Patent Application
20220023370
The present disclosure was made under Task No. HI16C0275 with the support of the Korean Ministry of Health and Welfare. The research management institution for the above project is the Korea Health Industry Development Institute, the research business name is “Guideline Center for Korean Medicine (Sharing Proven Health Technology and Medicine of Korean Medicine)”, and the research project name is “Optimization Research through Quality Index Establishment and Validation of Saegmaeksangagambang for Developing Korean Medicine for Treating Menopausal Syndrome”. The host institute is ViroMed CO., LTD. and the research period is from Jul. 2, 2018 to Nov. 30, 2018.
This application claims priority to and the benefit of Korean Patent Application No. 10-2018-0150175 filed in the Korean Intellectual Property Office on 28 Nov. 2018, the disclosure of which is incorporated herein by reference.
The present disclosure relates to a composition comprising a complex extract of Pueraria lobata and Platycodon grandiflorum as an active ingredient for preventing or treating metabolic bone diseases or menopausal symptoms.
The onset of metabolic bone diseases is accounted for by an imbalance in activity between osteoblasts and osteoclasts which are responsible for the formation and removal of bone tissues in the body, respectively. An osteoclast is a large multinuclear cell destroying or absorbing unnecessary osseous tissues during the metabolism of the bone. Mature osteoclasts are multinucleated, originating from a stem cell. After differentiation from mesenchymal hepatocytes, osteoblasts survive for about 34 months and form new bones at the site where activated osteoclasts break up old bones. A large number of osteoblasts form of an osteoid matrix which is gradually mineralized to complete osteogenesis. Thereafter, about 70% of osteoblasts die while some of osteoblasts differentiate into osteocytes and bone lining cells and survive. Since the amount of bones is maintained by balanced activities between osteoblasts and osteoclasts, it is important to develop a therapeutic agent targeting molecules which exerts significant influence on osteoclastic activity. That is, given an increased activity, osteoclasts responsible mainly for bone resorption accelerate the degradation of bones, causing osteoporosis characteristics of bone thinning and bone fracture. Therefore, studies have been focused on proteins regulatory of osteoclastic activity as targets for the therapy of bone diseases.
For example, osteopenia refers to a pre-stage of osteoporosis and the excessive resorption and formation of osteoclasts are known as a cause therefor. Bone atrophy in rheumatism is associated with excessive osteoclast resorption, as well. Fibrous dysplasia is results from vigorous osteoclastic activity. The suppression of osteoclast functions is used for therapy of Paget's disease and hypercalcemia. The suppression of excessive osteoclast formation and/or activation leads to the inhibition of neoplastic bone destruction. The increase in osteoclast resorption or differentiation causes osteolysis and osteoarthritis.
It is known that cancer cells having invaded into a bone proliferate in a microenvironment around the bone and stimulate osteoblast or osteoclast activities to determine whether to proceed to osteolytic bone metastasis or osteoblastic bone metastasis. Cancer cells having circulated along blood vessels settle to a bone and secrete osteolytic factors such as parathyroid hormone related protein (PTHrP), interleukin (IL)-1, IL-6, IL-8, and IL-11. The secreted factors induce osteoblasts to decrease in the expression of osteoprotegerin (OPG) and increase in the expression of a receptor activator of NF-kB ligand (RANKL). The increased RANKL binds to RANK of an osteoclast progenitor and matures a large number of osteoclast progenitors, resulting in bone destruction through an excessive bone resorption.
In addition, female menopause (climacteric), a type of endocrine syndrome, refers to a transitional period due to the decrease or loss of physiological and sexual function in women as the female hormone estrogen decreases due to the overall and gradual aging of the ovaries. Although not in the climacteric state, patients who have become deficient in estrogen due to other causes such as ovariectomy and hypo-ovarianism suffer from the same symptoms. Such menopausal symptoms include hot flashes, tachycardia, perspiration, or headaches, which are due to vascular changes, and muscle pain, arthralgia, and back pain, which are due to musculoskeletal changes. In addition, menopausal symptoms include urogenital change-related symptoms such as oliguria and incontinence and cranial nerve system change-related symptoms such as hypomnesia, depression, concentration decrease, and dizziness. Furthermore, menopausal women may suffer from amblyopia and skin and hair changes and even hormonal change-caused diseases such as menopausal osteoporosis and cardiovascular diseases, which may be fatal.
