Patent Application
20240366644
The present invention relates to the technical field of medicine, relates to an anti-inflammatory drug or health food/medicinal and edible food/functional food/dietary supplement, and more particularly, relates to an application of ginsenoside Rd in preparation of an anti-inflammatory drug or health food/medicinal and edible food/functional food/dietary supplement.
Inflammation is a protective response of the body against external infections or noxious stimulation, and is also a common pathological process of many different types of diseases. Long-term persistent and excessive inflammatory response or inflammatory storm will cause serious damages to the body itself, and even organ failure, so drug intervention is required to block the continuation and development of inflammations. At present, there are two main types of anti-inflammatory drugs commonly used in clinical practice: non-steroidal anti-inflammatory drugs and steroidal anti-inflammatory drugs (glucocorticoids). These two types of anti-inflammatory drugs have good clinical anti-inflammatory effects, but will produce a series of adverse reactions and tolerances after long-term massive use, e.g., the former causes gastric mucosal injuries, liver and kidney damages and cardiac toxic and side effects, and the latter causes water and sodium retention. In order to solve the tolerances and adverse reactions of drugs, the search for anti-inflammatory drugs with novel structures and unique mechanisms has always been a hot topic in the field of research and development of novel anti-inflammatory drugs. Ginsenoside Rd belongs to a diol type of ginsenoside, which is mainly found in Radix Ginseng, Radix Notoginseng, and Radix Panacis Quinquefolii. It has a wide range of biological activities, obvious pharmacological effects on heart and cerebral vessels, nervous systems, immune systems, etc., as well as the effects better than other monomeric saponins in analgesia and neuroprotection, but its anti-inflammatory and protective effects and mechanisms against acute inflammation (ALI) have not been reported in literatures.
A primary objective of the present invention is to provide anti-inflammatory properties and efficacy of ginsenoside Rd in view of the technical problems existing in the above-mentioned existing anti-inflammatory drugs or health food/medicinal and edible food/functional food/dietary supplement, and to provide new medicinal use of the ginsenoside Rd, that is, a novel application of the ginsenoside Rd in an anti-inflammatory drug or health food/medicinal and edible food/functional food/dietary supplement.
To fulfill said objective, in one aspect, the present invention provides an application of ginsenoside Rd in preparation of an anti-inflammatory drug or health food/medicinal and edible food/functional food/dietary supplement.
In the process of screening natural active ingredients with anti-inflammatory effects, the inventors have found that ginsenoside Rd in the chemical composition of Radix Ginseng has a significant anti-inflammatory effect.
The drug is used for the treatment of one or more of acute inflammations and chronic inflammations.
The drug or health food/medicinal and edible food/functional food/dietary supplement is composed of ginsenoside Rd and a pharmaceutically acceptable carrier.
The content of the ginsenoside Rd is 1% to 99.9%; preferably, 30 to 99.9%; further preferably, 60 to 99.9%; further preferably, 80 to 99.9%; and preferably, 98 to 99.9%. In particular, a ratio of a weight of the ginsenoside Rd to a total weight of the preparation is (0.01-100): 100, preferably (0.1-100): 100, and further preferably (10-100): 100.
In particular, the pharmaceutically acceptable carrier is often recognized by health professionals for this objective and as an inactive ingredient in pharmaceuticals. The compilation related to pharmaceutically acceptable carriers can be found in handbooks such as “Handbook of Pharmaceutical Excipients” (2nd Edition, edited by A. Wade and P. J. Weller; published by American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994).
In particular, the carrier includes an excipient, such as starch or water; a lubricant, such as magnesium stearate; a disintegrant, such as microcrystalline cellulose; filler, such as lactose; a binder, such as pregelatinized starch or dextrin; a sweetener; an antioxidant; an ointment base; a transdermal promoter; a preservative; a flavoring agent; spices, etc.; and the drug is an oral preparation, a topical preparation or an injection; the oral preparation is tablets, capsules, granules, pills, a solution, a suspension, an emulsion, an oral liquid, drops, or a syrup; the topical preparation is emplastrum, an ointment or a suppository; and the injection is an injection solution, a powder injection or a lyophilized powder injection.
The health food/medicine and edible food/functional food/dietary supplement is an oral preparation; and the oral preparation is tablets, a powder, granules, medicinal tea, pills, a paste, hard capsules, soft capsules, an oral liquid, medicated wine, a syrup, or drops. In another aspect, the present invention provides an anti-inflammatory drug or health food/medicinal and edible food/functional food/dietary supplement, wherein the drug or health food/medicinal and edible food/functional food/dietary supplement contains ginsenoside Rd.
