊断或治疗恶性肿 瘤，特别是早期恶性肿瘤试剂或药物上的用途， 以及一种诊断或治疗恶性肿瘤， 特 别是早期恶性肿瘤的装置。 背景技术
自 70年代， 已经证明恶性肿瘤组织中存在与正常组织的自体荧光不同的特征 荧光， 科学家试图通过荧光光语仪观察并记录肿瘤组织的特征荧光， 来鉴别、 诊 断恶性肿瘤。
筑波大学的蔡承熹等人认为， 在 5145A的氩离子激光器作为激发光源的激发 下， 正常标本和含肿瘤细胞标本产生不同的特征荧光， 二种荧光的光语不同， 因 此可以通过分析光谱来确定是否存在恶性肿瘤； 该方案的明显的缺陷是氩离子激 光器产生的蓝绿可见和被激发的荧光在可见光光奄段叠加。
CN85100242公开的也是一种光谱分析方法， 该方案希望克服因为激发光源能 量弱导致灵敏度低的缺陷， 其公开的方法改进了光源和传输系统， 光源采用 3000 - 4000A 的近紫外光， 既避免激发荧光光潘的叠加， 又可以大大提高激发光源的 能量， 最大程度地激发肿瘤组织的自体荧光， 便于肉眼观察和光谱诊断。
CN97106273认为， 光谱分析法太复杂， 提出一种通过正常和异常组织之间荧 光值的比值来诊断癌症的方法。
但如 CN85100242公开的凭借肉眼观察或光 i普分析的方法及装置在诊断恶性肿 瘤时， 用肉眼观察可能由于色彩失真而导致的误诊， 光 i普分析所用的装置存在着 成本高、 对操作者业务水平要求高的缺陷， 限制其使用， 特别是该装置中对于光 处理系统中的分色系统、 电路系统和扫描系统的要求较高， 容易使信号失真（参 见 ZL87105948背景技术部分）。 CN97106273中公开的方法及装置， 其检测过程及 结果一方面受到肿瘤自体荧光以及正常组织的自体荧光的干扰， 另一方面由于异 常組织的发展从初期到后期， 其荧光值和正常值之间差异性低， 因此准确性不高。 这就导致通过测定固有荧光正常值与同一被测者自身异常部位测出的固有荧光异 常值及它们的比率值来诊断肿瘤， 将受所设定的正常部位干扰， 导致严重的偏差 甚至影响检测和诊断结果。 EP1610616 , JP59 - 40869 提出利用光敏物质血 啉衍生物 ( HPD )在肿瘤细 胞中间的聚积， 采用荧光灯、 泵浦染料激光器照射得到 HPD被激发的荧光， 以此 来判断是否存在肿瘤。
CN87103403认为， 上述方法的缺陷是功率小， 不能照射深层； 该方案提出一 种新的医用脉冲激光装置， 并指出诊断用 405nm波长、 治疗用 630nm波长的激光 光源， 该方案同样使用光敏剂 HPD, 并再次指出， 405nm能够有效激发 HPD特有的 荧光， 且该组织输出的平均功率和峰值适中。
实际上， HPD的过敏性是很强的， 注入 HPD会在其后的数天内不能见光， 这 在一般性检查以及肿瘤检查中是令人无法接受的。
CN94106907认为，现有技术的气体激光很难有一致的波长和简单的波长调节； 因此提出了选用 HPD/PH1126/NPE6作为光敏剂， 采用波长可控的激光机产生激发 光， 通过像分析诊断肿瘤。
但是以激光为激励光源的缺陷是设备庞大、 造价高， 使用要求高。
现有技术和本发明的研究人员都认为， 选用对肿瘤具有亲和力的光敏剂比单 纯比较被激发的正常组织荧光和肿瘤组织荧光具有更多的优点， 因为这样就避免 了肿瘤特征荧光值在肿瘤发展的不同阶段之间的变化， 直接测量光敏剂的被激发 的荧光将使得检测更加灵敏。
即使这样， 如何得到尽可能大的灵敏度仍然是研究人员追求的目标。 获得尽 可能大的灵敏度是单纯通过光敏剂或检测仪器之一难以达到的。
蚕砂是蚕粪便的干燥物， 其中含有大量的叶绿素酸盐、 蛋白质和果胶， 是提 取叶绿素酸盐的便宜、 易得的原料。 一般蚕砂通过酸碱处理， 脱去镁及以酯键相 连的植醇等结构， 降解为相对稳定的叶绿酸混合物。 其水溶液在红光激发下能产 生 660nra左右橙红色荧光， 对于肿瘤具有选择性摄入和光敏特性（胡龙勤等.叶绿 素衍生物在医药上的应用 《国外医学.合成药、 生物药、 制剂分册》， 1987 , 8 : 146 )。 但该混合物不够稳定， 且其单体化合物难以得到。
CN92103649. 3公开了一种应用酶工程技术从蚕砂中制取叶绿素酸盐的方法， 主要通过将蚕砂预处理后在水解酶等作用下水解， 经皂化、 置换等工艺得到叶绿 素酸盐。
CN93103432. 9公开了一种用于光敏疗法的中药光敏剂及其生产工艺， 含有叶 绿三酸及其聚合物 60—80 %。 其生产工艺为以蚕砂为原料， 采用丙酮混合提取， 再经乙醇加 NaOH皂化， 然后酸化去镁， 最后先由硅胶 G拌和吸附， 再分別以石油 醚、 乙醇进行洗脱除杂质、 蒸馏水溶解蒸发精制得成品。 该光敏剂诊治效果好， 临床应用方便， 且生产工艺简单易行， 原料廉价易得。
CN00121046. 7公开了天然绿色着色剂叶绿素锌的制备工艺， 是将蚕砂用有机 溶剂浸提、 上层液浓缩后皂化， 用乙酸锌对皂化液进行置换， 经后处理得油溶叶 绿素锌。