In order to improve physical and mental health and life quality of middle-aged women, there has been a need for the development of a therapeutic agent for alleviating menopausal symptoms. A hormone replacement therapy and medicines such as non-steroidal drugs have been developed to alleviate such menopausal symptoms. However, most of those medicines are known to have side effects such as headaches and weight gain. Particularly, estrogen replacement therapy, which is accounted for by artificial injection of a hormone, is known to increasing the risk of onset of metrorrhagia, stroke, heart failure, breast cancer, and uterine cancer as well as a rejection response.
Due to the aforementioned demerits, much attention has been paid to a natural estrogen consumption in a food or additive form in place of an estrogen therapy, and development of a novel menopausal medicine which is excellent in terms of alleviation of the menopausal symptoms without side effects is demanded.
The present inventors made intensive research efforts to search for a material which can prevent or alleviate metabolic bone diseases or menopausal symptoms and thus completed the present disclosure.
Leading to the present disclosure, intensive and thorough research, conducted by the present inventors, into a natural substance for preventing, alleviating, and treating metabolic bone diseases or menopausal symptoms at excellent efficacy without side effects, resulted in the finding that a composition comprising a complex extract of Pueraria lobata and Platycodon grandiflorum is highly effective for preventing, alleviating, or treating metabolic bone diseases or menopausal symptoms.
Therefore, an aspect of the present disclosure is to provide a pharmaceutical composition for preventing or treating metabolic bone diseases or menopausal symptoms.
Another aspect of the present disclosure is to provide a food composition for preventing or alleviating metabolic bone diseases or menopausal symptoms.
Still another aspect of the present disclosure is to provide a method for preventing, alleviating, or treating metabolic bone diseases or menopausal symptoms.
An aspect of the present disclosure provides a pharmaceutical composition for preventing or treating metabolic bone diseases or menopausal symptoms, the composition including: (a) a complex extract of Pueraria lobata and Platycodon grandiflorum as an active ingredient; and (b) a pharmaceutically acceptable carrier thereof.
Leading to the present disclosure, intensive and thorough research, conducted by the present inventors, into a natural substance for preventing, alleviating, and treating metabolic bone diseases or menopausal symptoms at excellent efficacy without side effects, resulted in the finding that a composition comprising a complex extract of Pueraria lobata and Platycodon grandiflorum is highly effective for preventing, alleviating, or treating metabolic bone diseases or menopausal symptoms.
The term “Platycodon grandiflorum”, as used herein, refers to a root of Platycodon grandiflorum A. DC., which is a plant of the family Campanulaceae, called Kilkyoung in Korea, Jiegeng in China, and Kykyo in Japan, and has been used as a traditional oriental medicine. Platycodon grandiflorum has been used as an expectorant, an antitussive, and a therapeutic for treatment of coughing and bronchitis.
The term “Pueraria lobata”, as used herein, refers to a tuber of kudzu (Pueraria thunbergiana BENTH), which is a perennial climbing plant of the family Leguminosae, contains substances such as puerarin, puerarin xylose, daidzein, sitosterol, and the like, and is known to exhibit hyperkinemic, contraparetic, and antipyretic activities.
The composition of the present disclosure includes a complex extract of Pueraria lobata and Platycodon grandiflorum as an active ingredient. The complex extract of Pueraria lobata and Platycodon grandiflorum may be prepared (i) using a single extraction process in which a mixture of Pueraria lobata and Platycodon grandiflorum is subjected to extraction with an extraction solvent or (ii) by preparing single component extracts of Pueraria lobata and Platycodon grandiflorum separately and then mixing them.
As used herein, the term “extract” commonly refers to a crude extract in the art as stated above, but is intended to encompass, in a broad sense, a fraction which is formed by additional fractionation of the extract. That is, the complex extract of Pueraria lobata and Platycodon grandiflorum may be obtained not only using the solvent but also by additionally performing a purifying process thereon. For example, the complex extract of Pueraria lobata and Platycodon grandiflorum of the present disclosure includes a fraction obtained by passing the extract through an ultrafiltration membrane with a predetermined molecular weight cut-off value, or a fraction obtained by additionally performing various purification methods such as various chromatographies (designed for separation according to size, charge, hydrophobicity, or hydrophilicity).
According to an embodiment, the extract of the present disclosure is a polar organic solvent extract. The “polar organic solvent”, as used herein, includes (a) water, (b) an anhydrous or hydrated lower alcohol of 1-4 carbon atoms (e.g., methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, etc.), and (c) acetic acid, or a mixture of the polar aprotic solvents.
