Patent Application
20230165922
The present invention relates to the technical field of pharmaceuticals and daily care, and in particular to use of Radix Gentianae Macrophyllae and monomer compounds thereof, in particular, gentiopicroside, in killing mites.
Mites are a class of tiny animals belonging to Arthropoda, Arachnida, Latigastra, generally having a body size of about 0.5 mm, some as small as 0.1 mm, and most species have a body size of less than 1 mm. It is found that mites are closely related to the health status of human beings and animals (such as dogs, cats and other companion animals). For example, mites such as gamasid mites (Mesostigmata spp.), chiggers, itch mite (Sarcoptesscabiei), Demodex mites (Demodex spp.), flour mites (Acarus siro), dust mite and Pyemotes spp. are hematophagic and may harm the skin, causing “acne rosacea” or demodicosis, hypersensitivity, urinary acariasis, pulmonary acariasis, intestinal acariasis, scabies and the like, which seriously endanger the health of human beings and animals.
Demodex mites (belonging to Arachnid, Acarina) are microscopic ectoparasites that usually affect the pilosebaceous unit of the skin. In the wide range of reported species, D.folliculorum and D.brevis have been found on the body surface of humans, and mainly live in the hair follicles, sebaceous glands and meibomian glands. Demodex mites feed on epithelial cells of hair follicle, causing follicle dilatation and hair loss, manifesting clinically as blepharitis marginalis, meibomian gland dysfunction, madarosis, abnormal eyelash alignment, conjunctivitis and blepharoconjunctivitis, pterygium, keratitis, basal cell carcinoma of eyelid and the like. Demodex mites may also feed on lipids, causing xerophthalmia. In addition, Demodex mites may also cause mechanical obstruction of meibomian gland ducts, causing difficulty in lipid excretion and excessive retention of secretions, leading to the formation of chalazion. The clinical manifestations of the ocular diseases caused by Demodex infestation mainly include: recurrent red and itch eyes, dry eyes, burning eyes, foreign body sensation, photophobia and increased secretion; the diseases may also be accompanied by recurrent eyelash loss; blurred vision and decreased vision may occur in severe cases affecting the cornea. In addition to the above ocular diseases, a number of studies in recent years have reported that Demodex infestation is associated with a variety of common skin diseases, including seborrhoeic dermatitis, acne, acne rosacea, pityriasis folliculorum, perioral dermatitis, demodicosis, basal cell carcinoma and the like (see, e.g., Luo, X., Li, J., Chen, C., Tseng, S. & Liang, L, Ocular Demodicosis as a Potential Cause of Ocular Surface Inflammation, Cornea 36 Suppl 1, s9-s14; Karincaoglu, Y., Tepe, B., Kalayci, B., Atambay, M. & Seyhan, M, Is Demodexfolliculorum an aetiological factor in seborrhoeic dermatitis?, Clinical and experimental dermatology 34, e516-520; Chen, W. & Plewig, G, Human demodicosis: revisit and a proposed classification, The British journal of dermatology 170, 1219-1225).
Diseases caused by mites (such as ocular diseases and skin diseases) have not yet received sufficient clinical attention and are often misdiagnosed as bacterial diseases and the like, while the conventional antibacterial treatments usually have little curative effect.
In a first aspect of the present invention, provided is use of Radix Gentianae Macrophyllae in preparing a product for killing mites.
Specifically, the Radix Gentianae Macrophyllae can be used as the sole acaricidal active ingredient, or can also be used in combination with other ingredients with the same or different activities for killing mites.
In a second aspect of the present invention, provided is use of a Radix Gentianae Macrophyllae extract in preparing a product for killing mites.
Specifically, the Radix Gentianae Macrophyllae extract can be obtained by suitable extraction methods for traditional Chinese medicine known in the art, such as one or more of decoction, maceration, percolation, reflux, steam distillation, ultrasonic extraction and the like. In an embodiment of the present invention, the Radix Gentianae Macrophyllae extract can be obtained by decoction.
Specifically, the Radix Gentianae Macrophyllae extract is prepared by a method comprising:
In an embodiment of the present invention, the extraction solvent is water.
Specifically, the time for soaking is 1-24 hours (e.g., 1, 2, 3, 4, 5, 6, 12, 18 or 24 hours).
