Application of Herba Glehniae and Its Extracts

Abstract

The invention belongs to the field of biotechnology, and relates to the application of ophiopogonis and its extract, and specifically relates to the application of ophiopogonis or its extract in the preparation of medicine for preventing or treating systemic lupus erythematosus or its complications.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to application of ophiopogon serrata and an extract thereof. Background Art

[0002] Autoimmune diseases are a type of disease caused by the body's immune response to its own antibodies, resulting in damage to its own tissues.

[0003] Lupus is a chronic, recurrent autoimmune disease with a complex etiology. When it develops, it is characterized by immune abnormalities, the formation of a large number of autoantibodies, mainly antinuclear antibodies, and immune complexes, which accumulate in multiple organs throughout the body. And the immune complexes are deposited in the skin, mucous membranes, joints, kidneys and other tissues and organs throughout the body, causing organ damage and even endangering life. Therefore, lupus is also called systemic lupus erythematosus. Corresponding to it are drug-induced lupus erythematosus, cutaneous lupus erythematosus and neonatal lupus erythematosus. Although the clinical manifestations of drug-induced lupus erythematosus are similar to those of systemic lupus erythematosus, it will heal after about half a year of stopping the drug. Some patients (10-40%) with cutaneous lupus erythematosus will develop into systemic lupus erythematosus. The pathogenesis of neonatal lupus erythematosus is quite different from the above-mentioned lupus erythematosus, and most of them gradually improve and recover after birth.

[0004] Penthorum chinense Pursh, also known as water willow and water zelan, is the above-ground part of the Saxifraganceae family. The whole plant is used as medicine in folk medicine. It is warm in nature, sweet in taste, non-toxic, and has the effects of clearing away heat and detoxification, promoting blood circulation and removing blood stasis, promoting diuresis and reducing swelling, reducing jaundice, and calming the liver. It can treat amenorrhea, edema, metrorrhagia, leucorrhea, and traumatic injuries. Modern research shows that Penthorum chinense Pursh contains a variety of effective ingredients that have a protective effect on liver damage, and has a certain therapeutic effect on hepatitis A, hepatitis B, and chronic active hepatitis. The herb can be used as medicine to relieve liver fire, expel liver toxins, and has the effects of scavenging oxygen free radicals and delaying aging.

[0005] However, there is no related report on the use of Herba Chrysanthemi Indici or its extracts for treating or preventing systemic lupus erythematosus in the prior art. Summary of the invention

[0006] The purpose of the present invention is to provide the use of Herba Lycopodii or its extract in the preparation of medicine for preventing or treating systemic lupus erythematosus or its complications.

[0007] The invention discloses application of ophiopogon japonicus or its extract in preparing medicine for preventing or treating systemic lupus erythematosus or its complications.

[0008] In some embodiments, the systemic lupus erythematosus comprises cutaneous lupus erythematosus or other forms of lupus erythematosus.

[0009] In some embodiments, the cutaneous lupus erythematosus is selected from chronic cutaneous lupus erythematosus, subacute cutaneous lupus erythematosus, and acute cutaneous lupus erythematosus.

[0010] In some embodiments, the complications of systemic lupus erythematosus are selected from lupus cutaneous lesions, lupus nephritis, lupus heart disease, lupus pneumonia, lupus peritonitis, lupus vasculitis, lupus hepatitis, lupus eye lesions, and lupus-associated arthritis.

[0011] In some embodiments, the chronic cutaneous lupus erythematosus is selected from childhood discoid lupus erythematosus, generalized discoid lupus erythematosus, localized discoid lupus erythematosus, deep lupus erythematosus, lupus erythematosus panniculitis, mucosal lupus erythematosus, tumoural lupus erythematosus, pernio-like lupus erythematosus, lupus erythematosus-lichen planus overlap syndrome and verrucous lupus erythematosus.

[0012] In some embodiments, the extract comprises an aqueous extract of Herba Chrysanthemi Indici or an alcohol extract of Herba Chrysanthemi Indici. In some embodiments, the extract is an alcohol extract of Herba Chrysanthemi Indici. In some embodiments, the extract is an ethanol solution extract of 70-95% v / v. In some embodiments, the extract is an ethanol solution extract of 70-75% v / v.

[0013] In some embodiments, the extract contains flavonoids.

[0014] In some embodiments, the extract further contains at least one of chalcone compounds, saponins and polyphenolic acids.

[0015] In some embodiments, the extract contains chalcone compounds.

[0016] In some embodiments, the flavonoids include pyralidin and its glycosides and / or quercetin and its glycosides.

[0017] In some embodiments, the extract includes at least one of pine 7-O-glucose, pine 7-O-β-D glucose, 5-methoxy-pine 7-O-β-D-glucose, pine 7-O-[4″,6″-(S)-hexahydroxybiphenyl acyl]-β-D-glucose, pine 7-O-[3″-O-galloyl-4″,6″-(S)-hexahydroxybiphenyl acyl]-β-D-glucose, and pine 7-O-hexahydroxydibenzoyl-β-glucose.

[0018] In some embodiments, the quercetin glycoside compound includes at least one of quercetin-3-O-β-D-rhamnose, quercetin-3-OL-rhamnose and quercetin-3-O-arabinofuranosyl.

[0019] In some embodiments, the chalcone compound includes thonningianin dihydroxychalcone-7-O-[3″-O-galloyl-4″,6″-(S)-hexahydroxybiphenyl dicarboxyl]-β-D-glucose (thonningianin A) and / or thonningianin dihydrochalcone-7-O-[4”,6”-hexahydroxybiphenyl dicarboxyl]-β-D-glucose (thonningianin B).