但上述方法在预处理过程中， 都使用有机溶剂浸提， 再进一步降解、 处理， 影响产物在组织内的亲和力和潴留等综合性能。 此外现有技术的蚕砂提取物最佳 激发波长在 405 nm左右， 为了有效激发光敏剂， 要求能产生 405 nm左右光的激 发光源， 限制了使用， 因此开发在体内停留时间短、 在肿瘤组织内潴留浓度高、 激发波长适当的新型光敏剂， 对于癌症的早期诊断和及时治疗是非常有意义的。 发明内容
本发明的主要目的在于提供一种蚕砂提取物， 是一种检测荧光用的光敏剂， 该蚕砂提取物在体内肿瘤细胞中摄入好、 潴留部位明显 , 尤其在特定的光源激发 下产生强烈的特征荧光， 通过对该特征荧光的检测和亮度的测定， 能够识别细胞 的变异， 因而可以诊断组织的早期癌变以及恶性肿瘤。
本发明的另一个目的在于提供上迷蚕砂提取物的制备方法，该方法简单易行， 得到有效的具有检测特异性的光敏组合物。
本发明的再一个目的在于提供上述蚕砂提取物在制备诊断或治疗早期恶性肿 瘤试剂或药物上的用途。
本发明还有一个目的在于提供一种诊断或治疗早期恶性肿瘤的装置， 该装置 能够产生激发本发明蚕砂提取物产生特征荧光的激励光及接收、 检测和处理该特 征荧光的检测系统， 能够高效、 高准确率地分辨组织的早期癌变 （癌前期病变）； 另外该装置激发光源的辐照度可调， 使得检测结果重现性高， 此外该装置结构简 单、 生产成本低， 操作及诊断容易。
本发明的目的可以通过以下方式得以实现：
一种蚕砂提取物， 是蚕砂降解物的钠或钾盐， 其水溶液在 365 ± 15nm 的近紫 外光源激发下在 660nm ± 15nm的波长下具有最强的荧光。 优选激发波长为 365 ± 10nm, 发射波长 660nm ± 10nm。 更优选激发波长为 365nm, 发射波长 660nm。
本发明的蚕砂提取物中包括母核为二氢卟吩的具有环戊酮结构的二氢卟吩类 的钠或钾的配合物， 其含量约为 30- 58wt°/。，优选含量为 40- 50wt°/。。 本发明的蚕砂提取物的有效成分为脱镁叶绿酸 a、脱镁叶绿酸 b、 焦脱镁叶绿 酸&、 紫红素 -18、 二氢卟吩 IV 二氢卟吩^ 二氢卟吩 ^等脱镁叶绿环类及其盐 的混合物。
优选本发明的蚕砂提取物其水溶液在 365 ± 15nm 的近紫外光源激发下在 660nm土 15nm的波长范围内与空白对照相比具有至少 8 - 12倍的荧光值比率； 优 选的激发波长为 365土 l Onm,发射波长 660nm ± l Onm,至少 9 - 12倍的荧光值比率； 更优选激发波长为 365nffl， 发射波长 660nm, 至少 10 - 12倍的荧光值比率。
本发明的蚕砂提取物的制备方法包括： 将蚕砂或粗蚕砂提取物皂化、 后处理 和成盐， 其中的皂化在醇和碱的水溶液中加热下进行， 后处理为过滤、 浓缩并用 非极性有机溶剂提取皂化液以弃去不皂化物， 用酸酸化皂化液； 再成盐得到蚕砂 降解物的盐。
上述粗蚕砂提取物为采用现有技术的方法通过碱和 /或酸降解蚕砂制备的。 用非极性有机溶剂提取皂化液的浓缩液中的不皂化物是为了充分提取其中的 酯溶性及其他不不皂化物物质， 得到完全水溶的产物。
在本发明的一个优选实施例中， 蚕砂提取物是通过下述方法制备的蚕砂提取 物： 将蚕砂皂化、 提取、 酸化、 碱化成盐， 其中：
1 )蚕砂提取物皂化后过滤， 将滤液浓缩， 得浓缩液；
2 )用非极性有机溶剂提取浓缩液中的不皂化物， 去溶剂得皂化液；
3 )用酸酸化皂化液， 沉淀出酸化的皂化物；
4 )将沉淀出的酸化皂化物与碱或盐反应， 得到叶绿素酸盐后过滤， 将滤液浓 缩， 得浓缩的蚕砂提取物液；
制备过程中， 皂化是在蚕砂中加入 1-8倍的醇和 5- 40wt°/。碱的水溶液至 PH值 为 9-12 , 在加热回流下进行。
其中的碱为 NaOH或 KOH, NaOH或 K0H的用量为蚕砂重量的 3-30wt%。
皂化的时间为 4- 10小时。
将皂化液过滤后， 将滤液浓缩至原液体积的 1/2至 1/5。 其中的醇为甲醇、 乙醇、 丙醇或丁醇， 优选乙醇。
PH优选控制在 9. 5-10. 5 , 更优选 PH为 10。
在用非极性有机溶剂提取浓缩液中的酯溶性物质前， 还可以首先用水溶解浓 缩液， 然后再用非极性有机溶剂提取溶解液中没有被皂化的物质或酯溶性杂质， . 弃溶剂层， 取下层皂化液， 溶剂回收再利用。 . 溶解浓缩液的水的用量以及提取时溶剂的用量为浓缩液的卜 15倍，提取次数 为 1-4次， 或提取至提取液无色。
其中， 提取所用的非极性有机溶剂为石油醚、 乙醚、 正已烷、 环己烷、 四氯 化碳、 氯仿、 苯或取代苯， 优选石油醚、 乙醚、 正已烷、 环己烷； 最优选石油醚。
提取后用酸酸化下层皂化液， 至 PH为 3-5 , 弃上清液， 收取沉淀出的酸化的 皂化物。
酸化采用盐酸、硫酸、硝酸的稀溶液，优选盐酸。且优选酸化至 PH为 3. 5-4. 5。 最后将沉淀出的酸化皂化物与氢氧化钾、 氢氧化钠、 碳酸钾、 碳酸钠、 碳酸 氢钠、 碳酸氢钾反应成盐后过滤.， 将滤液浓缩或进一步干燥得到蚕 是取物。