In an embodiment, the polar organic solvent of the present disclosure is water, an anhydrous or hydrated lower alcohol of 1-4 carbon atoms, and acetic acid, or a mixture of two or more selected from the stated components.
In an embodiment of the present disclosure, the anhydrous or hydrated lower alcohol of 1-4 carbon atoms is at least one selected from the group consisting of methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, and normal-butanol.
The concentration of the organic solvent may be, but is not limited to, 1 to 100% (v/v), specifically 10 to 100% (v/v), 20 to 100% (v/v), 30 to 100% (v/v)), 40 to 100% (v/v), 50 to 100% (v/v), 60 to 100% (v/v), 70 to 100% (v/v), or 80 to 100% (v/v), and more specifically 25% (v/v), 50% (v/v), 75% (v/v), or 95% (v/v).
In order to prepare the extract according to an embodiment of the present disclosure, the amount of the solvent may be appropriately selected depending on an amount of medicinal herb components used therefor. Specifically, for example, the amount of the solvent may be, but is not limited to, a 1 to 20 volumes, more particularly, 2 to 20 volumes, even more particularly, 5 to 15 volumes, even far more particularly 7 to 12 volumes of the total weight of Pueraria lobata and Platycodon grandiflorum used to prepare the extract.
The extraction temperature for the extract of the present disclosure is not particularly limited and may be, for example, 0° C. to 120° C., and, specifically, 15° C. to 95° C.
In an embodiment of the present disclosure, the extraction temperature is preferably 80° C. or higher for water used as an extraction solvent, and 15° C. to 30° C. for a lower alcohol of 1-4 carbon atoms used as an extraction solvent.
No particular limitations are imparted to the extraction time. For example, the extraction time may be 1 hour to 10 days, 1 hour to 120 hours, and specifically 1 hour to 72 hours, 1 hour to 48 hours, 1 hour to 36 hours, 1 hour to 24 hours, 1 hour to 12 hours, 1 hour to 10 hours, or 1 hour to 6 hours, but is not limited thereto.
The extract used in the present disclosure can be obtained through hot water extraction, cold extraction, reflux cold extraction, ultrasonic extraction, or a conventional extraction method well known in the art. In an embodiment, the extract of the present disclosure can be obtained through cold extraction using a lower alcohol or through hot water extraction, and the extraction may be repeated for 1-10 rounds.
The complex extract of Pueraria lobata and Platycodon grandiflorum may be prepared into a powder form through an additional process such as a vacuum distillation and lyophilization, or spray drying.
In an embodiment of the present disclosure, active ingredients, Pueraria lobata and Platycodon grandiflorum are mixed at a weight ratio (w/w) of 1:20 to 20:1. In another embodiment, Pueraria lobata and Platycodon grandiflorum are mixing at weight ratio (w/w) of 1:15 to 15:1, 1:12 to 12:1, 1:10 to 10:1, 1:10 to 5:1, 1:10 to 3:1, 1:10 to 1:2, 1:10 to 1:1, 1:8 to 8:1, 1:8 to 5:1, 1:8 to 3:1, 1:8 to 1:2, 1:8 to 1:1, 1:6 to 6:1, 1:6 to 5:1, 1:6 to 3:1, 1:6 to 2:1, 1:6 to 1:1, 1:5 to 5:1, 1:5 to 3:1, 1:5 to 2:1, 1:5 to 1:1, 1:4 to 4:1, 1:4 to 3:1, 1:4 to 2:1, 1:4 to 1:1, 1:3 to 3:1, 1:3 to 2:1, 1:3 to 1:1, 1:2 to 2:1, 1:2 to 1:1, 1:1.5 to 1.5:1, or 1:1.
In an embodiment of the present disclosure, Pueraria lobata and Platycodon grandiflorum is mixed at a weight ratio of 1:1 to 1:8, more particularly 1:1, 1:2, 1:4, 1:8, 2:1, 4:1, or 8:1, and most particularly 1:1, but without limitations thereto.