[0020] In some embodiments, the polyphenolic acid comprises gallic acid and / or ellagic acid.

[0021] In some embodiments, the extract includes gallic acid, ellagic acid, thunbergin 7-O-glucose, thunbergin-7-O-β-D-glucose, 5-methoxy-thunbergin-7-O-β-D-glucose, dihydrochalcone-7-O-glucose, quercetin-3-O-β-D-rhamnose, thunbergin-7-O-[4″,6″-(S)-hexahydroxybiphenyl diol]-β-D-glucose, thunbergin-7-O-[3″-O-galloyl-4″,6″-(S)-hexahydroxybiphenyl diol]-β-D-glucose, thunbergin dihydroxychalcone-7-O-[3″-O-galloyl-4″,6″-(S)-hexahydroxybiphenyl diol]-β-D-glucose (Thonningianin A) and thonningianin dihydroxychalcone-7-O-[4″,6″-(S)-hexahydroxybiphenyl diol]-β-D-glucose (Thonningianin B).

[0022] In some embodiments, the extract includes pyralidin-7-O-[4″,6″-(S)-hexahydroxybiphenylyl]-β-D-glucose, pyralidin-7-O-[3″-O-galloyl-4″,6″-(S)-hexahydroxybiphenylyl]-β-D-glucose, and pyralidin dihydroxychalcone-7-O-[3″-O-galloyl-4″,6″-(S)-hexahydroxybiphenylyl]-β-D-glucose.

[0023] In some embodiments, the content of pine pinoside-7-O-[4″,6″-(S)-hexahydroxybiphenyl acyl]-β-D-glucose, pine pinoside-7-O-[3″-O-galloyl-4″,6″-(S)-hexahydroxybiphenyl acyl]-β-D-glucose and pine pinoside dihydroxychalcone-7-O-[3″-O-galloyl-4″,6″-(S)-hexahydroxybiphenyl acyl]-β-D-glucose in the extract is more than 30%, preferably 30-50%.

[0024] In some embodiments, the method for preparing the extract comprises the following steps:

[0025] Extract the yellow grass, filter it, and collect the filtrate;

[0026] Concentrating the filtrate to obtain a concentrate;

[0027] extracting the concentrated solution to obtain an extract;

[0028] The extract is purified to obtain the Herba Lycopodiellae extract.

[0029] In some embodiments, before soaking, the Herba Lycopodii is first made into Herba Lycopodii powder.

[0030] In some embodiments, the solvent used for the leaching is selected from water, methanol solution and ethanol solution.

[0031] In some embodiments, the macroporous resin used for purification is selected from HPD-500 macroporous resin and D-101 macroporous resin.

[0032] In some embodiments, the method for preparing the extract comprises the following steps:

[0033] Mix the yellow grass with 70-95% v / v ethanol solution for extraction, then filter and collect the filtrate;

[0034] Concentrating the filtrate to obtain a concentrate;

[0035] The concentrated solution is first extracted with petroleum ether and then extracted with ethyl acetate to obtain an ethyl acetate extract;

[0036] loading the ethyl acetate extract into a macroporous resin column;

[0037] The extract was eluted with 25-35% v / v ethanol solution and 75-85% v / v ethanol solution in sequence, and the effluent of 75-85% v / v ethanol solution was collected to obtain the Herba Glehniae Extract.

[0038] In some embodiments, the extract of Herba Glehniae is obtained by eluting with 25-35% v / v ethanol solution and 80% v / v ethanol solution in sequence, and collecting the effluent of 80% v / v ethanol solution.

[0039] In some embodiments, the ethyl acetate extract is diluted before loading into the macroporous resin column, for example, by diluting with 25-35% v / v ethanol solution.

[0040] In some embodiments, the medicament includes a pharmaceutically acceptable carrier.

[0041] In some embodiments, the carrier includes at least one of an excipient, a diluent, an absorbent, a filler, an absorption enhancer, an adsorption carrier, a stabilizer, a dispersant, and a preservative.

[0042] In some embodiments, the excipient is selected from at least one of calcium carbonate, lactose, calcium phosphate, and sodium phosphate.

[0043] In some embodiments, the diluent and absorbent are selected from at least one of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, dextran, colloidal silicon dioxide, gum arabic, gelatin, magnesium trisilicate and keratin.

[0044] In some embodiments, the filler is selected from at least one of starch, powdered sugar, calcium phosphate, calcium sulfate dihydrate, dextrin, microcrystalline cellulose, lactose, xylitol, sorbitol, maltose, erythrose, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, pregelatinized starch, mannitol (granular or powdered), microcrystalline cellulose, microcrystalline cellulose and lactose, and microcrystalline cellulose and calcium phosphate.

[0045] In some embodiments, the absorption enhancer is selected from at least one of quaternary ammonium salts and sodium lauryl sulfate.

[0046] In some embodiments, the adsorption carrier is selected from at least one of starch, lactose, bentonite, silica gel, kaolin and bentonite.

[0047] In some embodiments, the stabilizer is selected from at least one of polyethylene glycol, polypropylene glycol, mannitol, sucrose, trehalose, fructose, lactose, Tween 60, Span 80, cetearyl alcohol, myristyl alcohol, glyceryl monostearate, sodium lauryl sulfate and glyceryl distearate.

[0048] In some embodiments, the dispersant is selected from at least one of potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (cross-linked carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-soluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate and quaternary ammonium compounds.

[0049] In some embodiments, the preservative is selected from at least one of glycerin, methylparaben and propylparaben, benzoic acid, sodium benzoate, alcohol, and the like.

[0050] In some embodiments, the drug is in the form of an injection or oral administration form.

