CN110090210B

CN110090210B - Use of chlorophyll derivatives for improving mesenteric microcirculation disorders

Info

Publication number: CN110090210B
Application number: CN201810085103.0A
Authority: CN
Inventors: 刘朝胜; 吴瑜; 罗京; 付雯; 刘敏; 余明森; 王根才; 周红晖; 袁建新; 徐发新
Original assignee: Wuhan United Pharmacy Co ltd
Current assignee: Wuhan United Pharmacy Co ltd
Priority date: 2018-01-29
Filing date: 2018-01-29
Publication date: 2022-01-14
Anticipated expiration: 2038-01-29
Also published as: CN110090210A

Abstract

The invention belongs to the field of medicines, and relates to an application of chlorophyll derivatives in improving mesenteric microcirculation disturbance, in particular to an application of chlorophyll derivatives in preparing medicines for treating and/or preventing mesenteric microcirculation disturbance. The chlorophyll derivatives, such as chlorophyll extract and silkworm excrement extract, have good curative effect on improving mesenteric microcirculation disturbance. The chlorophyll derivative can also comprehensively prevent and improve mesenteric microcirculation disturbance through the action in multiple links. In addition, the chlorophyll derivatives have improved toxic and side effects, are suitable for a wide range of people and are safe, and can be used for treating children, the elderly, pregnant women and lactating women. In addition, the preparation method of the chlorophyll derivative and the process for preparing the medicine are simple and convenient, and the cost is effectively controlled.

Description

Use of chlorophyll derivatives for improving mesenteric microcirculation disorders

Technical Field

The invention belongs to the field of medicines, and relates to an application of chlorophyll derivatives in improving mesenteric microcirculation disturbance, in particular to an application of chlorophyll derivatives in preparing medicines for treating and/or preventing mesenteric microcirculation disturbance.

Background

Microcirculation refers to the circulation of blood in the capillaries between arterioles and venules. It is the direct contact part of the cardiovascular system with the tissue cells. The basic function of the microcirculation is to carry out metabolism and substance exchange, which are important components of the blood circulation and can be called as the "second heart" of the human body. Along with the improvement of living standard of people, the improvement of dietary structure, the change of life style, the influence of genetic genes and other factors, the incidence of microcirculation disturbance is in a trend of rising obviously, and the major characteristics of microcirculation disturbance disease are high morbidity and high recurrence rate. The microcirculation is sensitive to various pathogenic factors, such as physical, chemical and biological stimuli. Pathological changes of local or systemic microcirculation may occur under the action of pathogenic factors. For example, in the case of mesenteric microcirculation disorder, the intestinal absorption function is poor, and nutritional disorders, emaciation with sallow complexion, diarrhea or constipation occur, and in severe cases, duodenal bulbar ulcer and the like occur. The occurrence, development and outcome of many diseases are closely related to microcirculatory disturbance, and the microcirculation can directly affect the treatment of the diseases.

In the aspect of improving microcirculation function, many researches are made by traditional Chinese medicine and western medicine. The currently commonly used medicine for improving the microcirculation disturbance is mainly used for improving the microcirculation disturbance by protecting blood vessels, resisting blood coagulation, dissolving thrombus and removing fiber, resisting blood platelet, expanding blood vessels, reducing blood viscosity and promoting blood circulation to remove blood stasis. However, the therapeutic effect of the existing drugs on the mesenteric part needs to be improved. Furthermore, the existing medicine has obvious toxic and side effects, is easy to cause stomach discomfort, nausea, heartburn, appetite reduction, rash, bleeding and other adverse reactions and side effects, is limited in using population, and is not suitable or recommended for children, old people, pregnant women and lactating women. In addition, existing drugs tend to be expensive.

Therefore, there is a need to develop a novel drug which is effective in preventing and improving mesenteric microcirculation disorders, has improved drug efficacy, and/or avoids the above-mentioned drawbacks.

Disclosure of Invention

In order to ameliorate the above disadvantages, the present invention provides a use of a chlorophyll derivative for the manufacture of a medicament for the treatment and/or prevention of mesenteric microcirculation disorders.