The content of the ginsenoside Rd is 1% to 99.9%; preferably, 30 to 99.9%; further preferably, 60 to 99.9%; further preferably, 80 to 99.9%; and preferably, 98 to 99.9%. In particular, a ratio of a weight of the ginsenoside Rd to a total weight of the preparation is (0.01-100): 100, preferably (0.1-100): 100, and further preferably (10-100): 100. In particular, the drug or health food/medicinal and edible food/functional food/dietary supplement further includes one or more of a Radix Scrophulariae extract, a Rehmannia glutinosa Libosch extract, a Radix Ophiopogonis extract, a Radix Scutellariae extract, a Radix Ranunculi Ternati extract, a Fritillaria extract, a Menthae Haplocalycis Herba extract, a Semen Sesami Nigrum extract, a Zingiber officinale Roscoe extract, a grape seed extract, a pomegranate seed extract, a Rhizoma Cyperi extract, a Pericarpium Citri Reticulatae Viride extract, plant essential oil, arbutin, vitamin C and derivatives thereof, or vitamin E and derivatives thereof.
The drug may be prepared into various dosage forms by methods well-known in the art, such as tablets, capsules, pills, powder, granules, a syrup, emplastrum, an ointment, or a suppository.
Compared with the prior art, the present invention has the following obvious advantages.
The beneficial effects of the formula of the present invention are further described below by specific embodiments, and these examples include pharmacodynamic tests for capsules, tablets and soft capsules of ginsenoside Rd of the present invention. These examples are only exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention may be modified or replaced without deviating from the formula idea and the use scope of the present invention, but these modifications and substitutions fall within the protection scope of the present invention.
Ginsenoside Rd tablets were prepared in the following proportions:
Ginsenoside Rd and starch were mixed well and prepared into granules, and the granules were added with talcum powder and magnesium stearate, mixed well and pressed into 10,000 tablets.
1. Raw Materials were Prepared According to the Following Proportions:
Ginsenoside Rd and starch were mixed well and packed into 10,000 capsules.
1. Raw Materials were Prepared According to the Following Proportions:
Ginsenoside Rd and starch were mixed well and prepared into granules, which were then loaded into 10,000 bags.
1. Raw Materials were Prepared According to the Following Proportions:
Ginsenoside Rd, Rehmannia glutinosa Libosch extract, Radix Ophiopogonis extract and starch were mixed well and prepared into granules, and the granules were added with talcum powder and magnesium stearate, mixed well and pressed into 10,000 tablets.
1. Raw Materials were Prepared According to the Following Proportions:
Ginsenoside Rd, Fritillaria extract, Vitamin C and starch were mixed well and packed into 10,000 capsules.
Ginsenoside Rd, Radix Scutellariae extract, Vitamin C and powdered sucrose were mixed well and prepared into granules, which were then loaded into 10,000 bags.
Deionized water was finally added to the whole formula to 100%. Ginsenoside Rd, Rhizoma Cyperi extract and Pericarpium Citri Reticulatae Viride extract were dissolved with ethanol, and added with glucose syrup to 100% to obtain the finished product.
Ginsenoside Rd, Radix Scutellariae extract, Vitamin C and powdered sucrose were mixed well and prepared into granules, which were then loaded into 10,000 bags.
Ginsenoside Rd, xylitol, citric acid, aspartame and Menthae Haplocalycis Herba essence were mixed well and prepared into granules, and the granules were added with magnesium stearate, mixed well and pressed into 1000 tablets.
The aqueous phase was added to the oil phase, stirred, and added with ginsenoside Rd to obtain the finished product.
Male and female Kunming mice (SPF grade), weighing 18-22 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd., with quality certificate number: SCXK-(Liaoning) 2020-0001.
Twenty KM mice, weighing 18-22 g, were randomly grouped, with ten mice in each group. The groups include a model group, a positive control group (Indomethacin, 10 mg/kg), a ginsenoside Rd low-dose group (5 mg/kg), and a ginsenoside Rd high-dose group (10 mg/kg). After 30 min of gavage administration of mice (the model group was administrated with the equal volume of 0.5% sodium carboxymethylcellulose by gavage), 0.03 ml of xylene was evenly applied to both sides of the right ear of each mouse in each group to cause inflammations on both sides, and the left ear of the mouse was not applied as a control. After 4 h, the mouse was killed by dislocation of cervical vertebra, the two ears of the mouse were cut off along the auricle, round ear slices of the same size (a diameter of 6 mm) were taken at the same position of the left and right ears and weighed respectively, a mass difference of the two ear slices was used as an ear swelling degree, and a swelling inhibition rate was calculated.