Specifically, the time for decocting is 10-60 min (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 min).
Specifically, the method may further comprise:
heating the filter residue obtained in step (2) and an extraction solvent to boiling, then decocting and filtering; and combining the filtrates obtained in steps (2) and .
Optionally, the method may further comprise one or more of the processing procedures of mixing, concentrating, quantifying or sterilizing the obtained filtrate and the like.
Specifically, the Radix Gentianae Macrophyllae extract comprises one or more of gentiopicroside, loganic acid, sweroside, roburic acid, scopoletin, mollugin and swertiamarin.
More specifically, the Radix Gentianae Macrophyllae extract comprises gentiopicroside.
In a third aspect of the present invention, provided is use of a monomer compound of Radix Gentianae Macrophyllae, or a salt, an ester, an isomer, a solvate or a derivative thereof in preparing a product for killing mites.
Specifically, the salt, the ester, the isomer, the solvate and the derivative of the monomer compound of Radix Gentianae Macrophyllae are pharmaceutically acceptable salt, ester, isomer, solvate and derivative.
Specifically, the monomer compound is selected from one or more of gentiopicroside, loganic acid, sweroside, roburic acid, scopoletin, mollugin and swertiamarin.
Specifically, the monomer compound is gentiopicroside.
In a fourth aspect of the present invention, provided is use of gentiopicroside, or a salt, an ester, an isomer, a solvate or a derivative thereof in preparing a product for killing mites.
Specifically, the salt, the ester, the isomer, the solvate and the derivative of the monomer compound of Radix Gentianae Macrophyllae are pharmaceutically acceptable salt, ester, isomer, solvate and derivative.
Specifically, the product for killing mites described above can be used for therapeutic and/or prophylactic purposes, and also for non-therapeutic and/or non-prophylactic purposes.
Specifically, the mites described above can be one or more of Demodex spp., Dermatophagoidespteronyssinus, Sarcoptesscabiei. and the like. In an embodiment of the present invention, the mites described above are Demodex spp., such as D.folliculorum and D.brevis.
In an embodiment of the present invention, the product for killing mites described above is a pharmaceutical composition.
Specifically, the pharmaceutical composition described above further comprises a pharmaceutically acceptable excipient(s).
Specifically, the pharmaceutical composition described above is used for preventing and/or treating a disease caused by mite infestation.
Specifically, the disease described above may be an ocular disease, a skin disease, an allergic disease and the like.
Specifically, the ocular disease described above may be one or more of blepharitis marginalis, meibomian gland dysfunction, tarsitis, madarosis, abnormal eyelash alignment, glaucoma, cataract, ocular folliculitis, conjunctivitis, blepharoconjunctivitis, pterygium, keratitis, eyelid laxity and ectropion, basal cell carcinoma of eyelid, xerophthalmia, chalazion and the like; the ocular disease may have one or more symptoms selected from: red and itchy eyes, dry eyes, burning eyes, foreign body sensation, photophobia, increased eye discharge, loss of eyelashes, blurred vision, decreased vision and the like.
Specifically, the skin disease described above may be one or more of seborrhoeic dermatitis, acne, acne rosacea, pityriasis folliculorum, perioral dermatitis, demodicosis, gaile, basal cell carcinoma and the like.
Specifically, the allergic disease described above may be one or more of allergic asthma, allergic rhinitis, allergic dermatitis and the like.
Specifically, the pharmaceutical composition described above may be in any dosage form suitable for administration, such as a topical preparation, in particular, an ophthalmic preparation, a skin topical preparation and the like.
Specifically, the ophthalmic preparation described above may be an eye drop, an eye ointment, an ophthalmic gel, an ophthalmic emulsion, an ophthalmic suspension, an ophthalmic film, a collyrium, an intraocular injection and the like.
Specifically, the ophthalmic preparation may comprise a pharmaceutically acceptable excipient(s) such as a pH regulator, a co-solvent, an osmotic pressure regulator, a viscosity regulator, an antioxidant, a bacteriostatic preservative, a buffer, a suspending agent, a local anesthetic, a surfactant, a solubilizer, a wetting agent, an emulsifier, a stabilizer, a filler, a protective agent, a solvent and the like.
Specifically, the skin topical preparation described above may be an aerosol, a powder, a lotion, a tincture, a liniment, a film coating agent, an ointment, a gel, a paste, an emulsion and the like.