According to the present invention, the microcirculation disturbance may be mesenteric microcirculation disturbance caused by high-molecular dextran. For example, the microcirculatory disorder can be an acute microcirculatory disorder or a chronic microcirculatory disorder. For example, the microcirculatory disorder may be selected from, for example, mesenteric acute microcirculatory disorders.

According to the invention, the chlorophyll derivative may be a chlorophyll extract, such as a silkworm excrement extract.

According to the present invention, the chlorophyll derivative may comprise one or more of an alkali metal salt of transition metal-chelated chlorophyllin, an alkaline earth metal salt of transition metal-chelated chlorophyllin. Wherein, the transition metal can be one or more selected from iron, copper, zinc, manganese, cobalt, nickel, chromium and the like.

As an example, the transition metal chelated chlorophyllin alkali metal salt may be selected from one or more of sodium iron chlorophyllin, sodium copper chlorophyllin, sodium zinc chlorophyllin, sodium manganese chlorophyllin, sodium cobalt chlorophyllin, sodium nickel chlorophyllin, sodium chromium chlorophyllin, potassium iron chlorophyllin, potassium copper chlorophyllin, potassium zinc chlorophyllin, potassium manganese chlorophyllin, potassium cobalt chlorophyllin, potassium nickel chlorophyllin, potassium chromium chlorophyllin. The transition metal chelated chlorophyllin alkaline earth metal salt can be selected from one or more of calcium iron chlorophyllin, calcium copper chlorophyllin, calcium zinc chlorophyllin, calcium manganese chlorophyllin, calcium cobalt chlorophyllin, calcium nickel chlorophyllin, calcium chromium chlorophyllin, magnesium iron chlorophyllin, magnesium copper chlorophyllin, magnesium zinc chlorophyllin, magnesium manganese chlorophyllin, magnesium cobalt chlorophyllin, magnesium nickel chlorophyllin, and magnesium chromium chlorophyllin.

Alternatively, the chlorophyll derivative may be selected from one or more of the transition metal chelated chlorophyllin alkali metal salt, transition metal chelated chlorophyllin alkaline earth metal salt.

Preferably, the chlorophyll derivative contains sodium iron chlorophyllin in percentage by weightThe specific content is 5% or more, for example 10% or more, such as 15% or more, 22.5% or more, 25% or more, 28% or more, 30% or more, 35% or more, or 37% or more. At this time, as an example, other chlorophyll derivatives including, but not limited to, the above transition metal chelated chlorophyllin alkali metal salt except sodium chlorophyllin, and optionally additional chlorophyll derivatives may also be included in the chlorophyll derivative. The total weight percentage of the other chlorophyll derivatives is iron chlorophyllin (b) (C)₃₄H₃₀O₆N₄Fe) is 15% or more, for example 30% or more, such as 50% or more, 55% or more, 58% or more, 60% or more, 63% or more, 65% or more, or 70% or more. According to an embodiment of the present invention, the additional chlorophyll derivatives may include, but are not limited to, derivatives selected from the group consisting of: sodium chlorophyllin, iron chlorophyllin b, pheophorbide a, pyropheophorbide a, chlorin e6, chlorin e4, chlorin p6, chlorin f, purpurin-18, chlorophyllin, and the like.

The present invention also provides a pharmaceutical composition for treating and/or preventing mesenteric microcirculation disorders, comprising said chlorophyll derivative.

For example, the method for preparing the chlorophyll derivatives, such as chlorophyll extracts, comprises the steps of:

(1) extracting chlorophyll; preferably, the extract obtained by the extraction is further refined; (2) saponification; preferably, the saponification solution is further washed with an organic solvent; (3) carrying out iron melting; and (4) basification.

In the step (1), it is preferable that various biological materials (preferably silkworm excrement) containing chlorophyll and/or its derivative are extracted with an organic solvent or a mixture of an organic solvent and water, and the organic solvent is recovered to obtain chlorophyll. Preferably, the organic solvent may be acetone, ethanol, petroleum ether, n-hexane, gasoline (e.g., 120 # gasoline), toluene, or a mixture of two or more thereof; further preferably, the organic solvent is acetone, ethanol or a mixture thereof. Preferably, the extraction method is reflux, extraction, percolation, microwave or using ultra-micro vibration mill, etc.