The term “weight ratio (w/w)” as used in the context of mixing herein refers to a weight ratio of single ingredients before undergoing an extraction process. In the case of preparing the complex extract through a single extraction process in which a mixture of Pueraria lobata and Platycodon grandiflorum is extracted using an extraction solvent, for example, the mixing weight ratio refers to a weight ratio of respective single-components of Pueraria lobata and Platycodon grandiflorum included in the mixture. In the case in which the complex extract is prepared by separately extracting each of Pueraria lobata and Platycodon grandiflorum and mixing single extracts the mixing weight ratio refers to a weight ratio between “single component reference weights” calculated by a formula below:
Single component reference weight=weight of single component used for preparation of single extraction×(weight of single extract used for preparing final complex extract/weight of prepared single extract). [Formula]
The composition of the present disclosure can be produced as a pharmaceutical composition.
The term “prevention” in the present disclosure includes all kinds of actions of inhibiting or delaying the onset of metabolic bone disease or menopausal symptoms by administration of a composition including the complex extract of Pueraria lobata and Platycodon grandiflorum according to the present disclosure as an active ingredient.
The term “treatment” in the present disclosure includes all kinds of actions to improve or positively change metabolic bone diseases or menopausal symptoms by administrating a composition including the complex extract of Pueraria lobata and Platycodon grandiflorum according to the present disclosure as an active ingredient.
The pharmaceutical composition of the present disclosure includes the complex extract of Pueraria lobata and Platycodon grandiflorum at a pharmaceutically effective amount. The “pharmaceutically effective amount” refers to an amount sufficient to exhibit the efficacy or activity of the complex extract of Pueraria lobata and Platycodon grandiflorum, but it is not particularly limited as long as it achieves the purpose of the present disclosure.
The pharmaceutical composition of the present disclosure includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present disclosure pertains to a commonly used material for formulation and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystal cellulose, polyvinyl pyrrolidone, water, syrup, and mineral oil, but is not limited thereto. The pharmaceutical composition of the present disclosure may additionally include lubricants, humectants, sweetening agents, fragrances, emulsifiers, suspensions, preservatives, etc. Suitable and pharmaceutically acceptable carrier and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
The suitable level of an effective amount of the pharmaceutical composition of the present disclosure can be determined variously depending on factors such as formulation types, an administration method, an age and body weight of a patient, a gender of a patient, severity of patient's disease, diet, time of administration, route of administration, discharge rate, and responsive sensitivity. The general dosage of the pharmaceutical composition of the present disclosure is within a range of 0.001-1000 mg/kg on an adult basis. In addition, the dosage for a human body can be calculated on the basis of animal experiments.
The pharmaceutical composition of the present disclosure may be formulated into a unit dosage form or produced to be incorporated into a multi-dosage container by using pharmaceutically acceptable carriers and/or excipients according to methods which can be easily implemented by a person of ordinary knowledge in the art to which the present disclosure pertains. In this case, the formulation may be a form of solutions in an oil or aqueous medium, suspensions, or syrups or emulsions, or a form of extracts, powders, granules, tablets, or capsules, and may further include dispersants or stabilizers.
The “metabolic bone diseases” in the present disclosure refer to bone-related diseases triggered by the imbalance between osteoblasts and osteoclasts. Specific examples thereof include pathological bone diseases which accelerate bone destruction, such as osteoporosis, arthritis, periodontal disease, fractures, or Paget's disease which are caused by excessive bone resorption of osteoclasts, but is not limited thereto.
The “osteoporosis” refers to a state in which the amount of bone is reduced and the strength of bones is weaken due to a qualitative change thereof, leading to a high occurrence possibility of fractures. Bones protect various organs in the body, serve as a storage unit for materials required in the body, such as calcium, and maintain constancy by the balance of bone-degrading osteoclasts and bone-forming osteoblasts each present in osseous tissues. Osteoporosis may occur and proceed when the activity balance between the two cells is broken and excessive bone destruction by osteoclasts occurs. RANKL (receptor activator of nuclear factor kappa-B ligand) is a key element of osteoporosis and is coupled to a receptor thereof to activate various transcription factors, thereby promoting differentiation of osteoclasts.
“Arthritis” refers to a joint disease accompanied by inflammation on at least one joint area. A general form of arthritis is osteoarthritis in which cartilage protecting a joint is gradually damaged or bones and ligaments constituting a joint are damaged due to degenerative changes, causing inflammation and pain. Arthritis, with regard to the purpose of the present disclosure, is a disease accompanied by bone loss in a joint area and may include osteoarthritis or rheumatoid arthritis, but is not limited thereto.