Specifically, the dosage forms of the pharmaceutical composition described above can be prepared according to conventional production methods in the field of pharmaceuticals. For example, ophthalmic preparation techniques can refer to those found in Ophthalmic Drugs and Preparation Techniques (China Light Industry Press, 2010), Development and Production of Eye Drops (Chemical Industry Press, 2009), etc.
Specifically, the pharmaceutical composition described above may comprise 0.01%-99.5% (e.g., 0.01%, 0.1 %, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 99.5%) by weight of the active ingredient.
Specifically, the pharmaceutical composition described above can be used in humans, and can also be used as a veterinary drug in non-human animals, such as non-human mammals, e.g., companion animals (e.g., dogs, cats, rabbits, mice, etc.), livestock animals (e.g., horses, cows, sheep, pigs, dogs, rabbits, etc.) and the like.
In another embodiment of the present invention, the product for killing mites described above is a cosmetic product.
Specifically, the cosmetic product described above further comprises a cosmetically acceptable excipient(s).
Specifically, the cosmetic product described above may be a cosmetic product used for the face, such as a facial cleanser, a soap, a skin softener, a toner, a skin care lotion, a jelly lotion, a facial cream, a sunscreen cream, an essence, a facial mask, a gel, a foundation and a scrub.
Specifically, the cosmetic product described above may be a cosmetic product used for parts of the body other than the face, such as a neck cream, a shampoo, a shower gel, a hair conditioner, a body lotion, a scrub and the like.
Specifically, the cosmetic product described above may also be a cosmetic product used for the eyes and periocular region, such as an eye cream, a mascara, an eyeliner powder, a cream eyeliner, an eyeliner pencil, an eye shadow powder, an eye shadow cream, an eyebrow pencil, a brow powder and the like.
Specifically, the various forms of the cosmetic product described above can be prepared according to conventional production methods in the field of cosmetics.
Specifically, the cosmetic product described above may comprise 0.01%-99.5% (e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 99.5%) by weight of the active ingredient.
In another embodiment of the present invention, the product for killing mites described above is an acaricide which can be used for killing and controlling mites that may live in articles in the living environment (e.g., pillow case, pillow inner, bed sheet, bedding, mattress, clothing, carpet, cushion, sofa, summer sleeping mat, plush toy, air conditioner and the like).
Specifically, the acaricide described above may comprise any suitable excipient that can achieve the desired properties.
Specifically, the acaricide described above may be in a form of a spray, a lotion, a paster, a small packet and the like.
Specifically, the various forms of the acaricide described above can be prepared according to conventional production methods in the field of daily care products.
Specifically, the acaricide described above may comprise 0.01%-99.5% (e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 99.5%) by weight of the active ingredient.
The present invention further provides a method for preventing and/or treating a disease caused by mite infestation, comprising administering to a subject in need thereof Radix Gentianae Macrophyllae, a Radix Gentianae Macrophyllae extract, a monomer compound of Radix Gentianae Macrophyllae (in particular, gentiopicroside), or a salt, an ester, an isomer, a solvate or a derivative thereof, or the pharmaceutical composition described above.
Specifically, the disease described above may be an ocular disease, a skin disease, an allergic disease and the like.
Specifically, the ocular disease described above can be one or more of blepharitis marginalis, meibomian gland dysfunction, tarsitis, madarosis, abnormal eyelash alignment, glaucoma, cataract, ocular folliculitis, conjunctivitis, blepharoconjunctivitis, pterygium, keratitis, eyelid laxity and ectropion, basal cell carcinoma of eyelid, xerophthalmia, chalazion and the like; the ocular disease may have one or more symptoms selected from: red and itchy eyes, dry eyes, burning eyes, foreign body sensation, photophobia, increased eye discharge, loss of eyelashes, blurred vision, decreased vision and the like.
Specifically, the skin disease described above may be one or more of seborrhoeic dermatitis, acne, acne rosacea, pityriasis folliculorum, perioral dermatitis, demodicosis, gaile, basal cell carcinoma and the like.
Specifically, the allergic disease described above may be one or more of allergic asthma, allergic rhinitis, allergic dermatitis and the like.