[0051] In some embodiments, the injection dosage form is selected from injection solution, injection suspension, injection granule, injection gel and injection solid form. In some embodiments, the injection solid form can be a lyophilized powder injection.

[0052] In some embodiments, the oral dosage form is selected from the group consisting of tablets, capsules, caplets, pills, granules, microspheres, powders, syrups, decoctions, and paints.

[0053] The invention provides the herb or its extract, which can improve the content of immune cells in the blood, the concentration of inflammatory cytokines such as TNF-a and IFN, reduce the level of antinuclear antibodies in serum, improve glomerular inflammatory lesions, and have a positive effect on reducing the level of urine protein, thereby preventing or treating systemic lupus erythematosus or its complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The composition of PGF compounds characterized by HPLC is shown.

[0055] Figure 2 The effects of different treatments on the thymus index and spleen index of MRL / lpr mice are shown. Figure 2 A shows the effects of different treatments on the thymus index of MRL / lpr mice; Figure 2 B shows the effects of different treatments on the spleen index of MRL / lpr mice.

[0056] Figure 3 Shown are the effects of different treatments on the neutrophil content in the blood of MRL / lpr mice.

[0057] Figure 4The effects of different treatments on autoantibodies in the sera of MRL / lpr mice are shown. Figure 4 A shows the effects of different treatments on anti-double-stranded DNA antibodies in the serum of MRL / lpr mice; Figure 4 B shows the effects of different treatments on anti-snRNP / Sm antibodies in the serum of MRL / lpr mice.

[0058] Figure 5 Shown are the effects of different treatments on interferon IFN and tumor necrosis factor α (TNF-α) in the serum of MRL / lpr mice. Figure 5 A shows the effects of different treatments on cytokine IFN in the serum of MRL / lpr mice; Figure 5 B shows the effects of different treatments on the cytokine TNF-α in the serum of MRL / lpr mice.

[0059] Figure 6 Shown are the effects of different treatments on urinary protein in MRL / lpr mice. Figure 6 A shows the effects of different treatments on urine protein in MRL / lpr mice at week 4; Figure 6 B shows the effects of different treatments on urine protein in MRL / lpr mice at week 6.

[0060] Figure 7 Shown are the effects of different treatments on renal pathological changes in MRL / lpr mice. Figure 7 In the figure, the black arrows point to inflammatory cell infiltration; the gray arrows point to cell proliferation in the capillaries; and the white arrows point to matrix thickening in the mesangial area.

[0061] Figure 8 The effects of different treatments on the transcriptional expression of NF-κB, HIF-1α, and TLR4 in primary spleen cells of MRL / lpr mice are shown. Figure 8 A shows the effects of different treatments on the transcriptional expression of NF-κB in primary spleen cells of MRL / lpr mice; Figure 8 B shows the effects of different treatments on the transcriptional expression of HIF-1α in primary spleen cells of MRL / lpr mice; Figure 8 C shows the effects of different treatments on the transcriptional expression of TLR4 in primary spleen cells of MRL / lpr mice. Figure 8 In AC, PGF-L represents the group treated with 7.5% drug-containing serum (PGF); PGF-H represents the group treated with 10% drug-containing serum (PGF).

[0062] Fig. 9 Dual luciferase reporter gene assay was used to detect the activation effect of PGF on ARE transcription activity.

[0063] Fig.10The inhibitory activity of different doses of PGF-containing serum on HIF-1α, TLR4 and NF-κB overexpression, as well as NF-κB phosphorylation (P-NF-κB) in HK-2 cells. In the figure, LPS represents the LPS-treated group; PGF-L represents the 7.5% drug-containing serum (PGF) and LPS-treated group; PGF-H represents the 10% drug-containing serum (PGF) and LPS-treated group. DETAILED DESCRIPTION

[0064] The present invention is further described below by way of examples. It should be understood that the examples of the present invention are only used to illustrate the present invention rather than to limit the present invention. Simple improvements to the present invention based on the concept of the present invention all fall within the scope of protection claimed by the present invention.

[0065] definition

[0066] As used herein, the term "treating," ...

[0067] As used herein, the term "prevent" or "preventing" means at least reducing the likelihood of risk (or susceptibility) of acquiring a disease or condition (i.e., causing at least one clinical symptom of a disease that has not yet developed in a patient who may be exposed to or susceptible to the disease but does not yet experience or show symptoms of the disease).

[0068] The term "systemic lupus erythematosus" (SLE) or "lupus erythematosus" (LE) as used herein refers to an autoimmune disease characterized by the production of abnormal autoantibodies in the blood. These autoantibodies bind to their respective antigens to form immune complexes, which circulate and eventually deposit in tissues. This immune complex deposition can lead to chronic inflammation and tissue damage. Systemic lupus erythematosus or lupus erythematosus is a systemic autoimmune disease with the main clinical features of multi-system and multi-organ involvement throughout the body, repeated relapses and remissions, and the presence of a large number of autoantibodies in the body.

[0069] SLE or LE can manifest as a systemic disease (with cutaneous as well as other manifestations) or as a purely cutaneous disease (affecting only the skin). The cutaneous forms of SLE or LE are acute cutaneous lupus erythematosus, subacute cutaneous lupus erythematosus, and chronic cutaneous lupus erythematosus (ie, discoid lupus erythematosus).

[0070] The term "cutaneous lupus erythematosus" or "cutaneous lupus" as used herein refers to the skin manifestations of lupus erythematosus according to the Gilliam classification of lupus erythematosus skin diseases. Rothfield et al., Clinics in Dermatology 24:348-362 (2006). This system divides lupus skin diseases into specific lupus erythematosus skin diseases and nonspecific lupus erythematosus skin diseases, both of which show distinctive histological changes.