In a preferred embodiment, the purification is carried out by washing with deionized water or by adsorption with a macroporous adsorption resin. Preferably, refining is performed for removing non-chlorophyll-like substances.

In the step (2), it is preferable that the chlorophyll is dissolved in an organic solvent and saponified by adding an alkali solution; wherein, the organic solvent is preferably gasoline, acetone or a mixture thereof, and the gasoline is preferably No. 120 gasoline; the alkali solution is preferably an aqueous NaOH solution or an aqueous KOH solution, and more preferably, the concentration of the alkali solution is 25 to 35%. Saponification is to remove carotenoids.

In the step (3), preferably, the pH value of the saponification solution is adjusted to 2-4, precipitate is separated out, an organic solvent or a mixture of the organic solvent and water is added to dissolve the saponification solution, a ferrous salt is added, the solution is heated until the solution is dark green, and the organic solvent is recovered to obtain the iron substitute product. Preferably, the organic solvent may be acetone, ethanol or a mixture thereof; preferably, the ferrous salt may be ferrous sulfate, ferrous chloride or a mixture thereof; preferably, the heating keeps the solution slightly boiling.

According to the invention, the iron is ferrified to obtain iron chlorophyllin.

In the step (4), the iron substitute is preferably washed with deionized water until free iron ions are removed, an organic solvent is added to dissolve the iron substitute, and then alkali is added to alkalify the iron substitute. Preferably, the organic solvent may be acetone, ethanol or a mixture thereof; as an example, the base may be an aqueous or alcoholic solution of a base, such as an aqueous or ethanolic NaOH solution.

Preferably, the alkalization is carried out until no more precipitate is produced. Preferably, the concentration of the base in the aqueous or alcoholic solution of the base may be 25-35% by mass. According to the invention, preferably, the alkalization is carried out to obtain sodium iron chlorophyllin.

The invention also provides the use of the chlorophyll derivatives for the treatment and/or prevention of mesenteric microcirculation disorders.

Advantageous effects

The applicant unexpectedly finds that the silkworm excrement extract has a good curative effect on improving mesenteric microcirculation disturbance. The derivatives can also be used for comprehensively preventing and improving mesenteric microcirculation disturbance. In addition, the chlorophyll derivative has improved toxic and side effects, and is suitable for wide and safe crowds.

Detailed Description

The compounds of the general formula and the preparation and use thereof according to the present invention will be described in further detail with reference to the following examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

1. Test materials, instruments and methods

1.1 drugs and reagents

The faeces Bombycis extract is provided by Wuhan combined pharmaceutical industry, LLC, and is prepared according to the method disclosed in Chinese patent application 201110031023.5. During the test, the silkworm excrement extract is prepared into solutions with the concentrations of 16mg/ml (high dose), 8mg/ml (medium dose) and 4mg/ml (low dose) by pure water, and is prepared fresh every day.

Dextran T250 (molecular weight 25 ten thousand): specification: D-T250. Purchased from Huan Hua polysaccharide Biotech (Heyuan) Ltd, Hanbang. Batch number: d-15021.

Urethane (ullose): AR grade, purchased from chengdu corson chemical reagent plant, lot number: 20160811. the test was carried out by preparing a 20% strength solution with pure water.

1.2 method for measuring content of sodium iron chlorophyllin

Preparation of control solutions: precisely weighing 0.143g of spectrally pure ferric oxide dried to constant weight at 130 ℃, placing the spectrally pure ferric oxide in a beaker, adding 5ml of hydrochloric acid solution (1 → 2) to dissolve the ferric oxide, transferring the ferric oxide to a 100ml measuring flask, adding 4ml of hydrochloric acid solution (1 → 2), adding water to the scale, shaking up, precisely measuring 10ml, placing the hydrochloric acid solution in the 100ml measuring flask, adding water to the scale, and shaking up to obtain a solution containing 100 mu g of Fe per 1 ml. Precisely measuring the above solutions 0, 0.2, 0.4, 0.6, 0.8 and 1.0ml respectively, placing in a 10ml measuring flask, adding nitric acid solution (1 → 500) to scale, and shaking.