Compared with the model group, the ginsenoside Rd drug group could significantly inhibit ear swelling of mice caused by xylene, and had a significant anti-inflammatory effect. The ear swelling inhibition rate at a dose of 10 mg/kg was close to that of Indomethacin, achieving a more obvious anti-inflammatory effect.
ICR mice, weighing 20-30 g, laboratory animal supplier: Hunan SJA Laboratory Animal Co., Ltd., laboratory animal production license: SCXK (Xiang) 2019-0004, and feed supplier: Jiangsu Medicience Biopharmaceutical Co., Ltd.
Influenza virus (BSL-3 Laboratory, Guangzhou Customs Technology Center (Laboratory of Highly Pathogenic Microorganisms, State Key Laboratory of Respiratory Diseases). Ginsenoside Rd (content ≥95%), produced by Dalian Fusheng Natural Medicine Development Co., Ltd., detected by HPLC with Ginsenoside Rd provided by China Institute for Food and Drug Control as a reference substance, and having a content of 96.2%.
A lump of absorbent cotton was put into a beaker of 200-300 mL, and then poured with an appropriate amount of ether (till the absorbent cotton was wetted). The beaker filled with the absorbent cotton was inverted, and added with the mice for anesthesia. When the mice were extremely excited and then obviously weak, that is, the anesthesia depth was moderate, the mice were taken out. The ether-anesthetized mice were placed on their backs, with the mouse head facing upward, and then were subjected to intranasal infection. A diluted influenza virus was added to nostrils at 0.03 mL/nostril. A blank group was administrated with normal saline, instead of a virus suspension.
The mice were randomly divided into four groups, that is, drug groups (10 mg/kg, 5 mg/kg), a model group, and a blank group were administrated by routine gavage on the day before infection, once a day; and the blank group and the model group were perfused with the equal volume of normal saline.
On the Day 1, Day 3, Day 5 and Day 7 after influenza virus infection, venous blood was collected from the plexus ophthalmicus, and blood from mice in each group was preserved, subjected to serum separation, and stored in a refrigerator for later use.
According to the instructions of a test kit, various detection and analysis were carried out, and TNF-α and IL-6 concentrations were calculated based on the standard detection and standard curve plotting.
On the Day 1, Day 3, Day 5 and Day 7 after influenza virus infection, after the mice were killed by bleeding from the eyeballs, the whole lungs were picked, homogenized in a homogenizer according to a ratio of lung weight (g):normal saline (mL)=1:9, and centrifuged; and a supernatant was stored in the refrigerator for later use. According to the instructions of a test kit, various detection and analysis were carried out, and TNF-α and IL-6 concentrations were calculated based on the standard detection and standard curve plotting.
The results showed that inflammatory factors TNF-α and IL-6 increased to varying degrees after influenza virus infection in mice, and the ginsenoside Rd could down-regulate the related inflammatory factors, and had an anti-inflammatory mechanism that may be related to the regulation of inflammatory factor secretion.
hACE2 mice, aged 6-7 weeks and weighing 20-40 g, laboratory animal supplier: Jiangsu Gempharmatech Co., Ltd., laboratory animal production license: SCXK (Jiangsu) 2018-0008, and feed supplier: Jiangsu Medicience Biopharmaceutical Co., Ltd.
COVID-19 Delta virus (BSL-3 Laboratory, Guangzhou Customs Technology Center (Laboratory of Highly Pathogenic Microorganisms, State Key Laboratory of Respiratory Diseases).
Ginsenoside Rd (content ≥95%), produced by Dalian Fusheng Natural Medicine Development Co., Ltd., detected by HPLC with Ginsenoside Rd provided by China Institute for Food and Drug Control as a reference substance, and having a content of 96.2%.
Dexamethasone, purchased from Shanghai Chemical Reagent Company.
1.2.1 hACE2 transgenic C57BL/6 mice were divided into a normal group, a COVID-19 Delta virus infection group, drug groups (a 10 mg/kg low-dose group, and a 20 mg/kg high-dose group), and a positive control group (Dexamethasone, 5 mg/kg), with eight mice in each group. Except for the mice in the normal group who were administrated with PBS by nasal drops, the mice in the other groups were infected with COVID-19 Delta virus in 105 PFU by nasal drops. 2 h after infection, the mice in each drug group were administrated with the drug by gavage for five consecutive days, once a day. Dead or dying animals were dissected and lungs were picked after euthanasia; and surviving animals were dissected on the Day 5 after infection, and lungs were picked.