“Periodontal disease” refers to the inflammatory state of supportive tissue caused by bacteria, and can be separated into gingivitis and periodontitis. The cause of the disease is the formation of dental plaque by oral bacteria due to poor oral hygiene. Dental plaque refers to a mass of bacteria that grows after bacteria adhere to a tooth surface by using a sticky substance in the saliva as an adhesive. If left untreated, the plaque becomes inflamed and sometimes causes bleeding from the gums and bad breath. These symptoms are called gingivitis. As the gingivitis progresses further, the gap between the teeth and the gums becomes deeper, and a paradental cyst develops, and the periodontitis occurs because the bacteria causing the periodontal disease multiply. As periodontitis progresses, even weak stimuli such as brushing may cause bleeding and swelling of the gums, often turning into acute inflammation, causing pain. This inflammation lowers the function of making bones, the bone absorbing effect is increased, the alveolar bone becomes lower, and the alveolar bone is destroyed and eventually teeth are lost.
“Fractures” refer a fracturing of the bone in a state in which the continuity of the bone, the end plate, or the joint surface is abnormally broken. The causes of fractures include traumas such as traffic accidents, safety accidents caused by industrial accident, bone changes caused by diseases such as osteoporosis, bone cancer, metabolic disorders, and repetitive bone stress caused by sports or loads. In addition, a fracture status is, on the basis of a fracture line (line along bone tip generated by bone cutting), classified into crack fracture, greenstick fracture, transverse fracture, filamentous fracture, spiral fracture, segmental fracture, comminuted fracture, avulsion fracture, compression fracture, depression fracture, etc.
“Paget's disease” refers to a localized bone disease in which bone remodeling is excessively aggravated and the skeletal system of a wide area is invaded. The pathologic mechanism of Paget's disease is believed to be a combination of an excessive increase in bone resorption by osteoclasts with the ability to cleanse the bone and an increase in new bone formation by osteoblasts with bone-forming functions as a compensation thereto, and newly formed bones in the bone in Paget's disease are structurally disordered and are known to be very vulnerable to bone deformation and fracture.
“Menopausal symptoms” of the present disclosure include symptoms caused by vascular changes, such as hot flashes, tachycardia, perspiration, or headaches, and symptoms caused by musculoskeletal changes, such as muscle pain, arthralgia, and back pain. The menopausal symptoms also include symptoms caused by urogenital changes, such as oliguria or incontinence, and symptoms caused by changes in cranial nerve system, such as hypomnesia, depression, concentration decrease, and dizziness. Furthermore, symptoms such as amblyopia and skin and hair change may occur and diseases such as menopausal osteoporosis and cardiovascular diseases due to changes in hormones may occur to threaten women's health.
As identified as below, the complex extract of Pueraria lobata and Platycodon grandiflorum remarkably reduced the TRAP activity in an osteoclast differentiation experiment in which a macrophage cell line of a mouse was treated with RANKL, compared with a negative control group and a group treated with single extracts of Pueraria lobata and Platycodon grandiflorum, thereby identifying the treatment and alleviation efficacy for osteoporosis according to inhibition of osteoclast differentiation.
In addition, the complex extract of Pueraria lobata and Platycodon grandiflorum was identified to be remarkably excellent in terms of the vasorelaxation effects in a cardiovascular disease model using a thoracic aortic strip compared with a negative control group and a group treated with single extracts of Pueraria lobata and Platycodon grandiflorum respectively, thereby identifying the treatment and alleviation efficacy of menopausal cardiovascular diseases.
Furthermore, the complex extract of Pueraria lobata and Platycodon grandiflorum was identified to remarkably improve biochemical indicators for osteoporosis, such as ALP, Ca, MMP-9, and osteocalcin in an ovariectomized osteoporosis mouse model, compared with a negative control group and a group treated with single extracts of Pueraria lobata and Platycodon grandiflorum, thus identifying the treatment and alleviation efficacy of osteoporosis.
The complex extract of Pueraria lobata and Platycodon grandiflorum showed a synergistic effect for each indicator compared with the single extract of Pueraria lobata or Platycodon grandiflorum.
Another aspect of the present disclosure provides a food composition including the complex extract of Pueraria lobata and Platycodon grandiflorum as an active ingredient of the present disclosure for prevention or treatment of metabolic bone diseases or menopausal symptoms.
The term “alleviation” in the present disclosure includes all kinds of actions to at least reduce the level of pathogenesis of metabolic bone diseases or menopausal symptoms by administration of a composition including the complex extract of Pueraria lobata and Platycodon grandiflorum according to the present disclosure as an active ingredient.
