CN117064979A - External skin care oil for preventing and treating psoriasis and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of new indications of medicines, and in particular discloses external skin care oil for preventing and treating psoriasis as well as a preparation method and application thereof. The external skin care oil is prepared by mixing the following raw materials in parts by weight in a mode of batch heating, flash extraction and the like: 30-50 parts of astragalus membranaceus, 30-50 parts of angelica sinensis, 30-50 parts of garden burnet, 15-30 parts of frankincense, 15-30 parts of bletilla striata, 10-30 parts of radix scutellariae, 10-30 parts of coptis chinensis, 10-30 parts of cortex phellodendri, 10-30 parts of rheum officinale, 10-30 parts of gynura procumbens, 3-5 parts of borneol, 700-1000 parts of edible vegetable oil and 300-500 parts of vaseline. Scientific experiments prove that the external skin oil has better effects of tonifying qi and activating blood, purging fire and detoxifying, removing necrotic tissue and promoting granulation, can promote quick regression of the mouse psoriasis induced by Imiquimod (Imiquimod) in a short time, and has a control effect obviously superior to that of a control drug (dexamethasone combined with tacalcitol) commonly used in Western medicine.

Description

External skin-regenerating oil for preventing and treating psoriasis, and preparation method and application thereof

Technical Field

The invention relates to the technical field of new drug indications, and in particular to an external skin-regenerating oil for preventing and treating psoriasis, and a preparation method and application thereof.

Background Art

Psoriasis is a chronic skin disease characterized by erythema, scaling and hypertrophy, commonly known as psoriasis. Its clinical manifestations are erythema and scaling on the skin, so this type of erythema is also called infiltrative erythema. Scaly psoriasis is mica-like scaling, which is prone to occur on the scalp, elbows, knees and below the knees, buttocks, nails and other parts that are easily exposed to external stimuli. Of course, in addition to skin symptoms such as macules, itching and joint (pain, swelling, deformation, etc.) symptoms may also occur. The cause of psoriasis is not very clear. It is generally believed that it is caused by the internal genetic factors and the external environmental factors (such as bad eating habits, lack of sleep, smoking, drinking, infection, drugs, etc.), which make the body's immune balance abnormal (skin inflammation) and will not be transmitted to other people. However, due to its symptoms, which are sometimes good and sometimes bad and recurring, long-term treatment and care are very important.

Psoriasis is listed by the World Health Organization as one of the major diseases that affect human health, and it is also a key difficult disease in the dermatology departments of major hospitals. Modern medicine mostly uses topical medications (moisturizing emollients, glucocorticoids, vitamin D3 derivatives, calcineurin inhibitors, retinoic acid preparations and coal tar preparations, etc.), phototherapy, immunosuppressants, targeted biological agents, etc. for treatment, and has carried out multi-faceted exploration and made certain progress. However, the long course of psoriasis, high recurrence rate and comorbidities are still the key problems and treatment bottlenecks that plague psoriasis patients.

Traditional Chinese medicine has a long history and significant characteristics and advantages in treating psoriasis, but basic research is relatively weak. Therefore, research on traditional Chinese medicine for psoriasis has always been a hot topic in academia. From 2010 to 2021, the National Natural Science Foundation of China ( representing the country's highest, largest, and most authoritative technology platform for scientific research ) continuously funded 41.09 million yuan in research funds for the three major disciplines of traditional Chinese medicine, traditional Chinese medicine, and integrated traditional Chinese and Western medicine, focusing on the etiology and pathogenesis of psoriasis and the mechanism of action of traditional Chinese medicine in effectively intervening or alleviating psoriasis. However, it is very regrettable that despite the huge amount of funding and the extraordinary amount of time spent by thousands of scientists, there is still a lack of high-level papers and scientific and technological achievements. Project managers of the National Natural Science Foundation of China even pointed out that scientists should pay more attention to Chinese herbal external preparations with a long history and remarkable characteristics, emphasizing that the use of external medicines will play an indispensable role in the treatment of skin diseases, especially psoriasis. They suggested that researchers learn from the new technologies of modern biomaterials and preparations, identify scientific problems, and carry out preparation improvement research (Analysis of the Application and Funding of National Natural Science Foundation Projects in the Field of Traditional Chinese Medicine Research on Psoriasis in Recent Years. Journal of Guangzhou University of Chinese Medicine. Vol. 39, No. 7, July 2022, pp. 1688-1696; Corresponding author: Bi Minggang, male, researcher, director of the Tenth Division of the Medical Science Department of the National Natural Science Foundation of China; E-mail: bimg@nsfc.gov.cn).

Therefore, actively searching for a breakthrough treatment method for psoriasis has become the focus of many medical scholars and is also the main purpose of the present invention.

The research content of the present invention is the technical expansion of the National Natural Science Foundation Project "82174110". In recent years, the applicant team has been implementing the National Natural Science Foundation Project "82174110" (based on the β-catenin/COX-2 inflammatory circuit to explore the effect mechanism of skin oil in promoting rapid repair of diabetic foot disease). Although this project is essentially different from the prevention and treatment of psoriasis with skin oil, open wounds are a problem faced by both. At present, the prevention and treatment of psoriasis at the national level is facing a bottleneck. The applicant has found that although medical scientists consider "blood as the basis, blood heat as the first, and blood stasis as the result throughout the entire process of the occurrence and development of the disease", and treat it from the perspective of "heat", "toxicity" or "stasis", most experts and scholars ignore that "the damage of the natural barrier" is the gateway to the disease and the source of the continuous deterioration of the disease. The applicant found from the research on the prevention and treatment of diabetic foot ulcers with skin oil that skin oil can repair and rebuild the natural barrier of the skin in a short time, and applied it to the exploration of the prevention and treatment of psoriasis, which opened up the technical expansion of skin oil.

Summary of the invention

In view of the problem that there is a lack of specific drugs for preventing and treating psoriasis in the above-mentioned prior art:

The first object of the present invention is to provide an external skin-regenerating oil that is effective in preventing and treating psoriasis.

The second object of the present invention is to provide a method for preparing the above-mentioned external skin-regenerating oil for preventing and treating psoriasis.