Preparation of a test solution: taking a proper amount of sample, grinding, precisely weighing 10mg, placing in a 50ml beaker, adding 1.5ml of nitric acid and 3 drops of perchloric acid, covering a surface dish, heating to near dryness at 150-160 ℃, adding 2ml of nitric acid (1 → 2), covering the surface dish, continuously heating to near dryness, taking out the beaker, cooling, adding a proper amount of nitric acid solution (1 → 500) for dissolving, transferring to a 25ml measuring flask, adding nitric acid solution (1 → 500) to scale, and shaking uniformly to obtain the product.

The determination method comprises the following steps: taking the reference solution and the sample solution, measuring at 248nm wavelength according to atomic absorption spectrophotometry (first method for measuring content in the first version of Chinese pharmacopoeia 2015), calculating the percentage content of iron, and multiplying by 12.32 to obtain the final product. The product contains sodium iron chlorophyllin and sodium iron chlorophyllin (b) (C) calculated on dry product₃₄H₂₈O₆N₄FeNa₂) And (4) calculating.

1.3 content determination method of other chlorophyll derivatives

Taking about 0.18g of fine powder of a sample, precisely weighing, adding 10ml of dilute hydrochloric acid, carrying out ultrasonic treatment for 15 minutes, carrying out vacuum filtration, removing acid water, washing residues with water until a water washing solution is neutral (the methyl red indicator shows yellow, the bromothymol blue indicator shows yellow-green), shearing the residues and filter paper, transferring the residues and the filter paper into a beaker, precisely adding 20ml of sodium hydroxide titration solution (0.1mol/L), covering a watch glass, heating in a hot water bath for 15 minutes, cooling to room temperature, adding 5ml of water, shaking uniformly, and preparing a blank solution by the same method. Titrating with hydrochloric acid titration solution (0.1mol/L) according to potentiometric titration (the first part of Chinese pharmacopoeia 2015), correcting titration result with blank test, and calculating to obtain the final product.

V_{Air conditioner}: the volume (ml) of the blank solution consumed hydrochloric acid titration solution (0.1 mol/L);

V_{sample (A)}: the test solution consumes the volume (ml) of the hydrochloric acid titration solution (0.1 mol/L);

w: sample volume (g) of the test sample;

f: concentration correction factor of hydrochloric acid titration solution (0.1 mol/L).

The product contains other chlorophyll derivatives such as iron chlorophyllin (b) (C) calculated on dried product₃₄H₃₀O₆N₄Fe) is calculated.

1.4 test animals and rearing

1.4.1 the test animal New Zealand rabbit, male and female, the weight of 1.8 ~ 2.0kg, purchased from WUDO animal company Limited. Producing license numbers: SCXK 2014-. And (3) carrying out adaptive feeding in a constant-temperature environment, keeping the environment clean, and keeping drinking water and eating for 1 week.

1.4.2 feed

Name: compound feed for experimental rabbits (8011); the source is as follows: achievement of great achievements and experiments animal limited; the execution standard is as follows: GB 14924.3-2010; production batch number: 20160821, respectively; the nutrient components meet the requirements of the compound feed for the solid rabbits, and the nutrient components and pollutants meet the requirements of GB14924.1-2001 and Experimental animal feeding management regulations. Specific values (per kg feed) were: crude protein, not less than 140 g; 100-150 g of coarse fibers; crude fat, not less than 30 g; coarse ash content less than or equal to 90 g; water content less than or equal to 110 g; calcium to phosphorus ratio of 1.0-1.8 to 0.6-1.2; lysine is not less than 7 g; methionine + cystine, not less than 5 g.

The administration method comprises the following steps: the feed is placed in a feeding groove in front of the rabbit cages for free feeding.

1.4.3 Drinking Water

Name: domestic tap water; the quality requirement meets the sanitary standard of drinking water.

A water supply mode: the drinking water bottle is placed at a special position in front of the rabbit hutch for freely drinking water.

1.5 test apparatus

An electronic balance: BP221S type, manufactured by SARTORIUS, Germany.

Laser doppler PeriFlux 5000 system, produced by period, sweden.

