APPLICATION OF RADIX GENTIANAE MACROPHYLLAE AND MONOMER COMPOUND THEREOF IN KILLING MITES

Abstract

An application of Radix Gentianae Macrophyllae, a Radix Gentianae Macrophyllae extract, and a Radix Gentianae Macrophyllae monomer compound or a salt, an ester, an isomer, a solvate, and a derivative thereof in the preparation of an acaricidal product. The Radix Gentianae Macrophyllae monomer compound is selected from one or more of gentiopicrin, loganic acid, sweroside, roburic acid, scopoletin, rubimaillin, and swertiamarin.

Claims

1. Use of Radix Gentianae Macrophyllae, a Radix Gentianae Macrophyllae extract, 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, preferably, the Radix Gentianae Macrophyllae extract comprises one or more of gentiopicroside, loganic acid, sweroside, roburic acid, scopoletin, mollugin and swertiamarin; more preferably, the Radix Gentianae Macrophyllae extract comprises gentiopicroside; preferably, the monomer compound is selected from one or more of gentiopicroside, loganic acid, sweroside, roburic acid, scopoletin, mollugin and swertiamarin; and more preferably, the monomer compound is selected from gentiopicroside.

2. The use according to claim 1, wherein the mites are one or more of Demodex spp., Dermatophagoidespteronyssinus and Sarcoptesscabiei.

3. The use according to claim 1, wherein the product for killing mites is a pharmaceutical composition, a cosmetic product or an acaricide.

4. The use according to claim 3, wherein the pharmaceutical composition is a pharmaceutical composition for preventing and/or treating a disease caused by mite infestation; the disease is selected from: an ocular disease, a skin disease and an allergic disease; preferably, the ocular disease is selected from: 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 and chalazion; preferably, the skin disease is selected from: seborrhoeic dermatitis, acne, acne rosacea, pityriasis folliculorum, perioral dermatitis, demodicosis, gaile and basal cell carcinoma; and preferably, the allergic disease is selected from: allergic asthma, allergic rhinitis and allergic dermatitis.

5. The use according to claim 4, wherein the pharmaceutical composition is a topical preparation, preferably an ophthalmic preparation or a skin topical preparation.

6. The use according to claim 5, wherein the ophthalmic preparation is selected from: an eye drop, an eye ointment, an ophthalmic gel, an ophthalmic emulsion, an ophthalmic suspension, an ophthalmic film, a collyrium and an intraocular injection.

7. The use according to claim 5, wherein the skin topical preparation is selected from: an aerosol, a powder, a lotion, a tincture, a liniment, a film coating agent, an ointment, a gel, a paste and an emulsion.

8. The use according to claim 4, wherein the pharmaceutical composition is a human pharmaceutical composition or a veterinary pharmaceutical composition.

9. The use according to claim 3, wherein the cosmetic product is in a form selected from: 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, a scrub, a neck cream, a shampoo, a shower gel, a hair conditioner, a body lotion, 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 and a brow powder.

10. The use according to claim 3, wherein the acaricide is in a form selected from: a spray, a lotion, a paster and a small packet.

11. 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.

12. 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.

Description

DETAILED DESCRIPTION

[0075] 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.

[0076] 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.

[0077] (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.

[0078] 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.

[0079] 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.

[0080] 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.

Example 1

I. Methods

1. Subjects

[0081] 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.

2. Detection of Demodex Mites

[0082] 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).

3. In-Vitro Culture of Demodex Mites

[0083] On the basis of the above detection of Demodex mites, 35 .Math.L 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.

4. Medicine

4.1 7 Traditional Chinese Medicines

[0084] 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.

4.2 Method for Preparing Traditional Chinese Medicine Decoction

[0085] 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.

[0086] The treatment groups received different traditional Chinese medicine decoctions prepared by the above method, and the negative control group received sterilized water.

[0087] 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%.

5. Statistics

[0088] 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.

6. Results

[0089] 1. Radix gentianae macrophyllae can significantly shorten the in vitro survival time of demodex mites.

[0090] 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).