The third object of the present invention is to provide the medical use of the above-mentioned topical skin-regenerating oil for preventing and treating psoriasis. The inventive concept of the present invention is as follows:

In traditional Chinese medicine, psoriasis is called white psoriasis (it is named after the white skin color that itches and white skin that comes out when scratched), and it is also a disease that is favored by traditional Chinese medicine. In the past 12 years (2010-2021), the National Natural Science Foundation of China has funded more than 40 million yuan in scientific research funds for scientists in the medical field to focus on the etiology and pathogenesis of psoriasis and the mechanism of effective intervention or relief of psoriasis by traditional Chinese medicine. Unfortunately, the traditional Chinese medicine community still has no unified understanding of its dialectical cognition, lacks high-level research papers to improve theoretical cognition, and lacks effective means and topical drugs that can relieve patients' suffering (Analysis of the application and funding of the National Natural Science Foundation of China projects in the field of traditional Chinese medicine research on psoriasis in recent years. Journal of Guangzhou University of Chinese Medicine. July 2022, Vol. 39, No. 7, 1688-1696).

Based on the pathological characteristics of psoriasis, the applicant believes that psoriasis is mainly caused by the body's blood deficiency, or internal blood heat, as well as dryness and wind, which injure the human body's skin and muscles, stagnate qi and blood, cause the skin to lose nutrition and cause skin damage.

In other words, "blood is the basis, blood heat is the priority, and blood stasis is the result" runs through the entire process of the occurrence and development of the disease. Therefore, the applicant believes that the treatment of psoriasis should not be based solely on the symptoms of "heat", "toxicity", or "stasis", but should comprehensively consider the clinical symptoms presented by the same psoriasis patient at different times (clinical symptoms (mainly manifested in the damage of the skin barrier; therefore, repairing the natural barrier of the skin is the focus of the prevention and treatment of psoriasis) include ① when the skin lesions gradually increase, the skin lesions are red, and the patient feels severe itching, it is mainly blood heat; ② when there are no new skin lesions, the base of the skin lesions is light red, and the patient's subjective symptoms are stable, it is mainly blood dryness; ③ when the skin lesions have a tendency to subside, the base of the skin lesions is a little dark red, it is mainly blood stasis), the treatment method of invigorating qi and activating blood circulation, clearing away heat and detoxifying is adopted, so as to ultimately achieve the effect of treating both the symptoms and the root cause of the disease by removing dead tissue and regenerating new tissue (restoring the natural barrier of the skin).

In order to achieve the first purpose, the present invention adopts the following technical measures:

A topical skin-rejuvenating oil for treating psoriasis is prepared from the following raw materials in proportions in parts by weight: 30-50 parts by weight of astragalus, 30-50 parts by weight of angelica, 30-50 parts by weight of sanguisorba officinalis, 15-30 parts by weight of frankincense, 15-30 parts by weight of bletilla striata, 10-30 parts by weight of scutellaria, 10-30 parts by weight of coptis chinensis, 10-30 parts by weight of phellodendron, 10-30 parts by weight of rhubarb, 10-30 parts by weight of chrysanthemum procumbentum, 3-5 parts by weight of borneol, 700-1000 parts by weight of edible vegetable oil and 300-500 parts by weight of vaseline.

The edible vegetable oil is selected from at least one of sesame oil, coconut oil, peanut oil, soybean oil, linseed oil, castor oil and olive oil, preferably at least one of sesame oil, coconut oil and olive oil.

In order to achieve the second purpose, the present invention adopts the following technical measures:

A method for preparing the above-mentioned external skin-regenerating oil for treating psoriasis, the steps of which are as follows:

50%-90% of the total amount of edible vegetable oil is placed in a container, and then the dry powders of astragalus, angelica, sanguisorba officinalis, bletilla striata, scutellaria baicalensis, rhubarb, coptis chinensis, phellodendron amurense, and chrysanthemum notoginseng are added thereto and stirred evenly; after standing and soaking for 3-10 days, the oil is heated with stirring at a temperature of 55-65° C. for 1-2 hours and then stopped heating, and the dry powder of frankincense is added thereto, and the residue is filtered out and the filtered oil is collected; when the oil temperature drops to 30-40° C., borneol, the remaining edible vegetable oil and liquefied vaseline are added thereto, and the mixture is stirred evenly and cooled to obtain an external skin-regenerating oil for treating psoriasis.

Furthermore, the edible vegetable oil is selected from sesame oil and coconut oil. The preparation method of the external skin-regenerating oil for treating psoriasis comprises the following steps:

Put sesame oil into a container, then add dry powders of astragalus, angelica, sanguisorba officinalis, bletilla striata, scutellaria baicalensis, rhubarb, coptis chinensis, phellodendron amurense, and chrysanthemum notoginseng into the container, and stir evenly; after standing and soaking for 3-10 days, stir and heat the oil at an oil temperature of 55-65° C. for 1-2 hours, then stop heating, add dry frankincense powder into the container, filter to remove residue, and collect filtered oil; when the oil temperature drops to 30-40° C., add borneol, coconut oil and liquefied vaseline into the container, stir evenly, and cool to obtain a topical skin-regenerating oil for treating psoriasis.

Furthermore, the step of filtering and removing residues is: after passing through a flash extraction device at 20000rpm high-speed shearing for 3-5 times; and passing through a 5000-mesh sieve under negative pressure to remove residues.

In order to achieve the third purpose of the present invention, the present invention also adopts the following technical measures:

The present invention also provides the use of the external skin-regenerating oil in preparing a medicine for treating psoriasis caused by harmful stimulation.

Furthermore, the psoriasis includes psoriasis vulgaris and impetigo psoriasis.

The principles of the formulation of each Chinese medicinal raw material of the present invention are as follows:

Astragalus, Angelica, Sanguisorba officinalis, Bletilla striata, frankincense, borneol, etc. can promote blood circulation and remove blood stasis, reduce swelling and relieve pain, and are the main drugs (using drugs that invigorate Qi and promote tissue regeneration, promote blood circulation and remove blood stasis, so as to improve local microcirculation and enhance local resistance to injury and repair capabilities); Scutellaria baicalensis, Coptis chinensis, Phellodendron chinense, Rhubarb, etc. can detoxify and eliminate evil, and are the ministers (using drugs that kill miscellaneous bacteria to protect the ulcer surface from further damage and prevent further deterioration); Chrysanthemum and Panax notoginseng, etc. can astringe yin and promote tissue regeneration, remove rot and treat sores, reduce swelling and discharge pus, and are used as adjuvants (assisting the main and minister drugs to rebuild the skin's protective barrier); vegetable oil and vaseline (using their viscosity to cover the wound surface and prevent damage) harmonize the various drugs to achieve the effect of detoxification and tissue regeneration, and are the messengers.