1.2.2 In the infection group, four animals in each group were dissected for lung tissues for pathological study. Four animals were dissected, and lung tissues were picked. Total RNA was extracted from a homogenate supernatant of the lung tissues by a Trizol method, and inflammatory factor interleukin 16 (IL-1β) and monocyte chemoattractant protein 1 (MCP-1) were detected by RT-qPCR.
1.2.3 The specific operation steps of histopathological study of lung tissues were as follows:
Based on the comparison of the above lesions in various groups, the pathological changes of the surviving animals in various groups were milder than those of the dead animals; lesion degrees of the drug low-dose group and the drug high-dose group were milder than those of a virus group, and 10 mg/kg and 20 mg/kg drugs had good improvement effects on lung lesions caused by COVID-19 Delta virus; and the drug administrated to the high-dose group was better than Dexamethasone in the pathological improvement of pulmonary hemorrhage and interstitial pneumonia.
1.1.1 Experimental animals:
Balb/c mice, male, weighing 20-22 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd., with quality certificate number: SCXK-(Liaoning) 2020-0001.
Balb/c male mice, weighing 20-22 g, were randomly divided into five groups according to body weights, with eight mice in each group: a blank control group (propylene glycol-0.5% carboxymethylcellulose sodium), a model group (LPS, 15 mg/kg), a ginsenoside Rd low-dose group (10 mg/kg), a ginsenoside Rd high-dose group (20 mg/kg), and a Dexamethasone group (5 mg/kg). Mice in each group were intraperitoneally injected; after 1 h of administration, except for the blank control group, the mice in each group were intratracheally instilled with 20 μg of LPS for modeling; and the blank control group was intratracheally instilled with an equal volume of PBS.
After 1 h of administration, a mouse ALI model was established; each mouse was intraperitoneally injected with sodium pentobarbital at 30 mg/kg, anesthetized and fixed on an operating table dorsally; skin preparation was carried out on the neck; the skin was incised to expose the trachea; 20 μg of LPS was instilled into the trachea using a microglass syringe; and then the mouse was gently rotated head up on the operating table, such that the instilled liquid was evenly distributed in the mouse lungs, and the skin incision was sutured.
1.2.3 Determination of TNF-α, IL-1β, IL-6 and IL-18 content in lung tissues and BLAF
After 24 h of modeling, anesthetized mice were intraperitoneally injected with sodium pentobarbital at 30 mg/kg; lung tissues of the mice were taken and rinsed repeatedly in pre-cooled normal saline; the lung tissues were blood-removed, wiped with filter paper, weighed, and added with normal saline according to a ratio of 1:9 (W:V=1 g:9 ml); a tissue homogenate was prepared with a homogenization machine under an ice water bath; and a supernatant of mouse lung tissue homogenate was taken.
The mouse was anesthetized, and the trachea was separated; the whole lung was lavaged with 0.5 ml of pre-cooled normal saline, stayed for 1 min, and slowly pumped back with a syringe, and this process was repeated three times; and BLAF was collected, and centrifuged for 15 min at 4° C. and 1600 rpm/min.
In strict accordance with the operating instructions of an enzyme-linked immunosorbent assay kit (ELISA), the contents of TNF-α, IL-1β, IL-6 and IL-18 in the supernatant of mouse lung tissue homogenate and the BLAF supernatant were detected.
Compared with the blank control group, the contents of TNF-α, IL-1β, IL-6 and IL-18 in lung tissues and alveolar lavage fluid of each mouse in the model group were significantly increased (P<0.01); and compared with the model group, the ginsenoside Rd drug group and the Dexamethasone group could significantly reduce the contents of TNF-α, IL-1β, IL-6 and IL-18 in the lung tissues and BLAF of the mice, and the effect of the ginsenoside Rd high-dose group was better than that of Dexamethasone (P<0.01).
SD rats, male, weighing 200±20 g, laboratory animal supplier: Hunan SJA Laboratory Animal Co., Ltd., laboratory animal production license: SCXK (Xiang) 2019-0004, and feed supplier: Jiangsu Medicience Biopharmaceutical Co., Ltd.
Ginsenoside Rd (content ≥95%), produced by Dalian Fusheng Natural Medicine Development Co., Ltd., detected by HPLC with Ginsenoside Rd provided by China Institute for Food and Drug Control as a reference substance, and having a content of 96.2%.
Simvastatin tablets, produced by Zhejiang Jingxin Pharmaceutical Co., Ltd.
Dexamethasone, purchased from Shanghai Chemical Reagent Company.