成盐反应优选在醇和 1- 30wt%的碱或盐的水溶液中进行， 醇的用量为蚕砂的
1-5倍， 碱或盐的用量为蚕砂的 1- 25wt%。
反应在 PH 9-12下进行， 优选的 PH为 9-10。
反应后加热回收乙醇， 剩余物过滤、 浓缩得到膏状浓缩提取物， 再采用现有 技术中公开的方法干燥或制成各种制剂。
成盐反应一般在室温下充分搅拌就可以完成。 如果有必要， 也可以加热。 本发明的制备方法中， 由于直接将蚕砂皂化， 皂化后采用非极性溶剂提取其 中的酯溶性杂质， 然后酸化、 成盐等方法， 去除蚕砂中的镁、 蛋白和酯溶性杂质， 将蚕砂充分降解为脱镁叶绿酸 a、 脱镁叶绿酸 b、 焦脱镁叶绿酸 a、 紫红素 -18、 二氢卟吩 P6、 二氢卟吩 e6、 二氢卟吩 ^等脱镁叶绿环类盐的混合物， 得到叶绿素 酸盐含量高的蚕砂提取物， 在 365土 l5nm的近紫外光源激发下在 660nm ± 15nm的 波长下具有最强的荧光。 本发明的蚕砂提取物在水中能够完全溶解， 有利于在体 内肿瘤组织中特异的聚集、 吸收， 而在正常组织内则容易消化及排除， 无任何毒 副作用。
对于现有技术中公开的碱降解得到的各种粗提物， 将其用非极性有机溶剂提 取后酸化、成盐，也可以得到接近本发明性质的产品，如中国专利申请 93103432. 9 中公开的制备方法中， 在酸化前提取， 酸化后加入碱或盐成盐， 也可以得到本发 明的蚕 '提取物。
本发明的蚕砂提取物液采用现有技术的方法干燥， 可以得到干燥的蚕砂提取 物， 加入蒸熘水可以配制为一定的浓度的溶液， 并根据现有技术的方法， 辅以药 剂学上可接受的辅料， 制备为药剂学上可接受的任何剂型， 如散剂、 胶嚢、 丸剂、 片剂、 口月艮液、 注射液、 喷雾剂或涂敷剂。 在临床上， 早期的肿瘤组织很难和正常組织区分， 这就是采用自体荧光误诊 率高的原因， 虽然现有技术提出了在波长可调的激光激发下采用脱镁叶绿酸和叶 绿三酸作为光敏剂具有更高的灵敏度（CN94106907 ), 但是， 本发明提出的采用近 紫外光（例如汞灯产生）激发本发明所述的蚕砂提取物， 具有更加显著的特异性 和高灵敏度， 而且大大简化检测装置和对于操作者诊断水平的要求。
大量的动物实验表明， 本发明的蚕砂提取物作为光敏剂仅仅在一定时间内停 留在肿瘤细胞或癌前期病变等处， 因此， 理论上讲， 能否检测到荧光就意味着有 或没有肿瘤细胞， 但实际上正常细胞和肿瘤细胞本身也能够在近紫外光的激发下 发出荧光， 在近紫外光激发下， 注入的光敏剂的荧光值与正常细胞被激发产生的 荧光值的比值越大， 就意味着灵敏度更高、 检测结果更加准确， 本发明所述的光 敏剂在所述的近紫外光的激发下具有正常细胞至少 5 - 12倍甚至更高的荧光亮度 值。
作为一种光敏剂， 本发明的蚕砂提取物在检测的同时， 还可以通过杀灭癌细 胞进行光动力学治疗。
本发明提取物在同样强度的紫外光辐照强度下产生荧光亮度更高的原因有 二， 其一是所需的激发能量更低， 其二是所述的蚕砂提取物对肿瘤的特异性和亲 和力更强， 因此在肿瘤部位聚积的浓度更高。
当然， 获得这样的显著差异必不可少的是本发明所述诊断或治疗早期恶性肿 瘤的装置的配合。
本发明所述的装置包括： 辐照度可调的、 输出紫外光的光发生装置、 光传输 装置、 紫外辐照度检测装置和荧光检测装置， 光传输装置包括输入光导纤维、 荧 光采集光导纤维和输入光检测光导纤维； 光发生装置发出的紫外光经与之相连的 输入光导纤维和输入光检测光导纤维分别被传送到位于靶組织的输入光导纤维的 探测端部和荧光检测装置中用于检测光发生装置发出的紫外辐照度的光电探测器 内， 荧光采集光导纤维的输入端与接近靶组织的输入光导纤维的探测端部分同轴 混排为复合纤维， 并将靶組织产生的特征荧光经荧光采集光导纤维的输出端传送 至荧光检测装置中用于检测荧光亮度的光电倍增管， 光电倍增管输出的荧光信号 经电子线路处理后将荧光亮度以数字形式显示在荧光亮度显示窗口上， 或光电倍 增管输出的荧光信号经电子线路处理后输出到用于记录或数据处理的记录仪或计 算机。 紫外辐照度的光电探测器接收的紫外光经电路转换后， 将紫外辐照度以数字 形式显示在探测器中的紫外显示器上， 或输出到用于记录或数据处理的记录仪或 计算机内。
光电探测器探测紫外光的波长范围为 320- 400nm。
在光发生装置内， 或光发生装置与输入光导纤维连接之处或输入光导纤维的 输入端部设置一第一滤光装置。 经滤光片滤色后， 传送到输入光导纤维输入端的 为 365 ± 15nm的窄带紫外辐射， 优选输入 365 ± l Onra的窄带紫外辐射。
所述第一滤光装置为两个滤光片。
在荧光采集光导纤维的输出端与荧光检测装置中的光电倍增管之间设置一第 二滤光装置。 第二滤光装置为仅允许 600— 750腿波长的红荧光通过的滤光片， 其 目的在于滤除紫外辐射光、 自体荧光和其他散射或叠加等各种杂光对检测精度的 干扰， 避免紫外辐射光、 自体荧光干扰检测结果。 优选检测装置检测波长 610— 700nm处的荧光亮度， 更优选的波长为 650 ± 15nm。