[0071] The term "chronic cutaneous lupus erythematosus" or "CCLE" or "discoid lupus erythematosus" or "DLE" as used herein, is a chronic, often disfiguring disorder that primarily affects the skin through acute localized macules and plaques that exhibit erythema, follicular plugging, scaling, telangiectasia, and atrophy. The condition is usually precipitated by sun exposure, and the early lesions are erythematous, round, scaly papules that are 5 to 10 mm in diameter and exhibit follicular plugging. DLE lesions most commonly appear on the cheeks, nose, scalp, and ears, and they can also generalize to the upper trunk, on the extensor surfaces of the limbs, and on the mucous membranes of the mouth.

[0072] Chronic cutaneous lupus erythematosus (CCLE) is usually subdivided into classic discoid lupus erythematosus (DLE), childhood discoid lupus erythematosus, generalized discoid lupus erythematosus, limited discoid lupus erythematosus, deep lupus erythematosus, lupus erythematosus panniculitis (deep lupus erythematosus), mucosal lupus erythematosus, tumescent lupus erythematosus, pernio-like lupus erythematosus, lupus erythematosus lichen planus overlap syndrome, verrucous lupus erythematosus (hypertrophic lupus erythematosus) and other rare variants. Classical DLE is the most common form of CCLE, and most of the patients with classic DLE damage will not develop the characteristics of systemic lupus erythematosus. Classical DLE presents as papules with clear boundaries of red-purple macules with epidermal squamous scales. The size of the damage increases to coin-shaped or discoid plaques with peripheral hyperpigmentation. Adhesive squamous scales expand into expanded hair follicles. The center of the damage becomes flat along with scarring, depigmentation and telangiectasia. These plaques may fuse to form large, disfiguring lesions. The hair follicles may be clogged with thick scales that, when peeled off, show keratotic projections, which are referred to as carpettack signs. Histopathological features include hyperkeratosis and follicular plugging, loss of the tissue basal epidermis, and atrophic stratum spinosum. The substratum may also exhibit edema, liquefaction, thickening of the basement membrane, increased melanin staining, and pigment incontinence. Mononuclear infiltrates of macrophages and T lymphocytes are found in the dermis, with plasma cells in chronic lesions causing mucin deposition.

[0073] The term "acute cutaneous lupus erythematosus" or "ACLE" as used herein may be either localized or generalized. Localized ACLE is characterized by erythema on the malar protuberances and bridge of the nose (butterfly-shaped erythema), while the nasolabial folds are typically spared. The ACLE rash may have fine surface scaling and is associated with edema, although particularly severe cases may produce vesiculobullous skin changes. Histopathological changes include rare dermal cell infiltrates, focal liquefaction degeneration of the basal epidermis, and erythema of the upper dermis. Epidermal necrosis can occur in the most severe form.

[0074] The term "subacute cutaneous lupus erythematosus" or "SCLE" as used herein is subdivided into two morphological variants: annular SCLE and papulosquamous SCLE. Annular SCLE is also known as lupus marginatus, centrifugal symmetrical erythema, autoimmune annular erythema, and lupus erythematosus gyratum repens. SCLE presents as erythematous spots and papules, which subsequently develop into papulosquamous or annular plaques. Although some patients show both elements simultaneously, most patients tend to mainly develop into a type of damage. SCLE is very photosensitive, and damage is most common in the extensor surfaces of the neck and upper chest, upper back, shoulder, arm and forearm, and the back of the hand. Rarely, face and scalp are involved. Histopathological features include mononuclear cell infiltration in the hyperkeratosis of the basal cell layer, degeneration, and the junction of the dermis-epidermis and the dermis.

[0075] The term "drug-induced lupus erythematosus" or "DILE" as used herein, is a different autoimmune disease that occurs as a side effect of long-term use of certain drugs. The symptoms of DILE are similar to those of SLE and may include fatigue, low-grade fever, loss of appetite, muscle pain, arthritis, oral and nasal ulcers, facial rash, unusual sensitivity to sunlight, pleurisy, pericarditis, and Raynaud's phenomenon. Ulcers and rashes are examples of skin manifestations of DILE. After discontinuation of the causative drug, these symptoms subside within days to months in patients who do not have underlying immune system dysfunction. The most common drugs that cause DILE are hydralazine, procainamide, quinidine, isoniazid, diltiazem, and minocycline. Some of these drugs (e.g., procainamide, chlorpromazine, and quinidine) lead to the production of antinuclear antibodies against histone dimers H2A-H2B. Hydralazine forms antinuclear antibodies to H1 and H3-H4 complexes. DILE typically presents months to years after starting a drug, whereas SLE flares present hours to days after starting a drug.

[0076] The spontaneous lupus model mouse MRL / lpr used in this article is not conscious of any artificial treatment, occurs under natural conditions, and is more similar to human lupus. Spontaneous mouse lupus is essentially a genetic disease.

[0077] The term "lupus nephritis" used herein refers to a disease in which systemic lupus erythematosus (SLE) is combined with immune damage of different pathological types in both kidneys, accompanied by obvious clinical manifestations of kidney damage. Its onset is related to immune abnormalities such as immune complex formation, immune cells and cytokines. In addition to the systemic manifestations of SLE, the main clinical manifestations are hematuria, proteinuria, renal insufficiency, etc.

[0078] The term "neonatal" as used herein refers to an infant from the time the fetus is delivered from the mother and the umbilical cord is tied to the time the infant is less than 28 days after birth.

[0079] The term "child" used in this article is defined in the International Convention on the Rights of the Child as any person under the age of 18.