Compared with the prior art, the present invention has the following advantages and effects:

The research results show that the skin-regenerating oil prepared by the present invention can effectively prevent parakeratosis of the affected skin, inhibit excessive proliferation of keratinocytes, reduce psoriasis, reduce erythema, soften and thin the skin, make the skin texture clearer, and has anti-inflammatory and detumescent effects after being administered through the skin, significantly reducing the inflammatory response of the affected skin, thereby achieving the effect of treating psoriasis.

The skin-regenerating oil of the present invention has the following advantages in preventing and treating psoriasis: ① the drug cost is low, it is non-irritating, and has no toxic side effects; ② it is administered through the skin directly to the affected part of the skin, has a good healing effect, and takes a short time.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effects of the tested drugs on the skin of psoriasis mice, the Psoriasis Area and Severity Index (PASI) and the effects on the pathological structure of the skin of psoriasis mice and their pathological scores. A shows the effects of SFO and DXM+Tacalcitol on the skin of the affected part of the experimental mice; the scale in the figure is 1 cm. B shows the score of the Psoriasis Lesion Severity Index. C shows the score of scaling at the lesions of mice. D shows the score of erythema. E shows the score of epidermal thickening. F shows the HE staining of the skin tissue of psoriasis mice; the scale in the figure is 100 microns (objective lens is ×20). G shows the pathological score. In the figure, ** and *** indicate that the p values are less than 0.01 and 0.001 respectively compared with the blank group; in the figure, #, ##, and ### indicate that the p values are less than 0.05, 0.01, and 0.001 respectively compared with the model group.

Figure 2 shows the effects of the tested drugs on the skin of psoriasis mice, the psoriasis lesion severity index score, and the effects on the pathological structure of the skin of psoriasis mice and their pathological score. A shows the appearance of the skin of the experimental mice affected by the Frankincense extract group (Frankincense) and the G. procumbens extract group (G. procumbens); the scale in the figure is 1 cm. B shows the psoriasis lesion severity index score. C shows the scale score of the mouse lesions. D shows the erythema score. E shows the epidermal thickening score. F shows the HE staining of the skin tissue of psoriasis mice; the scale in the figure is 100 microns (objective lens is ×20). G shows the pathological score. In the figure, ** and *** indicate that the p values are less than 0.01 and 0.001 respectively compared with the blank group; in the figure, #, ##, and ### indicate that the p values are less than 0.05, 0.01, and 0.001 respectively compared with the model group.

Figure 3 is an external view of the effect of topical skin-regenerating oil on the skin of the feet of a patient with impetigo psoriasis.

Figure 4 is an external view of the effect of topical skin-regenerating oil on the skin of the right calf of a patient with impetigo psoriasis.

Figure 5 is an external view of the effect of topical skin-regenerating oil on the skin on the outer side of the left arm of a patient with psoriasis vulgaris (one of the typical cases).

Figure 6 is an external view of the effect of topical skin-regenerating oil on the skin on the upper inner side of the left arm of a patient with psoriasis vulgaris (one of the typical cases).

Figure 7 is an external view of the effect of topical skin-regenerating oil on the skin on the outer lower side of the right arm of a patient with psoriasis vulgaris (one of the typical cases).

Figure 8 is an external view of the effect of topical skin-regenerating oil on the skin on the upper outer side of the right arm of a patient with psoriasis vulgaris (one of the typical cases).

Figure 9 is an external view of the effect of topical skin-regenerating oil on the skin on the outer side of the right elbow of a patient with psoriasis vulgaris (typical case 2).

FIG. 10 is an external view of the effect of topical skin-regenerating oil on the skin on the outer side of the right arm of a patient with psoriasis vulgaris (typical case 2).

DETAILED DESCRIPTION

The applicant will further explain the technical solution and application of the present invention in detail below in conjunction with specific embodiments. It should be understood that the following content shall not limit the protection scope of the present invention in any way.

Embodiment 1: A method for preparing skin-regenerating oil, the steps are as follows:

700g of sesame oil is placed in a container, and then 50g of dry powder of Astragalus, Angelica, and Sanguisorba officinalis, 30g of dry powder of Bletilla striata, 10g of dry powder of Scutellaria baicalensis, Rhubarb, Coptis chinensis, Phellodendron amurense, and Panax notoginseng are added, and stirred evenly; after standing and soaking for 10 days, the oil is stirred and heated at an oil temperature of 55°C for 2 hours. After stopping heating, 30g of frankincense dry powder is added thereto, and then sheared 5 times (5 minutes each time) at 20000rpm by a flash extraction device; negative pressure is passed through a 5000 mesh sieve, and filtered oil is collected; when the oil temperature drops to 40°C, 5g of borneol, 300g of coconut oil, and 500g of liquefied vaseline are added thereto, and the mixture is fully stirred, and cooled to room temperature to obtain "skin-regenerating oil", which is divided into 100mL sterile bottles and sealed for storage.

In addition, for comparison:

Preparation of frankincense extract: 700g of sesame oil is placed in a container, and then 30g of dry frankincense powder is added and stirred evenly; the mixture is placed in a colloid mill and circulated and ground for 5h at room temperature. After stopping, the mixture is subjected to high-speed shearing at 20000rpm for 3 times (5min each time) in a flash extraction device; the mixture is passed through a 5000 mesh sieve under negative pressure, and the filtered oil is collected, 300g of coconut oil and 500g of liquefied vaseline are added thereto, and the mixture is stirred evenly, and the mixture is cooled to room temperature to obtain an external frankincense extract, which is then divided into 100mL sterile bottles and sealed for storage.

Preparation of the extract of Chrysanthemum notoginseng: 700g of sesame oil is placed in a container, and then 30g of powder of the dry stem of Chrysanthemum notoginseng is added and stirred evenly; the mixture is placed in a colloid mill and circulated and ground for 5h at room temperature. After stopping, the mixture is subjected to high-speed shearing at 20000rpm for 3 times (5min each time) in a flash extraction device; the mixture is passed through a 5000 mesh sieve under negative pressure, the filtered oil is collected, 300g of coconut oil and 500g of liquefied vaseline are added thereto, the mixture is stirred evenly, and the mixture is cooled to room temperature to obtain the extract of Chrysanthemum notoginseng for external use, which is then divided into 100mL sterilized bottles and sealed for storage.