2. Experimental models and methods

2.1 test grouping and administration

24 male and female half New Zealand rabbits were randomly divided into 4 groups of 6 rabbits each. Respectively as follows: negative control group, silkworm faeces extract high (80mg/kg/d), medium (40mg/kg/d), low (20mg/kg/d) dose group. The administration volume of the high, medium and low dose groups of silkworm excrement extract is 5ml/kg, and the administration is carried out once a day for 20 days continuously at 20 days before molding.

2.2 establishment of acute microcirculatory disturbance model

A dextran T250 (molecular weight is 25 ten thousand units) with the concentration of 10 percent is injected into the vein of the rabbit ear to establish an acute microcirculation disturbance model by 1 ml/kg. The specific operation is as follows: 5ml/kg of 20% ethyl carbamate is injected into the ear margin vein for anesthesia, the microcirculation perfusion amount (PU), the blood cell aggregation degree (CMBC) and the speed (V) of the moving blood cells of the mesentery are measured by a laser Doppler blood flow instrument, then dextran T250 is injected into the ear margin vein, and the PU, CMBC and V of the mesentery are measured by the laser Doppler blood flow instrument after administration for 30 min.

2.3 Observation index

After the negative control group and the silkworm excrement extract high, medium and low dose groups are continuously dosed for 20d, and 2h after the last dose, the change conditions of mesentery microcirculation (PU, CMBC and V) are measured before and 30min after the model is built respectively. Calculating the microcirculation change rate before and after modeling (PF 5010-LDPM unit of laser Doppler blood flow instrument is used for measuring local tissue microcirculation, and the microcirculation difference of different parts of different tissues is obvious, and in order to ensure the objectivity of data measured each time, we use the change rate to calculate the change condition of microcirculation).

2.4 statistical methods

Data for the measurement data in the analytical experiments are generally expressed as "mean. + -. standard deviation

"means. The significance test of the difference among the groups uses a nonparametric test or a single-factor variance analysis method provided by Prism5windows software; first, each group of data is subjected to normal test, and ONE-WAY analysis of variance (ONE-WAY ANOVA) is adopted when the data accords with normal distribution, and non-parametric test (Nonparametric Tests) is adopted when the data does not accord with normal distribution.

The count data is typically compared for differences between groups using a nonparametric test (e.g., chi-square test). "+", "#" (P <0.05) are statistically significant, and "+", "# #" (P <0.01) are statistically significant.

Example 1 protection of silkworm faeces extract against acute microcirculation disturbance of mesentery

According to the above method, the effect of faeces Bombycis extract containing sodium iron chlorophyllin about 37.6 wt% and other chlorophyll derivatives 60.0% or more in terms of iron chlorophyllin (b) on rabbit mesenteric acute microcirculation disturbance was tested, and the results are as follows.

TABLE 1 Effect of faeces Bombycis extract on the rate of change of rabbit mesenteric microcirculation: (

n＝6)

Note: comparison with negative control group: *: p <0.05, x: p <0.01

Table 1 shows that, compared with the negative control group, the blood Perfusion (PU), the blood cell aggregation level (CMBC) and the rate of change of the exercise blood cell rate (V) in the high-dose group of the silkworm excrement extract are all significantly reduced (P <0.01), and the evaluation indexes of the medium-dose and low-dose groups of the silkworm excrement extract are in a descending trend, which indicates that sodium iron chlorophyllin can improve mesenteric microcirculation disturbance.

Example 2 protection of silkworm faeces extract against acute microcirculation disturbance

The experiment of example 1 was repeated except that the experiment was carried out using silkworm excrement extract containing sodium iron chlorophyllin in an amount of about 29.1% by weight and other chlorophyll derivatives in an amount of 60.0% or more in terms of iron chlorophyllin (b). The results show that the blood flow Perfusion (PU), the blood cell aggregation degree (CMBC) and the change rate of the locomotor blood cell rate (V) of the high, medium and low dose groups are all reduced, and the mesenteric microcirculation disturbance of the tested rabbit is improved.

From the above examples, sodium iron chlorophyllin and chlorophyll derivatives (such as silkworm excrement extract) containing sodium iron chlorophyllin can effectively improve or regulate blood flow perfusion, concentration of motor blood cells and speed of motor blood cells, thereby improving acute microcirculation disturbance of mesentery and improving overall microcirculation disturbance.