[0080] The chemical structure of the term "branch pine 7-O-[4",6"-(S)-hexahydroxybiphenyl diol]-β-D-glucose" used herein is as follows:

[0081]

[0082] The chemical structure of the term "galactoside-7-O-[3"-O-galloyl-4",6"-(S)-hexahydroxybiphenyl diol]-β-D-glucose" used herein is as follows:

[0083]

[0084] The chemical structure of the term "chalcone dihydroxychalcone-7-O-[3"-O-galloyl-4",6"-(S)-hexahydroxybiphenyl diol]-β-D-glucose" used herein is as follows:

[0085]

[0086] Lupus patients generally have increased autoantibody concentrations in the blood, as well as increased concentrations of inflammatory cytokines such as TNF-α and IFN, an increase in the number of neutrophils, and an increase in urine protein levels, which are related to the dysregulated expression of HIF-1α, TRL-4 and NF-κB, as well as abnormal NF-κB phosphorylation. The inventors found that the herb or its extract can improve the content of immune cells in the blood, the concentration of inflammatory cytokines such as TNF-α and IFN, reduce the level of antinuclear antibodies in the serum, improve glomerular inflammatory lesions, and have a positive effect on reducing urine protein levels, thus having a significant effect on systemic lupus erythematosus or its complications.

[0087] The preparation method of the extract of the herbaceous chrysanthemum provided by the present invention obtains a high-purity extract of the herbaceous chrysanthemum. The extract of the herbaceous chrysanthemum includes flavonoids, chalcone compounds, saponins and polyphenolic acids, wherein the flavonoids and chalcone compounds are usually present in the plant in the form of glycosides. These compounds have positive effects on improving the content of immune cells in the blood, the concentration of inflammatory cytokines such as TNF-α and IFN, reducing the level of antinuclear antibodies in serum, improving glomerular inflammatory lesions, and reducing the level of urine protein, all of which have different degrees of effect, among which the effects of flavonoids and chalcone compounds are the most significant, and can not only prevent and treat systemic lupus erythematosus or its complications, for example, the therapeutic effect on lupus erythematosus is significant, but also the therapeutic effect on systemic lupus erythematosus and lupus nephritis is also obvious.

[0088] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0089] Example

[0090] Preparation Example Preparation of Herba Lycopodii Extract

[0091] The herb was naturally air-dried, and the herb was powdered with a powdering machine and passed through an 80-mesh sieve for standby use. 300g of the herb powder was taken, added to 3L of 70% ethanol aqueous solution by volume, mixed, and loaded into a round-bottomed flask, connected to a condenser, heated using a common electric heating jacket on the market, condensed and refluxed for 2 hours, filtered, and the first filtrate and the first filter cake were collected. The first filter cake was added with 3L of 70% ethanol aqueous solution by volume and condensed and refluxed again for 2h, and the second filtrate and the second filter cake were collected. The first filtrate and the second filtrate were combined, concentrated to one-tenth of the volume using a rotary evaporator, and an equal volume of petroleum ether was added for extraction. After removing the fat-soluble part, ethyl acetate was added to the water layer for extraction until the ethyl acetate layer of the last extraction was colorless and transparent. The ethyl acetate layer solution after extraction was spin-dried and weighed to obtain 7.93g of the herb ethyl acetate extract.

[0092] 2 g of the ethyl acetate extract of the herb was dissolved in 5 mL of a 30% ethanol aqueous solution by volume, and added to a macroporous resin (HPD 500) chromatographic column (3×150 mm) equilibrated with a 30% ethanol aqueous solution by volume, and eluted with 1 column volume of 30% ethanol aqueous solution, 80% ethanol aqueous solution and 95% ethanol aqueous solution by volume, respectively, and the 30% ethanol aqueous solution, 80% ethanol aqueous solution and 95% ethanol aqueous solution were collected. The three effluents were analyzed by HPLC on a C18 column ( Figure 1 ) It was concluded that the macrocyclic polyphenol pyruvic acid glycosides were concentrated in the 80% ethanol outflow, which was concentrated to dryness under reduced pressure to obtain the extract of P. chinensis (hereinafter referred to as PGF). The extraction efficiency of each step is shown in Table 1.

[0093] Table 1: Extraction rate of each part of macroporous resin

[0094] Part Name 30% ethanol flow-through 80% ethanol flow-through 95% ethanol flow-through Extraction rate 23.69±3.38％ 78.35±5.72％ 1.46±0.55％

[0095] Identified by UPLC-TOF-MS, Figure 1As shown, the main component in the effluent of 80% ethanol aqueous solution is compound S3, with a molecular weight of 874. The compound was identified as pyralidin dihydrochalcone-7-O-[3”-O-galloyl-4”,6”-(S)-hexahydroxybiphenyl diacyl]-β-D-glucose by comparison with the molecular weight and secondary spectrum of related compounds in the literature. Quantitative analysis of the peak area with the standard product showed that the content of the compound in PGF was 53.37±4.63%. In addition, the compounds pyralidin-7-O-[4”,6”-(S)-hexahydroxybiphenyl diacyl]-β-D-glucose (S1) and pyralidin-7-O-[3”-O-galloyl-4”,6”-(S)-hexahydroxybiphenyl diacyl]-β-D-glucose (S2) were also identified, with contents of 1.37±0.63% and 6.64±3.14%, respectively: see Table 2 for details.

[0096] Table 2 Chemical components attributed to PGF in UPLC-TOF-MS negative ion mode

[0097]

[0098]

[0099] Example 1 Oral administration of the extract of Herba Chrysanthemi sutchuenensis alleviates the autoimmune response in lupus model mice (MRL / lpr)

[0100] Eight-week-old spontaneous lupus model mice MRL / lpr were used as the research subjects (M group), the western medicine prednisone treatment group was used as the positive control (PAT treatment group) and the MRL / MPJ mice were used as the blank control group (CK group), and the preventive and therapeutic effects of oral gavage of Herba Glehniae extract (PGF treatment group) on MRL / lpr were evaluated.