When the composition of the present disclosure is prepared into a food composition, the food composition contains ingredients commonly added for food manufacturing and examples of the ingredients include proteins, carbohydrates, fats, nutrients, seasoning agents, and flavoring agents. Examples of the aforementioned carbohydrates include regular sugars, e.g., monosaccharides such as glucose, fructose, and so on; disaccharides such as maltose, sucrose, oligosaccharide, and so on; and polysaccharides such as dextrin, cyclodextrin, and so on; and sugar alcohol such as xylitol, sorbitol, erythritol, and so on. The flavoring agents may be a natural flavoring agent and a synthetic flavoring agent. For example, when the food composition of the present disclosure is prepared as a beverage, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and the like may be further included in addition to the active ingredients of the present disclosure.
The food composition of the present disclosure may use the complex extract of Pueraria lobata and Platycodon grandiflorum as it is included in the pharmaceutical composition of an aspect of the present disclosure, and thus descriptions of overlapping contents therebetween will be omitted to avoid excessive complexities.
Still another aspect of the present disclosure provides a method for preventing, treating, and alleviating metabolic bone diseases or menopausal symptoms, the method including a step of administering to a subject a pharmaceutical composition or food composition comprising the complex extract of Pueraria lobata and Platycodon grandiflorum described in the present disclosure as an active ingredient.
The definition for metabolic bone diseases or menopausal symptoms which are the target diseases of the treatment or alleviation method of the present disclosure is the same as the definition with respect to the target disease of the pharmaceutical or food compositions.
As used herein, the term “administration” or “administer” refers to direct administration of a therapeutically or alleviatively effective amount of a composition of the present disclosure to a subject (an individual) suffering from the target disease so that the same amount is formed in the body of the subject.
The “therapeutically effective amount” of a composition means a content of the composition that is sufficient to provide a therapeutic or prophylactic effect to a subject to which the composition is to be administered, and includes a “prophylactically effective amount”. Also, as used herein, the term “subject” includes mammals such as a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. Most specifically, the subject of the present disclosure is a human.
The method for preventing, treating, and alleviating metabolic bone diseases or menopausal symptoms includes a step of administering the pharmaceutical or food composition according to an aspect of the present disclosure, and thus descriptions of overlapping contents therebetween will be omitted to avoid excessive complexities.
The features and advantages of the present disclosure are as follows:
(a) The present disclosure provides a pharmaceutical composition for preventing or treating metabolic bone diseases or menopausal symptoms.
(b) The present disclosure provides a food composition for preventing or alleviating metabolic bone diseases or menopausal symptoms.
(c) The present disclosure provides a method for preventing, alleviating, or treating metabolic bone diseases or menopausal symptoms.
(d) The use of the pharmaceutical composition or the food composition according to the present disclosure can prevent, alleviate, and treat metabolic bone diseases or menopausal symptoms without side effects.
Hereinafter, the present disclosure will be explained in detail with reference to examples. These examples are provided only for the purpose of illustrating the present disclosure in more detail, and therefore, according to the purpose of the present disclosure, it would be apparent to a person skilled in the art that these examples are not construed to limit the scope of the present disclosure.
Washed and dried Pueraria lobata and Platycodon grandiflorum were mixed at a weight ratio (w/w) of 1:1, 2:1, 4:1, 8:1, 1:2, 1:4, and 1:8 and added with 10 volumes 70% aqueous ethanol, followed by extraction at 20° C. for 72 hours while stirring well. Thereafter, filtration was conducted before vacuum concentration at 45-50° C. Lyophilization afforded a complex extract powder. The yield is given in Table 1 below.
Pueraria lobata • Platycodon
grandiflorum (1:1)
Pueraria lobata • Platycodon
grandiflorum (1:2)
Pueraria lobata • Platycodon
grandiflorum (1:4)
Pueraria lobata • Platycodon
grandiflorum (1:8)
Pueraria lobata • Platycodon
grandiflorum (2:1)
Pueraria lobata • Platycodon
grandiflorum (4:1)
Pueraria lobata • Platycodon
grandiflorum (8:1)
Washed and dried Pueraria lobata and Platycodon grandiflorum were added with 10 volumes 70% ethanol aqueous solution to each medicinal herb weight, followed by extraction 20° C. for 72 hours while stirring well. Thereafter, filtration was conducted before vacuum decompression concentration at 45-50° C. Lyophilization afforded a total of two kinds of single extract powders. The yield is given in Table 2 below.