Embodiment 2: A method for preparing skin-regenerating oil, the steps are as follows:

500g of sesame oil is placed in a container, and then 30g of dry powder of Astragalus, Angelica, and Sanguisorba officinalis, 15g of dry powder of Bletilla striata, 30g of dry powder of Scutellaria baicalensis, Rhubarb, Coptis chinensis, Phellodendron chinense, and Panax notoginseng are added, and stirred evenly; after standing and soaking for 3 days, the oil is stirred and heated at 65°C for 1 hour. After stopping heating, 15g of frankincense dry powder is added thereto, and then sheared at 20000rpm by a flash extraction device for 3 times (5 minutes each time); negative pressure is passed through a 5000 mesh sieve, and filtered oil is collected; when the oil temperature drops to 30°C, 3g of borneol, 200g of coconut oil and 300g of liquefied vaseline are added thereto, and the mixture is fully stirred, and cooled to room temperature to obtain "skin-regenerating oil", which is divided into 100mL sterile bottles and sealed for storage.

Example 3 Effect of the SFO prepared in Example 1 on Imiquimod (IMQ)-induced psoriasis skin lesions in mice

The test animals were 20 male 6-week-old Kunming mice (provided by Hubei Provincial Center for Disease Control and Prevention). They were given free access to water and food, and the temperature was maintained at 25±1°C during the feeding period, with a light-dark cycle of 12 hours.

1) Grouping and drug administration

Twenty mice were randomly divided into 4 groups: blank group (Control), model group (Model), raw skin oil group (SFO), and dexamethasone combined with tacalcitol group (DXM+Tacalcitol).

After 7 days of adaptive feeding, each mouse was roughly cut off the hair of the experimental position on the back with a razor close to the skin, and the area of the exposed skin position was about 2.5 cm × 4 cm. Then the hair was removed again with a depilatory cream, and the skin was immediately washed thoroughly with warm water after depilation, so that the skin of the experimental position was exposed for use. One day after depilation, except for the blank group, which was not subjected to any damage treatment, 62.5 mg of imiquimod cream (Sichuan Mingxin Pharmaceutical Co., Ltd.) was applied to the back skin of each mouse in the other groups every day: during the experiment, the above operation was repeated every day, and 20 minutes were waited for the drug to be absorbed; the skin-skinning oil group and the dexamethasone combined with tacalcitol group were applied to the back skin of each group of mice 20 minutes after applying imiquimod cream, and then 0.2 mL of skin-skinning oil (prepared in Example 1) and 0.2 mL of compound dexamethasone acetate cream (China Resources Sanjiu Pharmaceutical Co., Ltd.) combined with tacalcitol cream (Iwakuni Manufacturing, Teijin Pharma Co., Ltd., Japan) were applied to the back skin of each group of mice; the drug was administered once a day. The model was successfully established when obvious scales, punctate blood spots and erythema appeared on the back of the mouse.

2) Effect of SFO on the gross and pathological changes of the skin of mice with psoriasis induced by imiquimod (IMQ) (HE staining)

At different time points, such as 1 day after administration, 5 days after administration, 9 days after administration, and 13 days after administration, scores were performed according to the Psoriasis Area and Severity Index (PASI) according to 5 degrees (Table 1), among which the mouse skin scales (Score of scaling), erythema (Score of erythema) and epidermal thickening (Score of thickness) were scored, and the three scores were added to obtain the total score (PASIScores), which was expressed as mean±SEM. In the evaluation process, in order to avoid subjective factors, the appearance of the mouse back skin was recorded with a Sony SLR macro camera 20 minutes after the application of the drug, and finally two people independently evaluated the changes in the skin appearance of the psoriasis model mice at each time period.

After 13 days of administration, all test animals were killed by anesthesia, and the skin tissue of the wound site was removed, wrapped with gauze, and fixed in formalin solution for 24 hours. The skin tissue was then rinsed with running water, dehydrated, and paraffin-embedded, followed by tissue sectioning, HE conventional staining, and observed and photographed under an optical microscope. Pathological scoring of mouse skin lesion tissue: Under an optical microscope, the pathological score (Pathological score) of each mouse skin tissue was performed according to the following inflammatory score table (Table 2), and expressed as mean ± SEM.

3) Experimental results

(1) Gross changes in the back skin of mice

One day after administration, as shown in Figure 1A-E, there was no significant difference in the appearance of the back skin of mice in each group.

After 5 days of administration, the back skin of mice in the model group (applied imiquimod) was covered with a small amount of scales, a small amount of erythema, slight thickening, dry and hard skin (compared with the blank group, the total appearance score was 3.25±0.48, P<0.001; the scale score was 1.20±0.20, P<0.01; the erythema score was 1.50±0.29, P<0.001; the thickening score was 0.75±0.25, P>0.01); the back skin of mice in the skin oil group had no obvious scales and erythema, no obvious thickening, and clear skin texture (compared with the model group, the total appearance score was 0.75±0.48, P<0.001; the The scales score was 0.50±0.29, P<0.05; the erythema score was 0.25±0.25, P<0.001), and the thickening score was 0.25±0.25, P>0.01); while the mice in the dexamethasone combined with tacalcitol group had slight fine scales on the back skin, red skin with slight erythema, no obvious thickening but dry and rough, with more wrinkles (compared with the model group, the total appearance score was 2.25±0.48, P>0.01; the scales score was 0.75±0.25, P>0.01; the erythema score was 0.75±0.25, P>0.01; the thickening score was 0.50±0.29, P>0.01).

After 9 days of administration, the back skin of mice in the model group was covered with thin scales, accompanied by flaky erythema, thickened (higher than the surrounding normal skin), red and swollen in the thickened area, and the skin texture was rough and hard (compared with the blank group, the total appearance score was 6.00±0.71, P<0.001; the scale score was 2.20±0.20; P<0.001), the erythema score was 2.25±0.25, P<0.001; the thickening score was 1.50±0.29, P<0.001). The back skin of mice in the skin oil group had no obvious thickening, slightly mild erythema, and no obvious scales (compared with the model group, the appearance score was 1.95±0.48, P<0.001). 0.001; among them, the scaling score was 0.75±0.25, P<0.001; the erythema score was 0.70±0.25, P<0.001; the thickening score was 0.50±0.20, P<0.01); while the back skin of mice in the dexamethasone combined with tacalcitol group showed a small amount of scaling, redness with erythema, thickening, dryness and dullness, and deeper wrinkles (compared with the model group, its appearance score was 4.25±0.75, P<0.05; among them, the scaling score was 1.75±0.25, P>0.01; the erythema score was 1.75±0.25, P>0.01; the thickening score was 0.75±0.38, P>0.01).