本发明的诊断或治疗早期恶性肿瘤的装置的中的荧光检测装置中的光电倍增管 的响应范围为 300— S50nm, 阳极灵敏度 100—300A/lm。
光发生装置输出辐照度在大于 0至小于 1. 999mW/cm2的辐照度可调的近紫外 光。 如果第一滤光装置设置在光发生装置之内， 则光发生装置输出波长为 365士 15nm、 辐照度为大于 0至 1. 999mW/cm2的辐照度可调的近紫外光。
光发生装置包括： 设置在其内的一发出光源的高压汞灯、 一抛物面镜、 一反 射镜和一滤光装置， 高压汞灯发出的辐射光经抛物面镜汇聚到反射镜上， 经反射 镜反射、 聚焦至滤光装置， 经滤光装置过滤后， 至输入光导纤维的输入端。 其中 高压汞灯、 抛物面镜和反射镜设置在机箱内， 滤光装置设置在机箱内、 输入光导 纤维的输入端或输入光导纤维内的某一位置。
为了便于连接， 在机箱的光输出的侧壁上设置一石英光棒， 石英光棒的一端 接收紫外光， 另一端与光导纤维的输入端直接相连或通过滤光装置与光导纤维的 输入端连接。 这样经反射镜反射的紫外光聚焦至石英光棒， 经第一滤光装置进入 输入光导纤維的输入端。 当然第一滤光装置也可以设置在石英光棒接收紫外光的 一侧。
抛物面镜的内表面覆盖有只反射紫外光而将其他长波的光都穿过的反射膜。 所述光发生装置还设置有增、 减光的控制器， 使光发生装置输出辐照度大于
0小于 1. 999mW/cm2的近紫外光。 在本发明的一个实施例中， 控制器类似一个可控 电源开关， 通过控制输入电流的大小实现光的增减， 在本发明的另一个实施方式 中， 在石英光棒的前部设置有可调节大小的窗口控制光的增减。 在本发明的优选 实施方式中， 通过控制抛物面反光碗光束会聚点上的楔形光栏进行紫外光量的调 节。
光发生装置还设置有冷却用的风扇。
其中输入光导纤维和输入光检测光导纤维具有一个共同的光输入端口。 所迷 光导纤维均为石英光导纤维， 但也可以将其中的荧光采集光纤改为玻璃纤维。
自输入光导纤维的接近耙组织的探测端部和荧光采集光导纤维的输入端部至 其上 0. 3- - 1腿处， 输入光导纤维和荧光采集光导纤维两束纤维同轴混排设置， 也 可以根据检测的被检测部位在体内的深度而定。
所述荧光检测装置检测的红荧光的亮度范围为 0-1999cd/m2。
优选荧光检测装置中光电倍增管输出的荧光的电信号经过电子线路 /D 转 换器）处理后， 将荧光的亮度显示在荧光亮度显示器上。 这时， 荧光检测装置中 还进一步包括一个荧光信号模拟输出端口， 模拟输出端口的一端与 A/D转换器的 输出端相连，另一端与记录仪或与计算机相连接， 用于通过记录仪记录或计算机 处理数据。
具体诊断过程为： 提前将本发明的蚕砂提取物通过注射、 口服或雾化等方式 注入到人体内 , 用复合纤维代替内窥镜的活检装置并探入体内或体表， 利用光发 生装置发出的、 经输入光导纤维传导的一定辐照度、 波长 365 ± 15nm的非激光光 源对可疑的部位（靶組织）进行照射， 激发进入异常组织 （靶组织） 的蚕砂提取 物产生强烈的特征荧光， 荧光釆集光导纤维将荧光传送至光电倍增管， 经过电子 线路（A/D 转换器） 处理后， 将荧光的亮度显示在荧光亮度显示器上， 通过对荧 光的识别和亮度测定来判别是否出现癌变、 结合内窥镜观察肿瘤的位置和大小， 实现早期癌症的诊断。 检测时， 可以在 0至 1. 999mW/cm2的范围内调整紫外光的 辐照度， 显示在整个量程内的紫外亮度， 也可以将紫外光的辐照度固定在一个固 定的数值， 这样可以得到高的重现率， 且便于同一患者的跟踪。
本发明的装置也可以单独使用。
本发明的研究人员意外地发现， 按本发明所述方法制备的， 不同于现有技术 指出的脱镁叶绿酸和叶绿三酸的蚕砂提取物， 配合本发明所述的装置并采用所述 的各项参数， 在动物实验中， 对于胃癌模型小鼠及健康小鼠对照实验发现， 肿瘤 组其组织的荧光值与正常组組织内的荧光值之间的比值在 8倍以上， 进一步的对 比实验发现， 现有技中的各种光敏剂对于胃癌模型小鼠及健康小鼠其荧光值之间 的比值小于 3倍。 这种差异突显出本发明装置和光敏剂的高度灵敏性。 一个月内 跟踪检测三次还发现， 采用本发明的蚕砂提取物及其装置在检测癌变的同时还具 有改善病变组织的作用， 特别是对于早期的组织变异， 效果尤其明显， 说明本发 明的蚕砂提取物同时还具有光动力学上的治疗效果。 另外使用本发明的蚕砂提取 物无须避光等其他条件， 使用方便、 无毒副作用， 患者容易接受。
本发明装置的特点之一是， 在传输近紫外光的光纤的适当部位， 设置一近紫 外光辐照检测装置， 该检测装置实际得到了光纤中近紫外光的辐照度， 经电路转 换后， 将紫外光的辐照度以数字形式显示在探测器中的紫外显示器上。 对同一组 织的不同部位或不同器官可以准确调节紫外光辐照强度， 并在同一或不同个体的 检测中始终将紫外光的辐照度固定在一个特定的数值上， 这样检测结果具有重现 性， 并有利于治疗效果及病变发展的跟踪检查。 特点之二是不彩用光谱分析的方 法诊断恶性肿瘤。