Pueraria lobata
Platycodon grandiflorum
Washed and dried Pueraria lobata and Platycodon grandiflorum were mixed at a weight ratio (w/w) of 1:1. For a hot water extract, the mixture was subjected to extraction under reflux at 90±5° C. for 3 hours. For an ethanol extract, the mixture was added with 10 volumes of 0, 25, 50, 75, 95% aqueous ethanol before extraction at a 20° C. for 72 hours while stirring well. Then, concentration in a vacuum at 45-50° C. was followed by lyophilization to give a complex extract powder. The yield is given in Table 3 below.
Pueraria lobata • Platycodon
grandiflorum (1:1)
Pueraria lobata • Platycodon
grandiflorum (1:1)
Pueraria lobata • Platycodon
grandiflorum (1:1)
Pueraria lobata • Platycodon
grandiflorum (1:1)
Pueraria lobata • Platycodon
grandiflorum (1:1)
Pueraria lobata • Platycodon
grandiflorum (1:1)
TRAP (tartrate-resistant acid phosphatase) is an enzyme used as an indicator of the osteoclast differentiation process, and the reduction in TRAP activity accounts for the inhibition of osteoclast differentiation. In this experiment, osteoclasts which were derived and differentiated by RANKL (receptor activator of NF-kappa B ligand, Sigma-Aldrich, US) from the mouse macrophage cell line RAW264.7 were used to identify effects of the single and complex extracts on osteoclast differentiation through measurement of TRAP activity, and effects of complex extracts on osteoclast differentiation for each solvent.
1-1. Cell Line Culture
First, RAW264.7 cells were incubated using a phenol-red free α-MEM (GIBCO, USA) medium supplemented with 10% charcoal-stripped fetal bovine serum (GIBCO) in a 5% CO2 incubator at 37° C. RAW264.7 cells were plated to a 96-well plate at a density of 1×103 cells per well, and stabilized for 24 hours. Thereafter, the cells were treated with 50 ng/ml of RANKL and 600 μg/ml of each of single and complex extracts of Pueraria lobata and Platycodon grandiflorum or 600 μg/ml of each of a hot water complex extract and ethanol complex extracts of Pueraria lobata and Platycodon grandiflorum for 72 hours before TRAP activity analysis.
1-2. TRAP Activity
In brief, the cells were incubated with 30 μl of TRAP activity assay solution (sodium acetate 600 mM, pH 5.5, L-ascorbic acid 17.6 mg/ml, sodium-tartrate dehydrate 9.2 mg/ml, 4-nitrophenylphosphate Na 3.6 mg/ml, Triton X-100 0.3%, EDTA 6 mM, NaCl 600 mM) for 30 minutes in 37° C. incubator. After the reaction was terminated with 300 mM NaOH absorbance was read at 450 nm wavelength.
TRAP activity is expressed as a ratio relative to the RANKL-treated group (negative control).
In the experiment for comparison between the single extracts and the complex extracts, treatment of RAW264.7 with RANKL increased TRAP activity, compared the untreated group, which was reduced in all of the extract-treated groups, as shown in Table 4 and
Pueraria lobata:Platycodon
grandiflorum ratio
100 ± 7.03
Pueraria lobata
Platycodon grandiflorum
As shown in following Table 5 and
Pueraria lobata:Platycodon
grandiflorum ratio
In addition, correction of the values in Tables 4 and 5 confirmed similar TRAP activity between Preparation Example 1-1 in Table 4 and Preparation Example 1-12 of Table 5.
The estrogen deficiency is a cause of blood pressure elevation in menopausal women by inducing vasoconstriction. Cerebrovascular constriction induces cerebral apoplexy. Therefore, a vasorelaxation efficacy is used as an important evaluation indicator in finding candidate materials of prophylactic and therapeutic drugs for menopausal cardiovascular diseases. In order to identify vasorelaxation efficacy of the complex extracts of Pueraria lobata and Platycodon grandiflorum of Preparation Example 1, phenylephrine (PE)-precontracted rat thoracic aortic strips were assayed for relaxation response in an organ bath.