TABLE-US-00001 Effects of traditional Chinese medicine decoctions on survival time of D.folliculorum in vitro Name of medicine Survival time (hours) Number of Demodex mites (N) Water 50.81 ±19.90 12 TTO 41.39 ± 19.33 22 T40 40.50 ± 13.12 21 Flos Lonicerae 37.06 ± 23.43 14 Flos Chrysanthemi 31.33 ± 13.26 15 Herba Houttuyniae 49.57 ± 16.40 11 Cortex Dictamni 32.57 ± 19.80 18 Radix Scutellariae 34.13 ± 21.90 16 Radix Gentianae Macrophyllae 21.44 ± 9.76 .sup.a’.sup.bc 28 Cortex Dictamni 31.84 ± 15.12 22

[0091] Multiple comparisons were performed by Bonferroni’s test. When compared with water, P.sup.A denotes P < 0.05, P.sup.a denotes P < 0.001, and P.sup.a’ denotes P < 0.001; when compared with TTO, P.sup.B denotes P < 0.05, P.sup.b denotes P < 0.01, and P.sup.b’ denotes P < 0.001; when compared with T40, P.sup.c denotes P < 0.05, P.sup.c denotes p < 0.01, and P.sup.c’ denotes P < 0.001.

Example 2: Preliminary Screening in Vitro of Monomer Compounds for Effect on Survival Time of Demodex Mites

Methods

1. Monomer Compound

[0092] 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.

TABLE-US-00002 Names and molecular formulas of monomer compounds No. Chinese Traditional Chinese medicine English name Molecular 1 Radix Gentianae Macrophyllae Gentiopicroside C16H20O9 2 Radix Gentianae Macrophyllae Loganic acid C16H24O10 3 Radix Gentianae Macrophyllae Sweroside C16H22O9 4 Radix Gentianae Macrophyllae Roburic acid C30H48O2 5 Radix Gentianae Macrophyllae Scopoletin C10H8O4 6 Radix Gentianae Macrophyllae Rubimaillin C17H16O4 7 Radix Gentianae Macrophyllae Swertiamarine C16H22O10

2. Preparation of Monomer Compound Solutions

[0093] 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%.

3. In-Vitro Culture of Demodex Mites

[0094] As in Example 1, 35 .Math.L 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.

4. Statistics

[0095] 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.

5. Results

[0096] 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).

TABLE-US-00003 Effects of monomer compounds in traditional Chinese medicine on survival time of D.folliculorum in vitro No. Monomer compound Survival time (hours) Number of Demodex mites (N) Control 10%DMSO 80.79 ±25.34 36 1 Gentiopicroside 37.31±36.06*** 15 2 Loganic acid 61.56±22.84 8 3 Sweroside 60.79±25.98 7 4 Roburic acid 63.41±12.20 9 5 Scopoletin 54.67±25.66 7 6 Mollugin 61.10±25.12 7 7 Swertiamarin 59.68±10.97 7 Multiple comparisons were conducted using Duncan’s test; as compared with the control group, P*:p < 0.05, P**: p < 0.01, P***: p < 0.001.

Example 3: Clinical Study on Treatment of Xerophthalmia Caused by Mite Infestation

(I) Inclusion and Exclusion Criteria

1. Inclusion Criteria

[0097] Conformance to the diagnostic criteria of xerophthalmia based on the medical history. [0098] (2) Aged 18-70 years; [0099] (3) Any gender; [0100] (4) Willingness to cooperate with treatment.

2. Exclusion Criteria

[0101] (1) Iridocyclitis confirmed by slit-lamp examination; [0102] (2) Ocular hypertension and hypotension; [0103] (3) Ulcerative wounds on eyelid skin and corneal epithelial infiltrating lesions on corneal surface; [0104] (4) Systemic disease, such as Sjogren’s syndrome in patients with impaired hepatic and renal function; [0105] (5) Aged <18 years or >70 years; [0106] (6) Mental state improper for evaluation; [0107] (7) Women in pregnancy and lactation.

3. Criteria for Withdrawal and Dropout

[0108] (1) Inability or unwillingness to continue the treatment due to other diseases accompanied in the treatment process; [0109] (2) Inability to cooperate or unwillingness to continue the treatment due to worsening symptoms in the treatment process; [0110] (3) Violation to the trial scheme and use of other medicines of this study; [0111] (4) Inability to determine efficacy due to incomplete final data.

(II) Endpoints

[0112] 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.

(III) Statistics

1. Estimation of Sample Size

[0113] 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.

2. Statistics and Data Analysis

[0114] 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.

[0115] 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.

[0116] The foregoing examples and methods described herein may vary based on the abilities, experience, and preferences of those skilled in the art.

[0117] The certain order in which the procedures are described in the present invention does not constitute any limitation to the order of the procedures.