After 13 days of administration, the back skin of mice in the model group accumulated flaky silvery-white scales, and the skin below and around the scales had flaky erythema and blood spots covered with a red film, with obvious redness and swelling, and obvious skin thickening (compared with the blank group, the total appearance score was 8.50±0.65, P<0.001); the scale score was 3.00±0.32, P<0.001; the erythema score was 2.75±0.25, P<0.001; the thickening score was 2.75±0.25, P<0.001). The back skin of mice in the skin oil group still had no obvious scales or thickening, with slight erythema, and the skin texture was clear and shiny (compared with the model group, the total appearance score was 1.95±0.51, P<0.00 1. Compared with the model group, the scores were significantly reduced; the scaling score was 0.75±0.25, P<0.001; the erythema score was 0.70±0.25, P<0.001; the thickening score was 0.50±0.20, P<0.001); while the dorsal skin punctate scaling of mice in the dexamethasone combined with tacalcitol group increased and was densely distributed, accompanied by flaky erythema, dry and dull skin, and deeper wrinkles (compared with the model group, the total appearance score was 5.00±0.58, P<0.001; the scaling score was 2.00±0.20, P<0.01; the erythema score was 1.75±0.29, P<0.01; the thickening score was 0.75±0.25, P<0.001).

Overall, compared with the abnormal changes in skin lesions in the model group, the scales, erythema, and thickening of the back skin of mice in other groups all improved to a certain extent, and their scores were lower than those in the model group; especially the back skin of mice in the Shengfu Oil group did not show scales during the entire medication process, only slight erythema; the dexamethasone combined with tacalcitol group also showed a certain recovery ability, but during the entire medication period, the skin of mice in this group had scales and erythema, and the skin wrinkles increased. In contrast, Shengfu Oil can not only effectively relieve skin scales, erythema, and skin bulges, but its effect is better than dexamethasone combined with tacalcitol.

(2) Pathological analysis of mouse skin

After continuous administration for 13 days, all the test mice were killed by anesthesia, and the skin tissues at the wound site on the back were obtained for HE staining. The pathological results were as follows (as shown in Figure 1F and Figure 1G).

a) Blank group: The cells of the epidermis and dermis of the back skin of the mice were arranged normally without edema, and a large number of hair follicles and other accessory organs were visible, but no other obvious abnormalities were observed.

b) Model group: Compared with the blank control group, the structure of the back skin tissue of mice in this group, including the epidermis and dermis, changed: ① The cutaneous protuberances were elongated and undulated, the stratum corneum was significantly thickened and the non-enucleated cells were arranged in layers, the stratum corneum was parakeratotic and hyperkeratotic, a large number of microabscesses were seen in the stratum corneum, and the acanthosis was thickened; ② The dermal papillae were seen in the dermis, the collagen bundles were partially disordered, the capillaries were dilated, a large number of red blood cells were extravasated and inflammatory cells were infiltrated; ③ The pathological score of the skin tissue (2.50±0.29, P<0.001) was significantly lower than that of the blank group (9.25±0.25).

c) Skin oil group: Compared with the model group, the back skin tissue of mice in this group showed the following: ① The epidermis had a clear structure and normal proportions, the cutaneous processes were slightly extended, the stratum corneum was thinner than that of the model group, and there was mild hyperkeratosis; ② The dermis had densely arranged collagen bundles, no obvious dilation of capillaries, and no obvious inflammatory cell infiltration; ③ The skin tissue pathology score was significantly higher than that of the model group (8.37±0.13, P<0.001).

d) Dexamethasone combined with tacalcitol group: Compared with the model group, the skin of mice in this group had not yet healed, showing certain effects: ① The structure of the epidermis was unbalanced, the skin processes were elongated and undulating, the stratum corneum was still thickened, there was excessive keratinization, and the granular layer disappeared, which was not significantly improved compared with the model group; ② Collagen bundles were disordered in the dermis accompanied by a large number of inflammatory cell infiltrations; ③ Its skin tissue pathology score was slightly higher than that of the model group (5.50±0.29, P<0.001).

Example 4 Effects of extracts of Frankincense and Gynura procumbens (Lour.) Merr.: G. procumbens in the skin-regenerating oil prepared in Example 1 on Imiquimod (IMQ)-induced psoriasis skin lesions in mice

The test animals were 20 male 6-week-old KM mice (provided by Hubei Provincial Center for Disease Control and Prevention). They were given free access to water and food, and the temperature was maintained at 25±1°C during the feeding period, with a light-dark cycle of 12 hours.

1) Grouping and drug administration

Twenty mice were randomly divided into four groups: blank group (Control), model group (Model), Frankincense extract group (Frankincense), and G. procumbens extract group (G. procumbens). After 7 days of adaptive feeding, the hair of the experimental position on the back of each mouse was roughly cut with a razor close to the skin, and the area of the exposed skin position was about 2.5cm×4cm. Then the hair was removed again with a depilatory cream. After depilation, it was immediately washed thoroughly with warm water to expose the skin of the experimental position for future use. One day after depilation, except for the blank group, which did not undergo any damage treatment, 62.5 mg of imiquimod cream (Sichuan Mingxin Pharmaceutical Co., Ltd.) was applied to the back skin of each mouse in the other groups every day; during the experiment, the above operation was repeated every day, and 20 minutes were waited for the drug to be absorbed; 20 minutes after applying imiquimod cream, 0.2 mL of frankincense extract and 0.2 mL of G. procumbens extract were applied to the back skin of each group of mice in the Frankincense extract group and G. procumbens extract group, respectively; the drug was administered once a day. The model was successfully established when obvious scales, punctate blood spots and erythema appeared on the back of the mouse.