Example 3 Long-term toxicity test of silkworm excrement extract and sodium iron chlorophyllin

Beagle dogs were orally administered at 1000, 500 and 250mg faeces Bombycis extract/kg, corresponding to 100, 50 and 25 times the maximum dose of the product for clinical adult severe patients, 1 time per day, with 1 day of rest per week for 180 days, and then stopped for 30 days. The symptoms of toxic reaction appeared during the period of Beagle dog administration and the period of drug withdrawal are observed, and the clinical adverse reaction possibly caused by the silkworm excrement extract is predicted, and the results are as follows:

(1) general observations

The Beagle dogs in each administration group have normal behaviors and do not have abnormal reactions such as abnormal screaming sound, restlessness, convulsion, tremor, ataxia, local muscle twitch, forced movement and the like; the reaction to the stimulation is not irritability, or apathy or depression, or hyperesthesia; the nerve reflex has no dull or lost phenomenon; muscle tone is not manifested as relaxation or stiffness.

The main abnormality during the administration is that the feces of the Beagle dog are black-stained by the silkworm excrement extract of the test article, and other abnormality is not found.

(2) Food intake

The intake of Beagle dogs in each group is not abnormal in the administration period and the withdrawal recovery period.

(3) Body weight

In the administration period and the recovery period, the weight average of Beagle dogs in each group is not abnormal, the measured weight value of the silkworm excrement extract in each administration group is not statistically different from that in a control group (p is more than 0.05), but the weight increase rate is about 10 percent higher than that in the control group.

(4) Electrocardiographic examination

The indexes of each administration group and the control group of the silkworm excrement extract have no obvious difference (p is more than 0.05) by the ECG examination of the Beagle dog after 180 days of administration and 30 days of drug withdrawal.

(5) Examination of body temperature

The body temperature of Beagle dogs is checked after 180 days of administration and 30 days of drug withdrawal, and the silkworm excrement extract has no obvious difference compared with each administration group and a control group (p is more than 0.05).

(6) Blood pressure examination

The blood pressure of Beagle dogs after 180 days of administration and 30 days of drug withdrawal is checked, and the blood pressure indexes of each administration group and the control group of the silkworm excrement extract have no obvious difference (p is more than 0.05).

(7) Ophthalmic examination

And (3) observing the eyelid, conjunctiva, cornea, iris, sclera, pupil, cornea, vitreous body, crystalline lens and eyeground of the Beagle dog in the administration period and the withdrawal recovery period, wherein all indexes are normal, and no abnormal condition is found.

(8) Routine examination of urine

After 90 days and 180 days of administration and 30 days of withdrawal, no obvious abnormality (p >0.05) is found in each index of each administration group compared with the control group.

(9) Routine examination of feces

Feces routine examination is carried out for 90 days and 180 days, black feces appear in each administration group of the silkworm excrement extract, and other abnormalities do not appear.

(10) Blood routine and reticulocyte examination

The RDWS (erythrocyte volume distribution breadth) of the high and medium dose groups which are administrated for 90 days is respectively obviously and obviously reduced (p <0.01 and p < 0.05). The dose group RDWS (red blood cell volume distribution width) was significantly reduced (p <0.05) during 180 days of administration, presumably independent of drug, and belonging to animal physiological value fluctuation. No obvious abnormality is found in other blood routine examination indexes.

(11) Blood coagulation function

Fluctuations in the blood coagulation function test values occurred, and there were no dose-response relationship and no time-response relationship, presumably drug-independent.