The present invention further provides a method for treating and/or preventing an ocular disease caused by mites, comprising administering to eyes of a subject in need thereof Radix Gentianae Macrophyllae, a Radix Gentianae Macrophyllae extract, a monomer compound of Radix Gentianae Macrophyllae, or a salt, an isomer, a solvate or a derivative thereof.
The present invention further provides use of Radix Gentianae Macrophyllae, a Radix Gentianae Macrophyllae extract, a monomer compound of Radix Gentianae Macrophyllae, or a salt, an isomer, a solvate or a derivative thereof in preparing a medicament for treating and/or preventing an ocular disease caused by mites.
In particular, reports where the association of mites with ocular diseases has been confirmed include:
Human Permanent Ectoparasites; Recent Advances on Biology and Clinical Significance of Demodex Mites: Narrative Review Article (Iranian journal of parasitology 12(1):12-21) has shown that D.brevis lives in the sebaceous glands and meibomian glands and D.folliculorum often occupies the hair follicle area of human eyelashes, of which both will cause ocular diseases after infecting the facial skin.
Ocular Demodicosis as a Potential Cause of Ocular Surface Inflammation (Cornea 36 Suppl 1(Suppl 1):s9-s14) demonstrates that D.brevis and D.folliculorum are two Demodex species that cause ocular demodicosis in humans, and there is a positive correlation between human age and the risk of the disease and a strong positive correlation between ocular demodicosis and ocular surface inflammatory conditions.
Demodex species in human ocular disease: new clinicopathological aspects (International ophthalmology 37(1):303-312) has shown that D.brevis and D.folliculorum are associated with the pathogenesis of external ocular diseases, and ocular demodicosis can cause an imbalance in the extraocular environment.
Ocular Demodex: a systematic review of the clinical literature (Ophthalmic & physiological optics: the journal of the British College of Ophthalmic Opticians (Optometrists) 40(4):389-432) has shown that Demodex infestation is a potential cause of several ocular surface diseases, and the parasitism of Demodex mites on the anterior structures of the eyes such as eyelids, eyelashes and the ocular surface can lead to the ocular demodicosis in humans, and the incidence is positively correlated with age.
The relationship between demodex and ocular discomfort (Investigative ophthalmology & visual science 51(6):2906-2911) suggested that the number of Demodex mites is positively correlated with the severity of ocular diseases and the age of patients.
Demodex mites (Clinics in dermatology 32(6):739-743) suggested that Demodex infestation can cause chronic blepharitis marginalis, and the prevalence of chronic blepharitis marginalis is positively correlated with the number of mites and the age of patients.
Quantitative Analysis of the Bacteria in Blepharitis With Demodex Infestation (Frontiers in microbiology 9:1719) has shown that D.brevis and D.folliculorum infestations can lead to the occurrence of ocular diseases, the prevalence is positively correlated with the number of mites and the age of patients, and the Demodex mites are carriers of Bacillus spp., which can play a role as co-pathogens in the pathogenesis of blepharitis marginalis.
Bacillus oleronius and Demodex mite infestation in patients with chronic blepharitis (Clinical microbiology and infection: the official publication of the European Society of Clinical Microbiology and Infectious Diseases 18(10):1020-1025) suggested that Demodex mites may cause the occurrence of blepharitis marginalis and are carriers of Bacillus spp., which can play a role as co-pathogens in the development of blepharitis marginalis.
Correlation between ocular Demodex infestation and serum immunoreactivity to Bacillus proteins in patients with Facial rosacea (Ophthalmology 117(5):870-877.e871) suggested that the incidence of ocular Demodex infestation in patients with xerophthalmia is lower than that in patients without xerophthalmia, and that the mite infestation and bacterial infection can coexist in ocular surface inflammation.
High prevalence of demodex brevis infestation in chalazia (American journal of ophthalmology 157(2):342-348.e341) has shown that the incidence of demodicosis caused by D.brevis is high in adults and pediatric patients with chalazion, and the ocular demodicosis is a major cause of chalazion.
Specifically, the subject described above can be any animal receiving the prophylaxis and/or treatment, in particular, a mammal, such as a human, a cat, a dog, a rabbit, a mouse, a horse, a cow, a sheep, a pig and the like. In one embodiment of the present invention, the subject described above is a human; and in another embodiment of the present invention, the subject described above is a non-human animal.