Lipopolysaccharide (LPS, lot number: 08168), purchased from Sigma Corp.
TNF-α enzyme-linked immunosorbent assay kit, Nanjing Jiancheng Biotechnology Co., Ltd.
IL-8 ELISA assay kit, Nanjing Jiancheng Biotechnology Co., Ltd.
After adaptive feeding (20-25° C., a humidity of 50-60%) for 1 week, the rats were randomly divided into a blank group, a model group, a positive control group, a ginsenoside Rd high-dose group, and a ginsenoside Rd low-dose group, with ten rats in each group.
Blank group: standard feeding, no intervention.
Model group: on the Day 1 and Day 14, each rat was intraperitoneally injected with 10% chloral hydrate (300 mg/kg) for anesthesia; the head was fixed in a low position; the glottis was exposed; lipopolysaccharide was quickly injected into the trachea; and the rat was kept upright, and swung left and right for 10-20 s, such that the lipopolysaccharide was evenly distributed in the two lungs. On the Days 2-13, and 15-28, the lungs were smoked in a closed organic glass box for 30 min every day, with 15 cigarettes per day.
Positive control group: the animal was anesthetized, and intratracheally injected with lipopolysaccharide, wherein smoking conditions and time were the same as those of the model group; and after tracheal injection of lipopolysaccharide for two weeks, the animal was administrated with Simvastatin by gavage (2.5 mg/kg), and killed after six weeks, and serum was collected.
In accordance with the instructions, the levels of TNF-α and IL-8 in the serum were detected by enzyme-linked immunosorbent assay. The data was expressed as
The detection results of TNF-α and IL-8 showed that different concentrations of drugs intervened in both TNF-α and IL-6. The efficacy of the high-dose group was considerable to that of Simvastatin.
Based on the comparison of the above lesions in various groups, the lesion degrees of the drug low-dose group and the drug high-dose group were milder than that of the model group; 10 mg/kg and 5 mg/kg drugs had good improvement effects on lung lesions; and the high-dose group had an effect considerable to the Simvastatin group.
BALB/c mice, female, weighing 22±2 g, laboratory animal supplier: Hunan SJA Laboratory Animal Co., Ltd., laboratory animal production license: SCXK (Xiang) 2019-0004, and feed supplier: Jiangsu Medicience Biopharmaceutical Co., Ltd.
ginsenoside Rd (content ≥95%), produced by Dalian Fusheng Natural Medicine Development Co., Ltd., detected by HPLC with Ginsenoside Rd provided by China Institute for Food and Drug Control as a reference substance, and having a content of 96.2%.
Ovalbumin (OVA), purchased from Sigma, Corp.
TNF-γ enzyme-linked immunosorbent assay kit, Nanjing Jiancheng Biotechnology Co., Ltd.
IL-4 ELISA assay kit, Nanjing Jiancheng Biotechnology Co., Ltd.
After adaptive feeding (20-25° C., a humidity of 50-60%) for one week, the mice were randomly divided into a blank group, a model group, a ginsenoside Rd high-dose group, and a ginsenoside Rd low-dose group, with ten rats in each group.
Except for the blank group, the mice in the other groups were intraperitoneally injected with 0.2 ml of sensitization solution (containing 50 μg of OVA) on the Day 1, Day 8 and Day 15, respectively. Starting on Day 22, the mice in each group were placed in a nebulizer and inhaled with 2% OVA for exciting asthma. This inhalation was performed once a day, 45 min each time, for seven consecutive days.
Normal saline was used in the blank group instead of OVA.
Administration by gavage was performed 30 min before each asthma stimulation, once a day for seven consecutive days. The blank group and the model group were administrated with normal saline by gavage, the high-dose group was administrated with 10 mg/kg ginsenoside Rd by gavage, and the low-dose group was administrated with 5 mg/kg ginsenoside Rd by gavage.
24 h after the last excitation, the mice were sampled, blood was collected from the eyeballs and separated to obtain serum, and IL-4 and INF-γ in the serum were detected.
The results showed that the ginsenoside Rd could increase the expression of INF-γ and reduce the expression of IL-4, thereby reducing airway inflammations in mice with chronic bronchial asthma and playing a role in the prevention and treatment of asthma.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210027815.3 | Jan 2022 | CN | national |
This application is a continuation-in-part application of PCT/CN2022/143825 having the international filing date of Dec. 30, 2022 and claiming the benefit of priority to Chinese patent application CN 202210027815.3 with the filing date of Jan. 11, 2022, the entire disclosure of both applications is incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2022/143825 | Dec 2022 | WO |
| Child | 18768402 | US |