2) Effects of Frankincense Extract and Panax Notoginseng Extract on the Gross Changes and Skin Pathological Changes of Imiquimod-induced Psoriasis Mice Skin (HE Staining)

At different time points, such as 1 day after administration, 5 days after administration, 9 days after administration, and 13 days after administration, scores were performed according to the Psoriasis Area and Severity Index (PASI) according to 5 degrees (Table 1). The mouse skin scales (Score of scaling), erythema (Score of erythema), and epidermal thickening (Score of thickness) were scored separately, and then the three scores were added to obtain the total score, which was expressed as mean ± SEM. In the evaluation process, in order to avoid subjective factors, the appearance of the mouse back skin was recorded with a Sony SLR macro camera 20 minutes after the application of the drug, and finally two people independently evaluated the changes in the skin appearance of the psoriasis model mice at each time period.

After 13 days of administration, all test animals were killed by anesthesia, and the skin tissue of the wound site was removed, wrapped with gauze, and fixed in formalin solution for 24 hours. The skin tissue was then rinsed with running water, dehydrated, and paraffin-embedded, followed by tissue sectioning, HE conventional staining, and observed and photographed under an optical microscope. Pathological scoring of mouse skin lesion tissue: Under an optical microscope, the pathological scoring of each mouse skin tissue was performed according to the following inflammatory scoring table (Table 2), and expressed as mean ± SEM.

3) Experimental results

(1) Gross changes in mouse skin

One day after administration, as shown in Figures 2A-E, there was no significant difference in the appearance of the back skin of mice in each group.

After 5 days of administration, the back skin of mice in the model group was covered with a small amount of scales, a small amount of erythema, slightly thickened, and the skin became dry and hard (compared with the blank group, the total appearance score was 4.00±0.45, P<0.001; the scale score was 1.40±0.25, P<0.001; the erythema score was 1.60±0.25, P<0.001; the thickening score was 0.90±0.10, P<0.01). The back skin of mice in the frankincense extract group had no obvious scales and erythema, but was slightly thickened, rough and less shiny (compared with the model group, the total appearance score was 2.20±0.58, P<0.0 5; among which the scaling score was 0.80±0.20, P<0.05; the erythema score was 0.60±0.25, P<0.01; the thickening score was 0.80±0.20, P>0.01); the back skin of the mice in the supine chrysanthemum notoginseng extract group had a small amount of scaling and mild thickening, but no obvious erythema, and the skin was dry and rough (compared with the model group, the total appearance score was 2.20±0.37, P<0.05; among which the scaling score was 1.00±0.20, P>0.01; the erythema score was 0.40±0.25, P<0.001; the thickening score was 0.80±0.20, P>0.01).

After 9 days of administration, the back skin of mice in the model group was covered with thin scales, accompanied by flaky erythema, thickened (higher than the surrounding normal skin), redness and swelling in the thickened area, and the skin texture was rough and hard (compared with the blank group, the total appearance score was 6.80±0.74, P<0.001; the scale score was 2.60±0.25, P<0.001; the erythema score was 2.60±0.25, P<0.001; the thickening score was 1.60±0.25, P<0.001). The back skin of mice in the frankincense extract group was covered with a small amount of scales, slightly thickened, with a small amount of needle-like fine erythema, and the skin texture was rough and less shiny (compared with the model group, the total appearance score was 3.3 The skin of the back of the mice in the supine position group was slightly thickened, with a small amount of erythema, dry and rough skin (compared with the model group, the total appearance score was 3.30±0.60, P<0.001; the scale score was 1.30±0.20, P<0.001; the erythema score was 1.00±0.32, P<0.001; the thickening score was 1.00±0.32, P>0.01).

After 13 days of administration, the back skin of mice in the model group was piled with flaky silvery-white scales, and the skin below and around the scales had flaky erythema and blood spots covered with a red film, with obvious redness and swelling, and obvious skin thickening (compared with the blank group, the total appearance score was 9.20±0.58, P<0.001; the scale score was 3.40±0.25, P<0.001; the erythema score was 2.80±0.20, P<0.001; the thickening score was 2.80±0.20, P<0.001). The back skin of mice in the frankincense extract group had a small amount of dense and fine scales, slight thickening, a small amount of needle-like fine erythema, and rough skin texture with less luster (compared with the model group, the total appearance score was 3 .60±0.49, P<0.001; among them, the scaling score was 1.40±0.20, P<0.001; the erythema score was 1.00±0.27, P<0.001; the thickening score was 1.20±0.20, P<0.001); the back skin of the mice in the supine position group had a small amount of scaling, slight thickening, increased erythema, and dry and rough skin texture (compared with the model group, the total appearance score was 3.70±0.60, P<0.001; among them, the scaling score was 1.30±0.20, P<0.001; the erythema score was 1.40±0.25, P<0.001), and the thickening score was 1.00±0.32, P<0.001).

Overall, compared with the model group, the scales, erythema, and thickening of the skin of mice in the other drug groups were improved to a certain extent; during the entire drug administration process, the mice in the frankincense extract group and the chrysanthemum and notoginseng extract group had mild skin damage symptoms such as a small amount of scales, slight erythema, and slight thickening. These results suggest that frankincense extract and chrysanthemum and notoginseng extract have a certain effect on relieving skin scales, inhibiting erythema, and inhibiting excessive skin growth and thickening.

(2) Pathological analysis of mouse skin

After 13 consecutive days of administration, all the test mice were killed by anesthesia, and the skin tissues at the wound site on the back were obtained for HE staining. The pathological results were as follows (as shown in Figures 2F and 2G).

a) Blank group: The epidermis of the mouse back skin was thin, the cells of the epidermis and dermis were arranged normally, there was no edema, and a large number of hair follicles and other accessory organs were visible, and no other obvious abnormalities were observed.

b) Model group: The structure of the skin tissue on the back of mice, including the epidermis and dermis, changed: ① The cutaneous protuberances were elongated and undulated, the stratum corneum was significantly thickened and the non-denucleated cells were arranged in layers, the stratum corneum was parakeratotic and hyperkeratotic at the same time, a large number of microabscesses were seen in the stratum corneum, the stratum spinosum was thickened, and the basal cell proliferation was embedded in the dermis; ② The dermis was partially disordered with collagen bundles, a large number of red blood cells extravasated and inflammatory cells infiltrated; ③ The pathological score of its skin tissue (2.60±0.24, P<0.001) was significantly lower than that of the blank group (9.00±0.27).

c) Frankincense extract group: ① The epidermis had a clear structure and normal proportions, the dermal processes were slightly extended, the stratum corneum was significantly thinner than that of the model group, but the epidermis was still thickened; ② The dermis had tightly arranged collagen bundles, a small amount of inflammatory cell infiltration in the lower part of the dermis, and no red blood cell extravasation; ③ Its skin tissue pathology score was significantly higher than that of the model group (6.50±0.50, P<0.001).

d) Prone group with Chrysanthemum notoginseng extract: ① The epidermal structure was clear, the skin processes were elongated and slightly undulating, the stratum corneum was thickened and hyperkeratotic, a small number of keratinocytes in the epidermis were not denucleated, keratinization was incomplete, and the stratum spinosum was hyperplastic; ② The dermis showed partial disorder and swelling of collagen bundles, moderate inflammatory cell infiltration around blood vessels, and a small amount of red blood cell extravasation; ③ The skin tissue pathology score was slightly higher than that of the model group (5.60±0.24, P<0.001).