Specifically, the dose of the Radix Gentianae Macrophyllae, the Radix Gentianae Macrophyllae extract, the monomer compound of Radix Gentianae Macrophyllae (in particular, gentiopicroside) or the salt, the ester, the isomer, the solvate or the derivative thereof or the pharmaceutical composition described above may vary according to the route of administration, the age and body weight of the subject, the disease to be treated in the subject and the severity, etc., and may be administered in one or more doses.
By comparing the killing effect of a plurality of traditional Chinese medicinal materials on mites, it was found that Radix Gentianae Macrophyllae can effectively shorten survival time of mites. A variety of monomer compounds of Radix Gentianae Macrophyllae were further screened in experiments of killing mites. It was found that these monomer compounds can shorten the survival time of the mites compared with a control group, and in particular, gentiopicroside can significantly shorten the survival time of the mites, and can be used for preparing a product (such as a medicament, a cosmetic product, a daily product and the like) for killing or controlling mites. The use disclosed herein can effectively utilize natural resources and improve the utilization value and commercial value of Radix Gentianae Macrophyllae.
Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention relates.
Unless otherwise indicated, in the present invention, the Radix Gentianae Macrophyllae refers to the traditional Chinese medicine material, which is a dried root of Gentianamacrophylla Pall., Gentianastraminea Maxim., Gentianacrassicaulis Duthie ex Burk. or Gentianadahurica Fisch.
(Fam. Gentianaceae). The processing steps for Radix Gentianae Macrophyllae comprises: removing impurities, washing, moistening, slicing into thick pieces and drying in the sun. Radix gentianae macrophyllae is pungent and bitter in taste, and neutral in nature, is beneficial to stomach, liver and gallbladder meridians, and has effects of dispelling wind and dampness, removing damp-heat, and relieving arthralgia and deficiency-heat.
Traditional Chinese medicine monomer refers to a compound obtained from a single traditional Chinese medicine (e.g., by extraction), in particular, a compound with therapeutic effects or health benefits. For example, the monomer compound of Radix Gentianae Macrophyllae described herein refers to a monomer compound obtained from Radix Gentianae Macrophyllae, such as, but not limited to, gentiopicroside (CAS: 20831-76-9), loganic acid (CAS: 22255-40-9), sweroside (CAS: 14215-86-2), roburic acid (CAS: 6812-81-3), scopoletin (CAS: 92-61-5), mollugin (CAS: 55481-88-4) and swertiamarin (CAS: 17388-39-5). The traditional Chinese medicine monomer compound described herein may be a commercially available product, or may be obtained from corresponding traditional Chinese medicines (for example, by extraction) before use, synthesized according to methods known in the art, or obtained by other suitable routes.
In the present invention, the term “animal” generally refers to vertebrates, in particular, mammals, including humans. The term “non-human animal” refers to any vertebrate except human beings, in particular, mammals. In some embodiments of the present invention, the non-human animal described herein is a domestic animal, i.e., an animal raised and domesticated by humans with artificially controlled reproduction for purposes such as food, labor, fur, companionship and experiment, such as an economic animal, a companion animal and a laboratory animal. The economic animal may be, for example, a livestock animal such as a pig, a cow, a sheep, a horse, a donkey, a fox, a raccoon dog, a mink, a camel and the like. The companion animal may be, for example, a dog, a cat, a rabbit, a murine (such as a guinea pig, a hamster, a gerbil, a chinchilla, a squirrel, etc.) and the like. The laboratory animal may be, for example, a monkey, a dog, a rabbit, a cat, a murine (such as a rat and a mouse) and the like.
The technical schemes of the present invention will be clearly and completely described below with reference to the examples of the present invention, and it is obvious that the described examples are only a part of the examples of the present invention but not all of them. Based on the examples of the present invention, all other examples obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.
The ethical guidelines of “The Declaration of Helsinki” and “Measures for the Ethical Review of Biomedical Research Involving Humans” were strictly followed in this study. Demodex mites from patients diagnosed with ocular Demodex infestation were collected in this study. Subjects who met the inclusion criteria were enrolled after giving an informed conversation and signing an informed consent.