该检测装置的优点是， 在逐步增加紫外光辐照强度过程中， 可以直接检测光 敏剂被激发的荧光值也相应增加； 另外对不同的部位或器官可以准确调节紫外光 辐照度， 确保在同一光源的使用期限内， 紫外光辐照度通过调整而能够始终保持 一致。
而这， 很好地克服了现有技术的缺陷， 即， 激发光的辐照度过大导致的本底 值过大无法检测， 和激发光的辐照度过小导致荧光亮度太低检测不到。 这两种情 况都直接影响检测结果的准确性。 此外本发明的装置还克服了现有技术中激发光 的辐照度不能够调整而导致的随着使用时间的增加， 紫外辐照度降低， 而装置中 其他条件不变， 检测结果不具有重现性或容易出现漏检的缺点。
进一步， 本发明中在检测装置中设置有一报警装置， 报警装置内预设定检测 模型。 当荧光亮度超过诊断模型规定量时， 报警装置通过其连接的峰鸣器报警， 即可诊断为癌变， 釆用这种方式， 能够简化检测过程， 进一步降低对于检测操作 人员业务水平的要求。
虽然生物体内存在自体荧光， 但至今为止， 尚未有正常组织的自体荧光超过 600nm 的报道。 本发明中在荧光采集光纤的输出端与检测装置中的光电倍增管之 间设置一仅允许 600- - 750nm 波长的红荧光通过的第二滤光片， 滤除了紫外辐射 光、 自体荧光， 排除了各种因素对于检测结果的影响， 因此可以确定所检测到的 荧光都是靶组织内光敏剂发出的荧光。 本发明装置对于荧光亮度的分辨率为 0. 01cd/m2, 又由于本发明光敏剂的在变异组织的强选择性亲和、 吸附， 正常组织 内的正常代谢， 因此即使对于早期的细胞变异， 检测的准确率可以达到 98°/。。
其中所述光敏剂优选采用本发明所述的蚕砂提取物。 用量为 0. 1- 100 μ g/kg 体重， 未吸收的蚕 、提取物 12小时后自然从体内排出。 在癌变部位潴留 12-24小 时。
本发明的装置配合内窥镜进行肉眼分辨， 由于其荧光值更高， 因此比现有技 术更加容易分辨和定位。
本发明可适用于各种癌症的早期诊断， 尤其是已经具有内窥镜检测的各器官 内癌变的检测及早期诊断， 如可疑肺癌、 胃癌、 膀胱癌、 子宫癌、 前列腺癌、 直 肠癌， 皮肤癌等部位的检测及癌前期病变的诊断。 特别是采用本发明的蚕砂提取 物作为光敏剂时， 由于其灵敏度高， 即使早期的轻微組织变异都能够产生潴留， 能极大低提高早期癌变组织的诊断率。
本发明的装置克服了现有技术中采用激光光源及光谱分析设备庞大、 价格昂 贵、 使用不便的缺陷， 以及紫外光源检测不准确的， 操作水平要求高的缺陷， 提 供的诊断或治疗早期恶性肿瘤的装置体积小， 价格便宜， 操作简单， 通过特征荧 光的绝对值诊断恶性肿瘤和早期癌变， 通过动物实 证明准确率极高。 具体实施方式
下面结合附图和具体实施例详细描述本发明， 所述实施例用于描述本发明， 而不是限制本发明。
附图说明
图 1是本发明诊断或治疗肿瘤装置的原理图
图 2是本发明诊断或治疗肿瘤装置的结构图
图 3是本发明诊断或治疗肿瘤装置的另一实施例的结构图
实施例 1
取 lkg蚕砂粉末， 先用 5000ml甲醇浸没， 再加入 20^%的 NaOH溶液 250ml , 控制 PH值在 10左右， 回流下搅拌 8小时， 抽滤， 收取滤液， 浓缩至原液的三分 之一， 加入浓缩液 5倍量的热水充分搅拌， 静止， 取上清液， 加入等体积的环已 烷萃取不皂化物至环己烷层无色， 分液， 取下层皂化液， 加入浓度为 20wt。/。的 HC1 调 PH至 3左右， 离心收取沉淀， 沉淀物加 15wt。/。的 ¾0H溶液 20 Oml及 95%的乙醇 2000ml搅拌溶解， PH控制在 11左右， 搅拌加热回收乙醇， 过滤， 弃沉淀， 滤液 浓缩， 浓缩物经干燥得 18g左右墨绿色蚕砂提取物粉末。
lg墨绿色蚕砂提取物粉末加 100ml蒸餾水溶解， 采用制剂学上的现有工艺制 成含量为 100mg/ml的注射液。
实施例 2
取 1kg 40目蚕 、粉末， 加 95%乙醇 3000ml和 10%的 NaOH 800ml溶液， 搅拌 回流， 反应过程中控制 Μ值在 11左右， 回流 6小时， 抽滤， 收取滤液， 浓缩至 原液的四分之一， 加入 10倍量的热水充分搅拌， 静止， 分取上清液， 加入等体积 的石油醚萃取不皂化物至石油醚层无色， 分液， 取下层皂化液， 加 30%HC1调 PH 至 4左右， 有沉淀析出， 离心收取沉淀， 沉淀物加 10%的 NaOH溶液 200ml及 95% 的乙醇 1500ml溶解， ra控制在 10左右， 充分搅拌后， 加热回收乙醇， 过滤， 滤 液浓缩， 浓缩物经干燥得 20g墨绿色蚕砂提取物粉末。
上述蚕砂提取物粉末加入淀粉等辅料， 根据现有技术的方法制备为有效成分 含量为 5rag/片的片剂或 5tng/袋的颗粒。