2-1. Preparation of Thoracic Aortic Strip
Thoracotomy was performed on the experimental animals male Sprague Dawley (SD) rats at 8 weeks of age that were anesthetized with ethylether inhalation. Immediately after being excised, thethoracic aorta was supplied with a gas of 95% of 02 and 5% of CO2 and cleaned of adherent tissues and fats in Krebs-Henseleit solution (K—H solution, composition, mM: NaCl, 118.0; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; NaHCO3, 25.0; and glucose, 11.1; pH 7.4) maintained at 37° C. Subsequently, the aorta was cut into strips in a ring form about 2 mm in length.
2-2. Isotonic Contraction
The prepared aortic strip was hooked at the opposite ends thereof by respective tungsten wires, after which the lower portion thereof was anchored to a hook installed on the bottom of a 10-mL organ bath while the upper portion thereof was connected to an isometric force transducer (AD Instrument Co., Australia) connected to a physiograph (AD Instrument Co., Australia) for the measurement of isotonic contraction changes, which was recorded using the PowerLab program (AD Instrument Co., Australia). The strip was stabilized for 15 minutes in the organ bath and then loaded with a passive tension of 1 g. An experiment was proceeded after re-stabilization for 1 hour. The Krebs-Henseleit solution in the organ bath was replaced with a fresh one every 20 minutes during stabilization.
In order to identify a vasorelaxation activity therein, the aortic strip was treated with 1 μM phenylephrine for 40 minutes in the organ bath to induce vasocontraction. The sufficiently contracted strip was treated with 1 mg/ml of each of the single and complex extracts of Pueraria lobata and Platycodon grandiflorum in Krebs-Henseleit solution in the organ bath to compare vasorelaxation activities. The negative control was treated with Krebs-Henseleit solution alone. The vasorelaxation rate (%) was calculated by the following equation.
A: strip contraction before PE-induced contraction
B: strip contraction after PE-induced contraction
C: strip contraction after K—H solution or extract treatment
As shown in Table 6 and
Pueraria lobata
Platycodon
grandiflorum
Estrogen deficiency, which is characteristically observed in menopausal women, induces differentiation of osteoclasts degrading bones, resulting in bone loss and osteoporosis. The ovariectomized (OVX) rat model is currently the best model to reflect estrogen deficiency-induced bone loss symptoms in menopausal women. An ovariectomized rat model was observed in have lowered blood concentrations of calcium (Ca) and inorganic phosphate, which are constituents of bones, and increased levels of MMP-9 (Matrix metallopeptidase-9), which is a main enzymes for degrading bones, ALP (alkaline phosphatase), which is a bone turnover marker, and osteocalcin. These markers are used as main indices for evaluating bone loss. In order to examine an alleviative effect of the complex extracts of Pueraria lobata and Platycodon grandiflorum of Preparation Example 1 on bone loss in terms of the markers, biochemical analysis was conducted on the blood of the ovariectomized female rat.
3-1. Construction of Ovariectomized (OVX) Rat
Female Sprague Dawley rats at 6 weeks of age (each weighing about 160 g±20%) were purchased from Orient (OrientBio, Gapyeong-gun, Gyeonggi-do) and acclimated for 14 days before experiments. The animals were anesthetized with Zoletil 50 (VIRBAC, France) and xylazine (Rompun®, Bayer AG, Germany) for ovariectomy. After the animals were shaved at both the abdominal and dorsal sides and disinfected at sites for surgery, incisions were made in the muscle and peritoneum to expose the uterus and ovaries to identify ovary loci. After exposure of the fallopian tubes and ovarian arteriovenous veins, the ovaries were excised using a cautery, followed by closing the wounds. The test substances, including single and complex extracts of Pueraria lobata (Comparative Example 1-1), Platycodon grandiflorum (Comparative Example 1-2), and Pueraria lobata and Platycodon grandiflorum (Preparation Example 1-1) were orally administrated once a day at a daily dose of 200 mg/kg for 7 days/week, 12 weeks. The normal group and the negative control were orally administered distilled water.
3-2. Blood Biochemical Test
After 12 weeks of administration, a serum separated from blood collected from the posterior vena cava was analyzed for alkaline phosphatase (ALP) and calcium, using a blood biochemical analyzer (7180 Hitachi, Japan) and an automatic electrolyte analyzer. In addition, the osteolysis markers MMP (Matrix metallopeptidase)-9 and Osteocalcin were measured by ELISA.
As shown in Table 7 and
Pueraria lobata
Platycodon
grandiflorum
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0150175 | Nov 2018 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/015988 | 11/20/2019 | WO | 00 |