The detection of Demodex mites was performed according to the conventional method of eyelash microscopy (12,20). 3 eyelashes were collected from each eyelid, with a total of 12 eyelashes. The collected eyelashes were immediately placed on glass slides with three eyelashes on each glass slide, and the glass slides were examined under a common optical microscope for observation. Demodex mites in all periods were counted and classified according to morphology (specific standard: D.brevis with a head-to-body ratio of 1:1, and D.folliculorum with a head-to-body ratio of 1:3 to 1:4). Only the adult mites completely exposed to the field of view were taken as experimental subjects (larvae and eggs were excluded from the study due to their fragility at early stage of life).
On the basis of the above detection of Demodex mites, 35 µL of the solutions was separately added to the slides, and the survival of the mites was observed every 2 hours. During the experiment, the Demodex mites were observed by two skilled operators separately for the activities (bodies, limbs and the like) through the microscope to determine whether they were dead or not. If the two operators gave different results, the survival status was determined independently by a third skilled operator. The in-vitro culture was conducted in a climatic chamber at a temperature of 20° C. and a humidity of 96%. During the observation, the glass slides were transported in a wet box to ensure the high humidity.
The traditional Chinese medicines prepared in ready-to-use forms (Flos Lonicerae, Cortex Dictamni, Radix Scutellariae, Herba Houttuyniae, Cortex Fraxini, Radix Gentianae Macrophyllae and Flos Chrysanthemi) selected in the experiment were purchased from the traditional Chinese medicine pharmacy of Guangdong Provincial Hospital of Chinese Medicine. The Flos Lonicerae, Radix Gentianae Macrophyllae, Radix Scutellariae, Herba Houttuyniae, and Flos Chrysanthemi were from Kangmei Pharmaceutical Co., Ltd., and the Cortex Dictamni and Cortex Fraxini were from Lingnan Traditional Chinese Medicine Tablets Co., Ltd.
60 g of dried ready-to-use forms was weighed using an electronic balance, and soaked in water with an amount of 10 times (600 mL) in a marmite overnight. The mixture was heated to boiling with strong fire, then decocted with small fire for 15-30 minutes (15 minutes for flowers and plants, and 30 minutes for rhizomes), and filtered with gauze. 500 mL of water was added to the filter residue, and the mixture was decocted in the same way and filtered. The two filtrates were combined in a beaker, and mixed well. The resulting mixture was heated and concentrated to a final volume of 100 mL, then cooled to room temperature, and transferred to a 100-mL volumetric flask and diluted to volume.
The treatment groups received different traditional Chinese medicine decoctions prepared by the above method, and the negative control group received sterilized water.
4.3 Positive control is tea tree oil and the major component terpinen-4-ol thereof Tea tree oil (TTO) and terpinen-4-ol (T4O) were purchased from Milwaukee (WI, USA), and diluted in sterilized water to a final concentration of 10%.
In this experiment, statistical analysis was conducted using SPSS22.0 statistical software, normality test was conducted using Shapiro-Wilk test, and homogeneity of variance test was conducted using Bartlett’s test. The data with normal distribution and homogeneous variance were subjected to One-way ANOVA, and when there was statistical significance, Bonferroni’s method was used for pairwise comparison among the groups; when the data did not converge to normal distribution, the Kruskal-Wallis test was adopted for statistical analysis with a test criterion α of 0.05.
1. Radix gentianae macrophyllae can significantly shorten the in vitro survival time of demodex mites.
The in-vitro culture of the Demodex mites was conducted in an environment at a humidity of 96% and a temperature of 20° C. The survival of the Demodex mites was observed dynamically and the survival time was recorded. The results are shown in Table 1. Compared with the negative control group, the survival time of the Demodex mites in the Radix Gentianae Macrophyllae group was significantly shortened (21.44 ± 9.76 vs. 50.81 ± 19.90, P < 0.001), and was shorter than that in the TTO group and the T40 group (21.61 ± 11.95 vs. 41.39 ± 19.33, 21.61 ± 11.95 vs. 40.50 ± 13.12, respectively, P < 0.01). The other 6 traditional Chinese medicines did not show significant differences in the in-vitro survival time of Demodex mites as compared with the control and the groups of TTO and T40 (P > 0.05). Interestingly, in this in-vitro culture experiment, TTO and its major active ingredient T40, which were previously considered to be able to kill mites in vitro, did not show significant differences in the in-vitro survival time of Demodex mites compared with the negative control sterilized water (P > 0.05).