实施例 3
取 1kg蚕 、粉末， 加 lOQml水软化， 再加 70%乙醇 8000ml和 30%的 K0H水溶 液 200ml , 搅拌回流， 反应过程中控制 PH值在 12左右， 回流 4小时， 抽滤， 收 取滤液， 浓缩至原液的一半， 加入 12倍量的热水充分搅拌， 静止， 分取上清液， 加入等体积的苯萃取不皂化物至苯层无色， 分液， 合并下层皂化液， 下层皂化液 加 5%硫酸调 PH至 5左右， 有沉淀析出， 离心收取沉淀， 沉淀物加 10%的 NaOH溶 液 100ml及 95%的乙醇 1000ml溶解， PH控制在 9左右， 搅拌， 加热减压回收乙 醇， 过滤， 滤液浓缩至得到膏状蚕砂提取物。
取膏状蚕砂提取物加蒸馏水， 采用制剂学上的现有工艺制备为 3-20mg/ml 口 服液。 采用荧光光度法测定含量。
实施例 4
取 1kg蚕砂粗粉， 加 500ml水软化， 再加 95%乙醇 1000ml和 5%的 NaOH 溶液
300ml , 搅拌回流， 反应过程中控制 PH值在 9. 5左右， 回流 10小时， 抽滤， 收取 滤液， 除乙醇， 加入 15倍量的热水充分搅拌， 静止， 分取上清液， 加入 3倍体积 的苯萃取不皂化物至苯层无色， 分液， 取下层皂化液， 加 30%的 NaOH (或 K0H ) 溶液 100ml及 95%的乙醇 3000ml,使 PH控制在 10左右，抽滤， 收取滤液， 减压下 加热除乙醇，加 30%盐酸调 PH至 5. 5左右， 析出沉淀， 离心， 收取沉淀， 水洗沉 淀物， 干燥,得到 30g墨绿色蚕砂提取物粉末。 上述蚕砂提取物粉末加入辅料， 根据现有技术的方法制备为片剂或喷雾剂。
实施例 5
取 1kg蚕砂粗粉， 加 95%乙醇 2000ml和 8%的 NaOH溶液， 搅拌回流， 反应过 程中控制 PH值在 9. 0左右， 回流 6小时， 抽滤， 收取滤液， 除乙醇， 加入 2倍量 的热水充分搅拌， 静止， 分取上清液， 加入 3倍体积的石油醚萃取不皂化物至石 油醚层无色，分液，取下层皂化液，加 30%的 NaC03溶液 300ml及 95°/。的乙醇 3000ml, 使 PH控制在 10左右，抽滤，收取滤液，減压下加热除乙醇，加 30%盐酸调 PH至 5. 5 左右， 析出沉淀， 离心， 收取沉淀， 水洗沉淀物， 干燥,得到 30g 墨绿色蚕矽、提 取物粉末。
上述实施例中， 成盐一步中也可以将 Na2C03改为 K2C03、 NaHC03或 KHC03,得到 相应的蚕砂降解的叶绿环类的 K或 Na盐。
实验例 1
含量测定：
称取蚕砂提取物粉末 l Dmg加生理盐水配制成 100ml , 得 Q. Img/ml的标准溶 液，分别配制成分别配置成以下浓度 50ug/ml、 30ug/ml、 1 Oug/ml、 8ug/ral、 5ug/ml、 3ug/mK lug/ml 0. lug/mK 0. 05ug/ml。
激发条件； 激发波长为 365腿， 带宽均为 10胆。 以 65 Onm处的荧光亮度为纵 坐标， 浓度为横坐标制作标准曲线， 分别用不同浓度测定其荧光强度值， 制成标 准曲线。
取样品 3. 5mg左右，精密称定，置 10ml量瓶中加生理盐水溶解并稀释至刻度， 摇匀， 精密吸取 0. 1ml供试溶液， 加生理盐水 9. 9ral , 摇匀， 测定荧光强度， 通 过查标准曲线来计算样品含量。
实验例 2
本实验例涉及本发明蚕砂提取物的毒性
采用实施例 1的方法制备的蚕砂提取物。
动物： 昆明种小鼠， 雌雄各半。
最大给药量测定， 取上述健康小鼠 60只， €机分为两组， 雌雄各半， 给药組 小鼠一次口服本发明的蚕砂提取物 25g/kg, 对照組不服用任何药物， 记录以上两 组给药前后第四天及第十天的体重与临床表现， 结果表明， 小鼠一次口服最大剂 量的蚕砂提取物 25g/kg, 未出现死亡， 未发现与给药有关的临床表现， 也未发现 给药对体重的影响， 说明人临床拟用剂量是一个安全剂量。 实施例 6
参见图 1、 2。 图 1中， 输入光导纤维和输入光检测光导纤维被统称为激发光 纤、 荧光采集光导纤维也就是收集光纤， 其中的光电转换所用装置为光电倍增管。 本发明的诊断或治疗恶性肿瘤的装置包括： 输出可调辐照度紫外光的光发生装置 1、 光传输装置 2和检测装置 3 , 检测装置 3中包括紫外辐照度检测装置和荧光检 测装置。 光传输装置包括输入光导纤维 4、 荧光采集光导纤维 5和输入光检测光 导纤维 6; 输入光导纤维 4和输入光检测光导纤维 6具有一个共同的光输入端部 21。 光发生装置 1发出的紫外光经与之相连的输入光导纤维 4和输入光检测光导 纤维 6的端部分别被传送到位于靶组织 7的输入光导纤维的探测端部 8和检测装 置 3中用于检测光发生装置 1发出的紫外光辐照度的紫外辐照度检测装置中的光 电探测器 9内，荧光采集光导纤维 5的输入端 10与接近靶组织 7的输入光导纤维 的探测端部 8部分同轴混排为复合纤维 11， 荧光采集光导纤维 5的输出端 12与 检测装置 3中用于检测荧光强度的荧光检测装置中的光电倍增管 13相连接，光电 倍增管 13输出的荧光信号经电子线路 14处理后显示在荧光亮度显示窗口 15上。