[0101] The PAT group was treated with 4.55 mg / kg prednisone acetate tablets per day, the PGF group was intragastrically administered with 150 mg / kg 1% sodium carboxymethylcellulose solution containing PGF per mouse per day, and the mice in the M and CK groups were intragastrically administered with the same volume of 1% sodium carboxymethylcellulose solution.

[0102] 1.1 Methods:

[0103] (1) Urine protein detection: Urine samples were collected at the 4th and 6th week of gavage and stored in a -80°C refrigerator. Urine protein detection was performed according to the instructions of the Bio-Tech Coomassie Brilliant Blue Staining Solution Kit. First, protein standards of different concentrations were prepared, and the protein standards and test samples were added to a 96-well plate. G250 staining solution was then added to each well. Finally, the absorbance at 595 nm was measured using an ELISA reader, and the urine protein content was calculated based on the standard curve.

[0104] (2) Cytokine and antinuclear antibody detection: After the last administration, blood was collected from the eyeball. The blood sample was allowed to stand for 1 hour and then centrifuged at 3000 rpm for 10 minutes. The upper serum was used to detect indicators such as inflammatory cytokines and autoantibodies. Mouse serum antinuclear antibodies were detected according to the instructions of the ELISA kit. The specific steps are as follows:

[0105] First, prepare standard solutions of different concentrations (1.5, 1, 0.75, 0.5, 0.25, 0.125, 0 mg / mL), add 50 μL to the standard wells, 10 μL to each sample well, and make up the rest with 40 μL of sample diluent; after sealing the plate with a sealing film, put it in a 37°C incubator for incubation for 30 minutes, wash with detergent, add enzyme labeling reagent and color developer to each well, mix well and incubate again for 10 minutes as above, finally add stop solution to each well, mix well and measure the absorbance at 450 nm with an enzyme reader, and finally calculate the content of antinuclear antibodies in serum samples according to the standard curve.

[0106] (3) Observation of pathological changes in mouse kidneys by H&E staining: After the embedded paraffin sections of the kidneys were dewaxed, hematoxylin was added to stain the cell nuclei, differentiated with 1% hydrochloric acid alcohol, rinsed with running water and then blued with 0.6% ammonia water. After rinsing again, the sections were placed in 85% and 95% alcohol for dehydration, and then placed in eosin staining solution for staining. After rinsing with distilled water, they were routinely dehydrated and transparentized, and finally sealed with neutral gum.

[0107] 1.2 Results and Analysis

[0108] Compared with lupus model mice (M group), the thymus and spleen indexes of mice in the PGF treatment group were reduced, but the effect was not as significant as that in the PAT treatment group (see Figure 2 A and Figure 2 B). However, the concentration of neutrophils in the blood of mice treated with PGF decreased more significantly than that of mice treated with PAT (see Figure 3 ); the concentration of anti-dsDNA antibodies in the serum of mice in the PGF treatment group was significantly lower than that in the M group (P<0.001) (see Figure 4 A), while PAT treatment can reduce the concentration of dsDNA antibodies but without significant difference. And the concentration of anti-snRNP / Sm antibodies was significantly lower than that of group M (P<0.05), while the effect of PAT treatment was more significant (P<0.01) (see Figure 4 B). The concentration of cytokine IFN in the serum of mice in the PGF treatment group was significantly lower than that in the M group (P<0.05) (see Figure 5A), and the significance of PAT treatment was more significant (P<0.01). PGF treatment can reduce the concentration of cytokine TNF-α in serum (compared with M group), but there is no statistical difference. PAT treatment can not only reduce the concentration of TNF-α, but also have statistical significance (see Figure 5 B). The urine protein of mice in the PGF treatment group was also improved (see Figure 6 A and Figure 6 B), and the activity of PGF is more stable. Pathological staining observations showed that compared with the lupus model group (M group), the inflammatory cell infiltration in the glomeruli and surrounding tissues, the proliferation of the glomerular mesangium and basement membrane, and the degree of tissue fibrosis in the kidneys of the PGF-treated mice were improved, and the glomerular contours were better restored than those of the PAT-treated group (see Figure 7 ). This shows that the effect of the extract of PGF (PGF) is similar to that of the Western medicine prednisone (PAT), which is manifested in that; compared with PAT, although the improvement of the activity of the lymphatic and kidney indexes of MRL / lpr mice by PGF treatment was not obvious, the degree of inflammatory infiltration of glomeruli, hyperplasia of the endomesangium and base, and tissue fibrosis were all improved, and the urine protein of mice was also improved, indicating that PGF can improve the systemic lupus erythematosus and lupus nephritis caused by MRL / lpr mice. In addition, the activity of PGF treatment in improving the neutrophil content in the blood and the dsDNA concentration in the serum of MRL / lpr mice is better than that of PAT. In addition, like PAT, PGF treatment can also reduce the concentrations of snRNP Sm, IFN and TNF-α in the serum. The above research results show that PGF has biological activity in preventing, treating or treating systemic lupus erythematosus.

[0109] Example 2 Mechanism of action of serum containing extract of Herba Chrysanthemi serratae protecting spleen and lymph node primary cells of lupus model mice (MRL / lpr)

[0110] Inflammation-induced cellular oxidative stress is one of the important mechanisms of autoimmune diseases. Therefore, this example uses primary cells of spontaneous lupus model mice as experimental materials to explore the mechanism of action of serum containing Herba Aspergillum extract in protecting systemic lupus erythematosus.