Multiple comparisons were performed by Bonferroni’s test. When compared with water, PA denotes P < 0.05, Pa denotes P < 0.001, and Pa’ denotes P < 0.001; when compared with TTO, PB denotes P < 0.05, Pb denotes P < 0.01, and Pb’ denotes P < 0.001; when compared with T40, Pc denotes P < 0.05, Pc denotes p < 0.01, and Pc’ denotes P < 0.001.
According to the results of Example 1, Radix Gentianae Macrophyllae can significantly shorten the in-vitro survival time of Demodex mites, and thus monomer compounds of this traditional Chinese medicine were selected for further experiments. The names and molecular formulas of the monomer compounds are shown in the following table: The monomer compounds were purchased from Chengdu Herbpurify Co., Ltd., and were standard references.
The powders of the monomer compounds shown in Table 2 were separately dissolved in dimethyl sulfoxide (DMSO), and then diluted with sterilized double-distilled water to make the final concentration of DMSO 10%, which could not shorten the survival time of Demodex mites as per a controlled study. The negative control group received DMSO mixed with sterilized water at a final concentration of 10%.
As in Example 1, 35 µL of each solution was added to a glass slide, and the survival of the mites was observed under an optical microscope every 2 hours. During the experiment, the Demodex mites were observed by two skilled operators separately for the activities (bodies, limbs and the like) through the microscope to determine whether they were dead or not. If the two operators gave different results, the survival status was determined independently by a third skilled operator. The in-vitro culture was conducted in a climatic chamber at a temperature of 20° C. and a humidity of 96%. During the observation, the glass slides were transported in a wet box to ensure the high humidity.
In this experiment, statistical analysis was conducted using SPSS22.0 statistical software, normality test was conducted using Shapiro-Wilk test, and homogeneity of variance test was conducted using Bartlett’s test. The data with normal distribution and homogeneous variance were subjected to One-way ANOVA, and when there was statistical significance, Bonferroni’s method was used for pairwise comparison among the groups; when the data did not converge to normal distribution, the Kruskal-Wallis test was adopted for statistical analysis with a test criterion α of 0.05.
Effects of monomer compounds of traditional Chinese medicine on survival time of D.folliculorum The in-vitro culture of the Demodex mites was conducted in an environment at a humidity of 96% and a temperature of 20° C. The survival of the Demodex mites was observed dynamically and the survival time was recorded. The results are shown in Table 3. Compared with the negative control group receiving 10% DMSO solution, the monomer compounds of Radix Gentianae Macrophyllae shown in Table 2 could shorten the in-vitro survival time of Demodex mites, and in particular, the survival time of Demodex mites was significantly shorter in the gentiopicroside group (37.31 ± 36.06 vs. 80.79 ± 25.34, P < 0.001).
Conformance to the diagnostic criteria of xerophthalmia based on the medical history.
Ocular surface discomfort scoring, general ophthalmic examinations (vision, intraocular pressure, slit-lamp examination), xerophthalmia measurements (conjunctival hyperemia scoring, BUT test, and Schirmer I test), ocular surface disease index (OSDI) scoring, xerophthalmia detector and ocular Demodex examinations were performed on patients before the treatment and weekly after treatment.
Considering that xerophthalmia is a common clinical ocular surface disease, with positive rate of Demodex up to 23.8%-90.0%. According to the sample size calculation formula of superiority test on the basis of quantitative data, 30 cases were enrolled in each of the treatment and control groups.
SPSS20.0 software and Excel were used for statistical analysis, normally distributed enumeration data were analyzed by paired sample t-test, measurement data were analyzed by χ2 test, and non-normally distributed data were analyzed by non-parametric test. The results were expressed as mean ± standard deviation, or x + s), and the differences were considered statistically significant when P < 0.05.
The above description is only for the purpose of illustrating the preferred example of the present invention, and is not intended to limit the scope of the present invention. Any modifications, equivalents and the like made without departing from the spirit and principle of the present invention shall fall within the protection scope of the present invention.
The foregoing examples and methods described herein may vary based on the abilities, experience, and preferences of those skilled in the art.
The certain order in which the procedures are described in the present invention does not constitute any limitation to the order of the procedures.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202010334100.3 | Apr 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/089107 | 4/23/2021 | WO |