(12) Biochemical examination of blood

[1] Liver function: the Beagle dog gamma-GT is obviously reduced in a low-dose group which is administered for 90 days and 180 days (p is less than 0.05). In the recovery period examination, the gamma-GT in the high, medium and low dose groups respectively shows obvious, remarkable and obvious reduction (p <0.05, p <0.01, p <0.05), and the examination value of the gamma-GT in the synchronous control group has larger fluctuation than that before the administration, so the gamma-GT is supposed to be unrelated to the medicament and belongs to the fluctuation of the physiological value of the animal. ② the TP of the high dose group is obviously increased (p <0.05) after 30 days of drug withdrawal, the TP check value of the high dose group after 30 days of drug withdrawal is almost consistent with the check value of the high dose group after 180 days of drug administration, but the TP check value is not statistically different (p >0.05) after 180 days of drug administration. Analysis shows that the TP value of the high-dose group is only increased by 5.8 percent after the drug is stopped for 30 days, the increase amplitude is small, the clinical significance is avoided, and the TP value of the control group is reduced by 5.7 percent after the drug is stopped for 30 days compared with the TP value of the control group after the drug is stopped for 180 days, which indicates that the abnormality is caused by the physiological value fluctuation of the control group and is irrelevant to the drug. ③ the albumin in the high dose group after 90 days of administration is reduced by 3.7 percent, the reduction range is smaller, and is higher than the measured value before administration, but no statistical difference or reduction effect exists before administration, and the analysis shows that the measured value of the albumin in the control group after 90 days of administration is 10.8 percent higher than the measured value before administration, possibly related to the increase, and belongs to physiological value fluctuation. The 180-day albumin measurements were higher in the high dose group than in the 90-day measurement indicating that no time-response relationship was present, independent of drug.

[2] Renal function

The Crea of the low-dose group is obviously increased (p is less than 0.05) after 90 days of administration, the increasing amplitude is 5.4 percent, but the Crea of the high-dose group is not obviously increased, and dose-response relation does not exist; the Crea measurements were lower in the 180-day low dose group than in the 90-day group, indicating no time-response relationship, presumably independent of drug. ② after 30 days of recovery, Crea in the high dose group is obviously increased (p is less than 0.05), the measured value in the recovery period is lower than the measured value in 90 days of administration, which indicates that physiological value fluctuation exists and is irrelevant to the medicine.

[3] Blood sugar, blood fat and myocardial zymogram

The Beagle dog, given for 90 days, showed a significant reduction in GLU (p <0.05) in the low dose group, with no dose-response relationship, presumably drug-independent. The administration lasts 180 days and the withdrawal is 30 days, and each index has no obvious abnormality (p is more than 0.05) when each administration group is compared with a control group, and is supposed to be unrelated to the medicament and belong to the fluctuation of the physiological value of the animal.

(13) Serum electrolyte examination

Compared with a control group, each administration group has no obvious abnormality (p is more than 0.05) after administration for 90 days, administration for 180 days and withdrawal for 30 days, but the inspection value in the quarantine period fluctuates, and the dosage-response relation and the time-response relation are not existed, so that the animal physiological value is supposed to fluctuate regardless of the medicament.

(14) Examination of bone marrow

After the Beagle dog is administrated for 180 days, the number of megakaryocytes of bone marrow of the high, medium and low dose groups is obviously increased (p is less than 0.05), the recovery period is finished, and the number of megakaryocytes of the low dose group and a control group is obviously increased (p is less than 0.05), and the time-response relationship is formed, so that the judgment is related to the hematogenesis function of the medicine.

(15) Systematic autopsy

The Beagle dogs are checked after 180 days of administration and 30 days of withdrawal, the nutritional state, the cadaver characteristics and the physical examination of each group of Beagle dogs have no abnormal expression, abnormal secretion is not found in each natural orifice, and obvious abnormal expression is not found in oral cavity, nasal cavity and main visceral tissues. The trachea and the bronchus are not obviously abnormal in respiratory tract examination, and lung tissues are not affected by adhesion, emphysema, swelling or atrophy and the like.

(16) Examination of visceral indices

After 180 days of administration and 30 days of withdrawal, the visceral organ indexes of heart, liver, spleen, lung, kidney, adrenal gland, uterus, ovary, testis, epididymis, thyroid gland, thymus, brain, bladder, prostate and pancreas are checked, and no obvious difference is seen between each administration group and the control group (p > 0.05).

(17) Histopathological examination

No statistically different pathological changes (p >0.05) were observed in each tissue by histopathological examination 180 days after administration of the silkworm excrement extract and 30 days after withdrawal of the drug.