[0111] 2.1 Methods:

[0112] (1) Preparation of drug-containing serum

[0113] Ten SD rats were randomly divided into a blank group and a PGF group, with 5 rats in each group. The blank group was given 1% sodium carboxymethyl cellulose, and the PGF group was given 1% sodium carboxymethyl cellulose solution containing PGF. The dosage concentration was calculated based on the body weight of the rats, and the drug was administered twice a day for 3 consecutive days. One hour after the last administration, the rats were anesthetized with carbon dioxide to draw blood from the heart. After standing at room temperature for 4 hours, the blood was centrifuged at 3000 rpm for 15 minutes, the serum was drawn, and the blood was inactivated in a 56°C water bath for 30 minutes. Finally, the blood was sterilized with a 0.22μm microporous filter membrane on a clean bench, and the blood was placed in a -80°C ultra-low temperature refrigerator for storage.

[0114] (2) Extraction of primary spleen cells from lupus model mice (MRL / lpr)

[0115] After the mouse is anesthetized, follow the principle of aseptic operation and remove the spleen. After removing the excess tissue and capsule, wash it repeatedly with PBS solution. Place the cell filter on the culture dish, place the spleen or lymph node in the sieve and grind it gently, and add serum-free 1640 culture medium to rinse it. Finally, aspirate the cell suspension in the culture dish into a centrifuge tube, centrifuge at 1000rpm for 10min to collect the cells, discard the supernatant, add appropriate red blood cell lysis solution, centrifuge again at 1000rpm for 5min, discard the supernatant, resuspend the cells with 1640 culture medium containing serum, count the cell suspension and evenly spread it in a 6-well plate. After 2 to 4h, give 7.5% and 10% drug-containing serum (PGF) for 6h respectively.

[0116] (3) RNA extraction and real-time fluorescence quantitative PCR

[0117] Use 1mL Trizol to blow the cells in the 6-well plate thoroughly. After the cells are fully lysed, add 200μL chloroform, mix thoroughly, transfer to a 1.5mL EP tube and let stand for 5min, centrifuge at 12000rpm at 4℃ for 10min. Transfer the supernatant to a new 1.5mL EP tube, then add an equal volume of isopropanol to precipitate RNA, mix thoroughly, let stand at -20℃ for 30min, centrifuge at 12000rpm at 4℃ for 10min to precipitate total RNA. Add 1mL pre-cooled 75% ethanol to wash the precipitate, desalt it, and evaporate the ethanol. Finally, use 50μL DEPC water to re-dissolve the RNA. Determine the concentration of total RNA using the Onedrop instrument. After reverse transcription of mRNA into cDNA, perform real-time fluorescence quantitative PCR amplification.

[0118] 2.3 Results and Analysis:

[0119] SLE is an autoimmune disease, and the abnormality of the immune system directly leads to multi-organ involvement. The spleen, as the main site of specific immunity in the body, is not only closely related to the onset of SLE, but its oxidative stress response and released inflammatory factors are also one of the important signs for the diagnosis of SLE. The spleen primary cells of MRL / lpr mice were treated with PGF-containing serum, and it was found that compared with the control serum (CK group), the medicated serum could significantly inhibit the transcriptional expression of NF-κB, HIF-1α, and TLR4 (P<0.05) (see Figure 8 A. Figure 8 B and Figure 8 C).

[0120] Existing clinical studies have shown that NF-κB (nuclear factor κB) is involved in cellular inflammatory response and immune response. Abnormal activation is closely related to the occurrence of systemic lupus erythematosus and is regulated by HIF-1α (hypoxia-inducible factor-1) and TLR4 (toll-like receptor 4).

[0121] In this study, compared with the control serum, low concentrations of PGF-containing serum treatment can significantly improve the transcription of the above three proteins, while high concentrations of PGF serum, although it can also inhibit HIF-1α and TLR4, have no inhibitory effect on the expression of NF-κB. This study shows that PGF can inhibit the abnormal expression of intracellular NF-κB by improving the expression of HIF-1α and TLR4 in the spleen primary cells of MRL / lpr mice.

[0122] Example 3 Mechanism of action of the extract of Herba Aspergillume var. erythematosus in improving systemic lupus erythematosus by alleviating cellular oxidative stress

[0123] Inflammation-induced cellular oxidative stress is one of the important mechanisms of autoimmune diseases. Therefore, this example discusses the mechanism of action of Herba Chrysanthemi Indici in relieving cellular oxidative stress.

[0124] 3.1 Methods

[0125] (1) Dual luciferase reporter gene assay

[0126] Dual luciferase assay reference ReporterAssaySystem kit detects changes in transcriptional activity of related transcription factors. Specific steps: press 1×10 5293T cells were evenly spread in a 48-well plate per well and cultured in a 37°C, 5% CO2 incubator. After the cells have grown to about 80% of the well area, the Renilla internal reference vector and the ARE vector to be detected were transfected. The next day, 20 μL of H2O2 was added to the transfected cell culture medium to a final concentration of 600 μmol / L. At the same time, 20 μL of PGF extract was added to the final concentrations of 10, 20, and 40 μg / mL, respectively, and the cells were continued to be cultured in a constant temperature incubator. After 24 hours, the well plate was removed, the cell culture medium was discarded, 65 μL of 3×Passive Lysis Buffer was added to each well, and the cells were quickly lysed on a horizontal shaker for 15 minutes. The cell lysate in each well was transferred to a 1.5 mL centrifuge tube, and after thorough mixing, it was centrifuged at 14000 rpm for 30 seconds. Take 20 μL of supernatant and transfer it to a 96-well plate. Add 25 μL of Luciferase Assay Buffer Ⅱ. After mixing, immediately place the 96-well plate into the microplate reader to measure the firefly luciferase activity value F (Firely Luciferase). After the test, add 25 μL of 1×Stop& Buffer, and immediately measure the Renilla luciferase activity value R after mixing, and finally calculate their ratio.