Example 5: One of the typical cases of the skin-regenerating oil prepared in Example 1 promoting the repair of impetigo-type psoriasis. One of the typical cases: Mr. Zhan, a retired leader of a design institute (from Shimen, Hunan). 30 years ago, he went to hospitals across the country to seek medical treatment for impetigo-type psoriasis. According to Mr. Zhan, this disease almost exhausted his energy and life savings, and he learned from his fellow patients that the 195 Hospital was special in treating psoriasis, so he bought a house near the 195 Hospital ten years ago and sought rehabilitation in the 195 Hospital (special hot springs + pine oil encapsulation). However, the condition was not satisfactorily controlled. On the contrary, it had affected the whole body, and the feet and legs were more serious. As a last resort, he began to receive injections of biological agents five years ago. Unfortunately, the drug resistance and systemic side effects of biological agents forced Mr. Zhan to change biological agents regularly. Starting from June 12, 2023, the skin-regenerating oil prepared in Example 1 was applied to both feet (including toenails and toes) for 2 weeks (Figures 3 and 4; applied twice a day, a thin layer each time to maintain the moist dimension, and no encapsulation, the total duration is about four hours). It was found that the effect was more obvious than that of pine oil encapsulation (although the biological agent injection was used a month ago, the skin of the affected area of the skin-regenerating oil was obviously better than that of the unapplied skin; in other words, the application of the skin-regenerating oil was better than the effect of the simple use of biological agent injection): (1) Figure 3 is a comparison photo of both feet (including toenails and toes) before and after the application of the skin-regenerating oil. As can be seen from the left photo, the toenails of both feet are dark yellow in color, the surface of the toenails is rough and uneven, with dense sunken groove-shaped nail erosion points, irregular shape, and nail meat separation. After applying the oil for 1 week (middle photo), the toenails were yellow-gray, the skin of the feet was peeling and cracking, and the skin of both feet had obvious scales; after applying the skin-regenerating oil for 2 weeks, the scales on the red patches on the foot lesions were reduced and the red patches became lighter. (2) Figure 4 is a comparison of the skin of the right calf before and after applying the skin-regenerating oil. The photo on the left shows that the skin of the calf has a large range of erythema and scales, the skin texture is unclear, and the skin of the entire calf is thick and hard; after applying the skin-regenerating oil for 1 week (middle photo) and 2 weeks (right photo), the range of erythema on the calf skin has decreased, the red patches have subsided, the scales have also significantly decreased, and the skin texture has gradually become clearer and the skin has become softer. Long-term effects are expected.

Example 6: Typical cases 1 and 2 of the skin-regenerating oil prepared in Example 2 promoting the repair of psoriasis vulgaris. Typical case 1: Mr. Chen, a retired leader of a university, found a lesion with a diameter of 2 cm on his forehead in 1995. He was diagnosed with psoriasis vulgaris in the Department of Dermatology of Wuhan First Hospital. He was prescribed Enfu Cream for external use and Huodan Pills for oral administration, but there was no improvement. Then dexamethasone was taken orally to cause large-area skin disease. After one month of treatment with pine oil + vaseline encapsulation in the 195th Hospital of the People's Liberation Army, the lesions improved, but it was difficult to fundamentally eliminate them, so it was changed to 5% coal tar application, which was maintained until May this year (there is a 29-year history of psoriasis so far). From May 21, 2023, the skin on the outside of the left arm was locally applied with the skin-regenerating oil prepared in Example 2 (a thin layer was applied without encapsulation; two to three times a day, each time for about 2-4 hours), and other parts of the skin were selected for skin application after seeing the effect. The therapeutic effect is shown in Figures 5-8: (1) Figure 5 is a comparison of the skin on the outer side of the left arm before and after applying the skin-regenerating oil. From the first to the fourth photo from the left, it can be seen that the scales gradually disappeared, the red patches gradually lightened, and the skin texture gradually became clearly visible. In other words, the psoriasis lesions on the skin in this area were basically eliminated in less than 25 days; (2) Figure 6 is a comparison of the skin on the inner upper side of the left arm before and after applying the skin-regenerating oil. Mr. Chen experienced significant improvement after applying the skin-regenerating oil on the outer side of his left arm for one week, so he started to use the affected skin on the inner upper side. From the first to the third photo from the left, it can be seen that the surface scales gradually disappeared, the lesion site did not bulge, the red patches gradually faded, and the skin texture basically recovered with the application time (after 27 days); (3) Figure 7 is a comparison of the skin on the outer lower side of the right arm before and after applying the skin-regenerating oil. From the first photo from the left, it can be seen that before applying the skin-regenerating oil, the skin on the affected part was obviously raised, red in color, and with unclear skin texture; after applying the skin-regenerating oil, the symptoms gradually alleviated; in the fourth photo, it can be seen that the skin area of the affected part became smaller, the raised part disappeared, and the color became obviously lighter, with clear texture; (4) Figure 8 is a comparison of the upper outer side of the right arm before and after applying the skin-regenerating oil. From the first to the fourth photo from the left, it can be clearly seen that before applying the skin-regenerating oil, the skin on the affected part was red (with bleeding spots), scaly, and raised, and with unclear texture; after applying the skin-regenerating oil, the symptoms of the skin on the affected part gradually alleviated. In particular, after applying it for 18 days and 25 days, the recovery effect of the skin on the affected part was good (the skin had no scales, no raised part, and became smooth with clear texture). During the use of the skin-regenerating oil, Mr. Chen did not feel any discomfort, and did not need to use complicated encapsulation or oral medication as in the previous treatment, and it did not interfere with his daily work at all.