And (4) conclusion: the silkworm excrement extract and sodium iron chlorophyllin have no toxic reaction except that excrement black staining and bone marrow megakaryocyte proliferation reaction are caused. The dosage of 1000mg silkworm excrement extract/kg and below is the safe dosage range.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a chlorophyll derivative for the manufacture of a medicament for the treatment and/or prevention of mesenteric microcirculation disorders;

among the chlorophyll derivatives: the weight percentage content of the sodium iron chlorophyllin is more than 29.1 percent; the chlorophyll derivative is silkworm excrement extract;

the total weight percentage content of other chlorophyll derivatives, calculated as iron chlorophyllin (b), is more than 60%.

2. Use according to claim 1, wherein the mesenteric microcirculation disorder is selected from the group consisting of mesenteric microcirculation disorders caused by high molecular dextran.

3. Use according to claim 2, wherein the microcirculatory disorder is selected from acute microcirculatory disorder or chronic microcirculatory disorder.

4. Use according to claim 1 or 2, characterized in that the chlorophyll derivative has a sodium iron chlorophyllin content of more than 30% by weight.

5. Use according to claim 1 or 2, characterized in that the chlorophyll derivative has a sodium iron chlorophyllin content of 35% by weight or more.

6. Use according to claim 1 or 2, characterized in that the chlorophyll derivative has a sodium iron chlorophyllin content of 37% by weight or more.

7. Use according to claim 1 or 2, characterized in that the other chlorophyll derivatives comprise transition metal chelated alkali metal salts of chlorophyllin in addition to sodium chlorophyllin, and optionally additional chlorophyll derivatives.

8. Use according to claim 7, characterized in that the additional chlorophyll derivative is selected from the following derivatives: sodium chlorophyllin, iron chlorophyllin b, pheophorbide a, pyropheophorbide a, chlorin e6, chlorin e4, chlorin p6, chlorin f, purpurin-18, and chlorophyllin.

9. Use according to claim 1 or 2, characterized in that the total content in weight percentage of the other chlorophyll derivatives, calculated as iron chlorophyllin (b), is above 63%.

10. Use according to claim 1 or 2, characterized in that the total content in weight percentage of the other chlorophyll derivatives, calculated as iron chlorophyllin (b), is above 65%.

11. Use according to claim 1 or 2, characterized in that the total content in weight percentage of the other chlorophyll derivatives, calculated as iron chlorophyllin (b), is at least 70%.

12. Use according to claim 1 or 2, characterized in that the preparation process of the chlorophyll derivative comprises the steps of:

(1) extracting chlorophyll, and further refining the extract; (2) saponifying, and further washing the saponified solution with an organic solvent; (3) iron melting: regulating the pH value of the saponification solution to 2-4, precipitating, adding an organic solvent or a mixture of the organic solvent and water to dissolve the saponification solution, adding ferrous salt, heating until the solution is dark green, and recovering the organic solvent to obtain an iron substitute product; and (4) basification: washing the iron substitute product with deionized water until no free iron ion exists, adding an organic solvent to dissolve the iron substitute product, and adding alkali to alkalify the iron substitute product.

13. The use according to claim 12, wherein in step (1), silkworm excrement is extracted with organic solvent or mixture of organic solvent and water, and chlorophyll is obtained after recovering organic solvent; the organic solvent is acetone, ethanol or mixture thereof; the extraction method comprises refluxing, extracting, percolating, and microwave or ultra-micro vibration milling;

refining by washing with deionized water or by adsorption with macroporous adsorbent resin.

14. The use according to claim 12, wherein in step (2), chlorophyll is dissolved in an organic solvent, and an alkali solution is added to saponify the chlorophyll; wherein the organic solvent is gasoline, acetone or mixture thereof; the alkali solution is NaOH water solution or KOH water solution, and the concentration of the alkali solution is 25-35%.

15. Use according to claim 12, wherein in step (3), the organic solvent is acetone, ethanol or a mixture thereof; the ferrous salt is ferrous sulfate, ferrous chloride or a mixture thereof.

16. Use according to claim 12, wherein in step (4), the organic solvent is acetone, ethanol or a mixture thereof; the alkali is NaOH aqueous solution or NaOH ethanol solution.