参见图 3 , 在本发明的另一个实施方式中， 检测装置 3 包括两个独立设置的 紫外辐照度检测装置 300和荧光检测装置 301。 而图 2 中， 紫外辐照度检测装置 和荧光检测装置共同设置在检测装置 3中。
在荧光采集光导纤维的输出端与荧光检测装置中的光电倍增管之间设置一第 二滤光装置 45。 第二滤光装置 45为仅允许 600- - 750nm波长的红荧光通过的滤光 片， 以防止紫外辐射进入光电倍增管冲淡荧光信号的纯度。
光发生装置包括一发出光源的高压汞灯 16、 一抛物面镜 17、 一反射镜 18、 石英光棒 1 和滤光装置 20 , 高压汞灯 16发出的辐射光经抛物面镜 17汇聚后投 射至呈 45度角设置的反射镜 18上， 经反射后至石英光棒 19 , 经光过滤装置 20 过滤后， 波长为 365 + 15nm的窄带紫外辐射进入具有共同输入端口的输入光导纤 维 4和荧光检测光导纤维 6的输入端。 抛物面镜 17的 表面覆盖有只反射
330-400nm波长的近紫外光而将其他光透过的反射膜。 滤光装置 20包括两个滤光 片， 经滤光片滤色后， 传送到.输入光导纤维输入端的为 365 + 15nm的窄带紫外辐 射， 优选 365 ± 10nm。
在本发明的一个实施例中，光发生装置内的高压汞灯 16电连接一个可调控制 开关 （图中未示）， 通过控制输入电流的大小调整输出近紫外光的辐照度从 0 到 1. 999mW/cm2。 在本发明的另一个实施例中， 在石英光棒 19 的前部设置有类似于 照相机快门的可调节进光量的窗口以控制光的增减， 也可以参照图 1所示在石英 光棒 19的前部设置一挡光片 30, 挡光片 30呈扇形、 半圆形或楔形光栏， 通过面 板上的旋扭 31的旋转带动挡光片 30转动， 以控制输出紫外光的辐照度。
作为一种变换， 还可以在反射镜 18与其反射光束的汇聚点之间的光路内， 设 置一遮光板， 通过旋钮控制遮光板动作， 部分遮挡光线， 以控制输出紫外光的辐 照度（图中未示）。
输入光导纤维 4、 输入光检测光导纤维 6和荧光采集光导纤维 5为石英光导 纤维。 输入光导纤维的接近靶组织的探测端部和荧光采集光导纤维的输入端部至 其上 0. 5m处二者同轴混排设置为复合纤维 8 , 光纤被放置在软管内， 复合纤维 8 部分的直径为 2士0. 15nm。检测装置 3的光电倍增管 13优选响应范围为 300- 850nm, 倍增极 8-15级，阳极灵敏度 100-300A/ lm,电压 1000V,暗电流卜 Ι ΟηΑ的光电倍增 管，在本发明的优选实施方式中， 光电倍增管为北京核仪器厂生产的型号为 GBB24 的光电倍增管， 光电倍增管 13检测的红荧光的亮度范围为 0- 1999cd/m2。 检测装 置 3中光电倍增管 13输出的荧光的电信号经过电子线路 14 /D转换器）处理后， 将荧光亮度显示在荧光亮度显示器 15上。 其中荧光亮度的分辨率为 0. 01 cd/m2。 荧光亮度显示器 15指示的准确度为： tl O%。 '
在本发明的另一种实施方式中， 荧光采集光导纤维 5为玻璃纤维。
紫外光强度的光电探测器 9通过输入光检测光导纤维 6接收光发生装置 1发 出的紫外光， 经电路转换（ A/D转换器 46 ), 将该紫外光的辐照度以数字形式显示 在检测装置 3中的紫外显示器 11上。探入人体分支的传光束出端 lcffl处的紫外辐 照度从 0至 1. 999mW/cm2可以按需要调节。 紫外显示器 22指示的准确度为 ±10%。 进一步检测装置 3 中紫外辐照度检测装置中还设置有紫外信号模拟输出端口， 用 于通过记录仪记录或计算机处理数据。
光电探测器 9 为硅光电管， 进入硅光电管的光经过 ZTO、 QB21两个滤光片滤 光。 在本发明的优选实施方式中， 光电探测器 9为北京师范大学光电仪器厂生产 的型号为 UV- A光电探测器。
在本发明的另一个实施方式中， 检测装置 3包括一个荧光信号模拟输出端口 40, 模拟输出端口的一端与 A/D转换器 14的输出端相连，另一端与记录仪或计算 机相连接（图中未示）， 用于通过记录仪记录或计算机处理数据。 也可以模拟输 出端口 40和荧光亮度显示器 15同时设置。 本发明中在检测装置中设置有一阀值可调的报警装置 41 ,报警装置的电路 43 内预设定检测模型。 当荧光亮度超过诊断模型规定量时， 报警装置通过其连接的 峰鸣器 42报警， 即可诊断为癌变， 采用这种方式， 能够简化检测过程， 进一步降 低对于检测操作人员业务水平的要求。
在本发明的另一个实施方式中， 荧光亮度检测分为三个量程， 通过检测装置 的面板上的按扭控制选择量程。