Typical case 2: Mr. He (from Beijing), a retired leader of a submarine unit. Thirty-five years ago, he developed psoriasis due to exposure to chemical substances at work. After that, he sought medical treatment nationwide and found that the treatment effect of Hospital 195 was good. However, due to recurrence after treatment, he had to buy a house near Hospital 195 to facilitate improvement treatment (special hot springs + pine oil encapsulation). From June 12, 2023, he chose to locally accept the application of the raw skin oil prepared in Example 2 for 2 weeks (Figures 9 and 10; apply a thin layer without encapsulation; apply twice a day, and keep it for a total of about 4 hours). It was found that compared with pine oil encapsulation, the effect was more obvious: (1) Figure 9 is a comparison of the skin on the outside of the right elbow before and after the application of raw skin oil. The left photo shows the state before application, with severe scales (thick and numerous), red and swollen edges, obvious bulges, and thick and hard skin on the affected area, accompanied by strong itching; after applying for one week (middle photo) and two weeks (right photo), the scales on the affected area were significantly reduced, the redness and swelling of the edges and bulges were also reduced, the thickness of the rash softened and thinned, and the skin texture gradually appeared; (2) Figure 10 is a comparison of the skin on the outer side of the right arm before and after applying the skin-skinning oil. The left photo shows the state before application, with a large area of severe scales (thick and numerous), red and swollen edges, bulges, and obvious hard shell-like wrinkles and creases caused by thick scales, and the skin on the affected area was thick and hard, accompanied by strong itching; after applying for 1 week (middle photo) and 2 weeks (right photo), the thickness of the scales on the affected area softened and thinned, the wrinkles and creases caused by thick scales disappeared, the color of the skin lesions changed from red to dark, and the normal skin texture gradually increased. It is recommended to apply it at night before going to bed to improve the efficacy and recovery speed.

The above scientific experiments prove that the external skin-regenerating oil prepared by the present invention has good effects of "invigorating qi and activating blood circulation, purging fire and detoxifying, removing rot and regenerating muscles", and can promote the rapid disappearance of imiquimod-induced psoriasis in mice (including scales, erythema and skin thickening and other body surface behavioral lesions; pathological changes in tissue structure include epidermal hyperkeratosis, thickening of the stratum corneum and granular layer, and hypertrophy of the stratum spinosum; thickening of the dermis, accompanied by severe inflammatory cell infiltration and red blood cell overflow; fewer hair follicles and other skin appendages, etc.) in a short period of time, and its preventive and therapeutic effects are significantly better than the control drugs commonly used in Western medicine clinics (dexamethasone combined with tacalcitol) and frankincense extracts and Panax notoginseng extracts in traditional Chinese medicine. The external skin-regenerating oil prepared by the present invention also has better therapeutic effects on psoriasis vulgaris and impetigo psoriasis with a history of decades than Western medicine or combined Chinese and Western medicine (such as encapsulation of drugs such as pine oil and coal tar, dexamethasone, and biological preparations) of famous hospitals and famous doctors.

Table 1. Psoriasis Area and Severity Index (PASI) scores

Table 2. Pathological scores of mouse skin lesions.

Claims (8)

1. The external skin care oil for treating psoriasis is characterized by being prepared from the following raw materials in parts by weight: 30-50 parts of astragalus membranaceus, 30-50 parts of angelica sinensis, 30-50 parts of garden burnet, 15-30 parts of frankincense, 15-30 parts of bletilla striata, 10-30 parts of radix scutellariae, 10-30 parts of coptis chinensis, 10-30 parts of cortex phellodendri, 10-30 parts of rheum officinale, 10-30 parts of gynura procumbens, 3-5 parts of borneol, 700-1000 parts of edible vegetable oil and 300-500 parts of vaseline.

2. The topical skin care oil for treating psoriasis according to claim 1, wherein the edible vegetable oil is selected from at least one of sesame oil, coconut oil, peanut oil, soybean oil, linseed oil, castor oil and olive oil.

3. A method for preparing the topical skin care oil for treating psoriasis according to any one of claims 1 to 2, characterized by comprising the following steps:

placing 50% -90% of the total edible vegetable oil into a container, adding dry powders of radix astragali, radix Angelicae sinensis, radix Sangusorbae, rhizoma Bletillae, radix Scutellariae, radix et rhizoma Rhei, rhizoma Coptidis, cortex Phellodendri, and radix Gynostemmatis, and stirring; standing for soaking for 3-10 days, stirring at 55-65deg.C for 1-2 hr, stopping heating, adding Olibanum dry powder, filtering to remove residue, and collecting filtrate; when the temperature of the oil is reduced to 30-40 ℃, adding borneol, the rest edible vegetable oil and liquefied vaseline into the mixture, fully stirring the mixture, and cooling the mixture to obtain the skin care oil for external use for treating psoriasis.

4. A method for preparing topical skin care oil for treating psoriasis according to claim 3, comprising the steps of:

placing sesame oil into a container, adding dry powders of radix astragali, radix Angelicae sinensis, radix Sangusorbae, rhizoma Bletillae, radix Scutellariae, radix et rhizoma Rhei, rhizoma Coptidis, cortex Phellodendri, and radix Gynostemmatis, and stirring; standing for soaking for 3-10 days, stirring at 55-65deg.C for 1-2 hr, stopping heating, adding dry powder of milk, filtering to remove residue, and collecting filtered oil; when the oil temperature is reduced to 30-40 ℃, adding borneol, coconut oil and liquefied vaseline into the mixture, fully stirring the mixture, and cooling the mixture to obtain the skin care oil for external use for treating psoriasis.

5. The method for preparing topical skin care oil for treating psoriasis according to claim 3 or 4, wherein the step of filtering to remove the residue comprises: shearing for 3-5 times at 20000rpm by flash extraction device; sieving with 5000 mesh sieve under negative pressure to remove residue.

6. Use of a topical skin care oil according to any one of claims 1-2 or a topical skin care oil prepared by a method according to any one of claims 3-5 in the manufacture of a medicament for the treatment of psoriasis.

7. Use of a topical skin care oil according to any one of claims 1-2 or a topical skin care oil prepared by a method according to any one of claims 3-5 in the manufacture of a medicament for the treatment of psoriasis caused by noxious stimuli.

8. The use according to claim 6 or 7, wherein the psoriasis comprises psoriasis vulgaris, psoriasis pustulosa.