[0127] (2) Experiment on the protection of PGF-containing serum against oxidative stress in cells

[0128] HK-2 cells, a normal kidney proximal tubule cell line, were inoculated into 6-well plates and cultured in a DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, in a 5% CO2, 37°C incubator. After the cells grew to about 80% of the well area, equal volumes of PGF-containing serum were added to the wells to make the serum concentrations reach 7.5% and 10%, respectively. After continued culture in a constant temperature incubator for 24 hours, lipopolysaccharide (LPS) solution (final concentration 1 μg / mL) was added for stimulation for 2 hours, and the cells were lysed to extract total protein to detect the expression of HIF-1α, NF-κB and TLR4, and the phosphorylation degree of NF-κB.

[0129] (3) Protein extraction and western blotting

[0130] Use 200 μL cell lysis buffer to lyse the cells in 6 empty plates, repeatedly blow until the cells are completely lysed, collect them into 1.5 mL centrifuge tubes, centrifuge at 12000 rpm for 15 minutes at low temperature, and absorb the supernatant. Use the BCA protein concentration kit to measure the protein concentration, then add 2× loading buffer to mix thoroughly, heat it in a metal bath to fully denature the protein sample, and then centrifuge it at 12000 rpm for 5 minutes. The proteins in the sample were separated by 10% SDS-PAGE gel electrophoresis and transferred to a cellulose acetate membrane. After the transfer, it was first blocked with 5% skim milk powder at room temperature, and then incubated with a specific antibody prepared with 3% skim milk powder at 4°C overnight. After washing three times with 1×TBST solution, it was incubated with a detection antibody prepared with 5% skim milk powder at room temperature in the dark for 2 hours. Finally, after washing three times with 1×TBST solution, add the color developing solution and scan the membrane with the Odyssey fluorescence imaging system, and the image was analyzed using Odessey V3.0 software and Image J software.

[0131] 3.2 Results

[0132] The results of Example 2 showed that PGF may improve spontaneous lupus lesions by inhibiting cellular oxidative stress response. The dual fluorescence reporter gene experiment proved that high concentration of PGF activated the transcription of cellular antioxidant response element (ARE). Compared with the blank control group, #P<0.05; compared with the H2O2 treatment group, *P<0.05 (see Fig. 9 ). In addition, under inflammatory conditions (LPS treatment), PGF-containing serum inhibited the overexpression of HIF-1α, NF-κB and TLR4, and the phosphorylation of NF-κB (P-NF-κB) (see Fig.10 ).

[0133] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0134] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0135] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. Use of the extract of Herba Lycopodii in the preparation of a medicament for preventing or treating systemic lupus erythematosus or its complications, The extract is a 70-75% v / v ethanol solution extract. The extract includes gallic acid, ellagic acid, thunbergin-7-O-glucose, thunbergin-7-O-β-D-glucose, 5-methoxy-thunbergin-7-O-β-D-glucose, dihydrochalcone-7-O-glucose, quercetin-3-O-β-D-rhamnose, thunbergin-7-O-[4″,6″-(S)-hexahydroxybiphenyl diol]-β-D-glucose, thunbergin-7-O-[3″-O-galloyl-4″,6″-(S)-hexahydroxybiphenyl diol]-β-D-glucose, thunbergin-dihydroxychalcone-7-O-[3″-O-galloyl-4″,6″-(S)-hexahydroxybiphenyl diol]-β-D-glucose (Thonningianin A) and pyrocatechol dihydroxychalcone-7-O-[4″,6″-(S)-hexahydroxybiphenyl diol]-β-D-glucose, The content of pine pinoside-7-O-[4″,6″-(S)-hexahydroxybiphenyl diol]-β-D-glucose, pine pinoside-7-O-[3″-O-galloyl-4″,6″-(S)-hexahydroxybiphenyl diol]-β-D-glucose and pine pinoside dihydroxychalcone-7-O-[3″-O-galloyl-4″,6″-(S)-hexahydroxybiphenyl diol]-β-D-glucose in the extract is 30-50%; The complications of systemic lupus erythematosus are selected from lupus cutaneous lesions, lupus nephritis, lupus heart disease, lupus pneumonia, lupus peritonitis, lupus vasculitis, lupus hepatitis, lupus eye lesions and lupus associated arthritis.

2. The use according to claim 1, characterized in that: The preparation method of the extract comprises the following steps: Extract the yellow grass, filter it, and collect the filtrate; Concentrating the filtrate to obtain a concentrate; extracting the concentrated solution to obtain an extract; Purifying the extract to obtain the Herba Lycopodii extract, The solvent used for the leaching is selected from ethanol solution.

3. The use according to claim 2, characterized in that: The macroporous resin used in the purification is selected from HPD-500 macroporous resin and D-101 macroporous resin.

4. The use according to claim 1, characterized in that: The medicament includes a pharmaceutically acceptable carrier.

5. The use according to claim 4, characterized in that: The carrier includes at least one of an excipient, a diluent, an absorbent, a filler, an absorption promoter, an adsorption carrier, a stabilizer, a dispersant and a preservative.

6. The use according to claim 1, characterized in that: The dosage form of the drug is selected from the group consisting of injection solution, injection suspension, injection granule, injection gel, injection solid form, tablet, capsule, caplet, pill, granule, microsphere, powder, syrup, decoction and paint.

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