本发明的紫外光辐照度检测装置釆用紫外标准辐照仪定标， 荧光亮度检测装 置定标光源为 A光源加 1. 5mm厚的 CB550滤光片。
至于光源内各结构的固定方式， 可以参考 ZL97106273. 0 ZL85100424 Z100216974. 6中的内容。
经动物实验及临床实验表明， 本发明的光敏剂及检测装置， 对于恶性肿瘤检 测的准确率可达 92-98%,对于癌前期病变的检出率可达 89-93%。该仪器结构筒单、 操作容易， 成本低， 是一种有前途的医用诊断、 治疗仪。

Claims

权 利 要 求
1.一种蚕砂提取物， 其特征在于所述的蚕砂提取物是蚕砂降解物的钠或钾盐， 其水溶液在 365 土 15nra的近紫外光源激发下在 660nm ± 15nm的波长范围内与空白 对照比具有 8 - 12倍的荧光值比率；优选的激发波长为 365 ± l Onm，发射波长 Onm ± 10nm, 9 - 12倍的荧光值比率； 更优选激发波长为 365nm, 发射波长 660nm, 10 - 12倍的荧光值比率；
2.根据权利要求 1所述的蚕砂提取物， 其特征在于所述的提取物包括脱镁叶 绿酸 a、 脱镁叶绿酸 b、 焦脱鎂叶绿酸 a、 紫红素 -18、 二氢卟吩 P6、 二氢卟吩 e6、 二氢卟吩 e4等母核为二氢卟吩的具有环戊酮结构的二氢卟吩类的钠或钾的配合 物， 配合物的含量约为 30- 58wt%, 优选含量为 40- 50wt%。
3.—种蚕砂提取物， 是通过下述方法制备的： 将蚕砂皂化、 提取、 酸化、 成 盐， 其中：
1 )蚕砂提取物皂化后过滤， 将滤液浓缩， 得浓缩液；
2 )用非极性有机溶剂提取浓缩液中的不皂化物， 去溶剂得皂化液；
3 )用酸酸化皂化液， 沉淀出酸化的皂化物；
4 )将沉淀出的酸化皂化物与碱或盐反应， 得到叶绿素酸盐后过滤， 将滤液浓 缩， 得浓缩的蚕砂提取物液；
4.根据权利要求 3所述的蚕砂提取物， 其中皂化是在蚕砂中加入 1-8倍的醇 和浓度为 5- 40wt°/。的碱的水溶液至 PH值为 9-12 , 及加热回流下进行， 反应时间为
4-10小时。
5. 根据权利要求 3或 4所述的蚕砂提取物， 其中步驟 2 ) 中， 用 3-15倍的 水溶解浓缩液， 用非极性溶剂提取溶解液上清液中没有被皂化的物质， 取下层皂 化液。
6. 根据权利要求 5所述的蚕砂提取物， 步骤 3 ) 中， 用酸酸化皂化液， 至 HI 至 3-5 , 弃上清液， 收取沉淀出的酸化皂化物。
7. 根据权利要求 6所述的蚕砂提取物， 将沉淀出的酸化皂化物与氢氧化钾、 氢氧化钠、 碳酸卸、 碳酸钠、 碳酸氢钠、 碳酸氢钾反应成盐后过滤， 将滤液浓缩 或进一步干燥得到蚕砂提取物。
8. 一种诊断或治疗肿瘤的装置， 包括： 辐照度可调的、 输出紫外光的光发生 装置、 光传输装置、 辐照度检测装置和荧光检测装置， 光传输装置包括输入光导 纤维、 荧光采集光导纤维和输入光检测光导纤维； 光发生装置发出的紫外光经与 之相连的输入光导纤维和输入光检测光导纤维分别被传送到位于靶组织的输入光 导纤维的探测端部和荧光检测装置中用于检测光发生装置发出的紫外辐照度的探 测器内， 荧光采集光导纤维的输入端与接近靶组织的输入光导纤维的探测端部分 同轴混排为复合纤维， 并将靶组织的光敏剂产生的特征荧光经荧光采集光导纤維 的输出端传送至荧光检测装置中的光电倍增管中， 光电倍增管输出的荧光信号经 电子线路处理后将荧光亮度以数字形式显示在荧光亮度显示窗口上， 或光电倍增 管输出的荧光信号经电子线路处理后输出到用于记录或数据处理的记录仪或计算 机。
9. 根据权利要求 8所述的诊断或治疗肿瘤的装置， 其中紫外辐照度的探测器 接收的紫外辐照度经电路转换后， 将紫外辐照度以数字形式显示在探测器中的紫 外显示器上， 或输出到用于记录或数据处理的记录仪或计算机内。
10. 根据权利要求 8或 9所述的装置，在光发生装置内， 或光发生装置与输入 光导纤维连接之处或输入光导纤维的输入端部设置一第一滤光装置。
11. 根据权利要求 8或 9所述的装置， 根据权利要求 1或 2所述的装置， 在 荧光采集光导纤维的输出端与荧光检测装置中的光电倍增管之间设置一第二滤光 装置。
12. 根据权利要求 8 - 10所述的诊断或治疗肿瘤的装置， 其中光发生装置输出 的近紫外光大于 0至 1. 999mW/ cm2范围内可调，检测装置的光电倍增管检测的红荧 光的亮度范围为 0- 1999cd/m2。
PCT/CN2003/000465 2003-06-16 2003-06-16 Extrait de faeces bombycis, et sa methode de preparation, et appareil pour diagnostiquer et pour traiter des tumeurs malignes WO2004110465A1 (fr)

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