US20130131159A1 - Plectranthus amboinicus fraction having anti-arthritis activity - Google Patents

Plectranthus amboinicus fraction having anti-arthritis activity Download PDF

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US20130131159A1
US20130131159A1 US13/680,689 US201213680689A US2013131159A1 US 20130131159 A1 US20130131159 A1 US 20130131159A1 US 201213680689 A US201213680689 A US 201213680689A US 2013131159 A1 US2013131159 A1 US 2013131159A1
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extract
plectranthus amboinicus
arthritis
absorption peak
hexane
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Feng-Nien Ko
Jen-Wei Chen
Wen-Ling Yang
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ONENESS BIOTECH Co
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ONENESS BIOTECH Co
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Assigned to ONENESS BIOTECH CO. reassignment ONENESS BIOTECH CO. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHEN, JEN-WEI, KO, FENG-NIEN, YANG, WEN-LING
Publication of US20130131159A1 publication Critical patent/US20130131159A1/en
Priority to US14/328,475 priority Critical patent/US20140322365A1/en
Priority to US15/496,521 priority patent/US11517604B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps

Definitions

  • the present invention relates to a plant extract, which in particular, is a fraction of Plectranthus amboinicus (Lour.) Spreng enriching anti-arthritis activities.
  • Arthritis is the most common autoimmune disease.
  • the hallmarks of the disease are inflammation of the synovial tissues with progressive erosion of bone leading to malalignment of the joint and disability.
  • the persistent nature of arthritis suggests that not only local but also systemic immune dysfunction, consisting in predominance of the pro-inflammatory response and over production of inflammatory cells and inflammatory substances, such as CD4+ T cells, B cells and inflammatory cytokines. All of which cause long-term destruction to joint tissues.
  • TNF- ⁇ is a key player in the proinflammatory cytokine cascade by stimulating the production of prostaglandin E, collagenase as well as other cytokines such as interleukin-1 (IL-1) and interleukin-6 (IL-6).
  • IL-1 interleukin-1
  • IL-6 interleukin-6
  • TNF- ⁇ and IL-1 ⁇ also stimulate the secretion of matrix metalloproteinases and exert a direct effect on the multiple tissues inside the joint including chondrocytes, macrophages, synovial fibroblasts, and osteoclasts leading localized joint destruction.
  • IL-6 can further increase inflammatory cells in the joint tissue, stimulate the proliferation of osteoclasts and strengthen the role of IL-1 ⁇ .
  • the substantial cross-talk between pro-inflammatory cytokines IL-1 ⁇ , TNF- ⁇ , IL-6, and IL-17 is essential to induce joint destruction, chondrocyte inhibition, and disturbance for restoration of degenerated tissues in arthritis.
  • the current clinical majority is to develop biological antagonists specifically aiming at the above-mentioned inflammatory factors or cells, such as TNF- ⁇ and IL-6 antagonists, to reduce or prevent joint damage and maintain functionality.
  • biological antagonists specifically aiming at the above-mentioned inflammatory factors or cells, such as TNF- ⁇ and IL-6 antagonists, to reduce or prevent joint damage and maintain functionality.
  • TNF- ⁇ and IL-6 antagonists such as TNF- ⁇ and IL-6 antagonists
  • Plectranthus amboinicus (Lour.) Spreng, a perennial herb mainly originated from Malaysia, Brazil, China and India, belongs to the Labiatae family, the alias of which are Agastache rugosa, Lysimachia capillipes Hemsl, Spearmint, Patchouly, Indian peppermint, or Pogostemon Cablin.
  • Plectranthus amboinicus has leaves that are thick, opposite, widely ovate, toothed on the margin of a little volume with apex rounded or pure sharp. The herb is about 15 to 30 cm high, covered with fine hairs, and carries a strong pungent smell.
  • Plectranthus amboinicus is a type of Chinese herbal medicine with the efficacy to prevent colds, strengthen body immunity, and reduce symptoms such as ear swelling, inflammation, and fever.
  • As a traditional Chinese medicine it is anti-inflammatory, carminative and capable of detoxification and relieving symptoms like fever, tonsillitis, sore throat, pneumonia, chills and heat, headache, nausea of chest and abdomen, vomiting, diarrhea, feeble stomach, spleen cold and others.
  • Fresh juice of Plectranthus amboinicus is also effective for abrasions, cuts, burns, insect bites, unknown swelling, boils and sores, ear inflammation, sore throat, swollen poison, bruises and the like.
  • Taiwan Patent Application No. 092135016 disclosed a method to prepare crude extracts of Plectranthus amboinicus (Lour.) Spreng comprising steps of: (a) pressing the plant leaves to obtain juice; (b) adding alcohol to the juice of step (a) to reach a final alcohol concentration at 70-80%, and then placing at a low temperature; (c) adjusting the pH value of the alcohol-containing juice of step (b) to pH5.0 ⁇ 7.0, and then placing at a low temperature; (d) filtering to remove the impurities in the alcohol-containing juice of step (c); (e) distilling and concentrating the resulted pure juice in step (d) to collect the distillate, and adjusting the pH value of such distillate to pH5.0 ⁇ 7.0; and (f) vacuum filtering the distillate in step (e) to obtain dark brown and clear extracts of Plectranthus amboinicus.
  • Taiwan Patent Application No. 093134346 provided a water extract of Plectranthus amboinicus leaf having the efficacy to treat cancer and/or tumor which has a molecular weight more than 50 kD.
  • Taiwan Patent Application No. 086118191 also disclosed a type of water extract of Pogostemon cablin or Agastache rugosa capable of preventing and treating infection by Haemophilus influenzae .
  • the preparation of such extract involves an elution by a reagent composed of 90.5% ethanol, 4.5% methanol and 5.0% isopropanol.
  • Taiwan Patent Application No. 096145943 i.e. Taiwan Invention Patent No. 1-335225
  • Taiwan Invention Patent No. 1-335225 also provided an extract of Plectranthus amboinicus (Lour.) Spreng for treating skin disorders and promoting wound healing, in particular, wound healing in patients with diabetes.
  • the invention as disclosed in this patent is characterized by the use of a combination of the solid-liquid separation (stir separation) and specific processing, which comprises stiffing adsorption resin (e.g. DIAION) in the extract of Plectranthus amboinicus , and isolating various fractions of such extract by using different solvents in respective separation steps.
  • stiffing adsorption resin e.g. DIAION
  • Taiwan Patent Application No. 095134243 i.e. Invention Patent No. 1320714, disclosed a water extract of Plectranthus Amboinicus Benth having the efficacy to treat rheumatoid arthritis.
  • the extract was soaked in an appropriate amount of high-polar solvent, filtered, condensed under reduced pressure by a rotary concentrator, diluted in a solvent, and then separated by in a column
  • four segments of different solvents from high polarity to low polarity referred to as high-polar solvent, sub high-polar solvent, medium-polar solvent and low-polar solvent
  • high-polar solvent, sub high-polar solvent, medium-polar solvent and low-polar solvent could be used for elution continuously.
  • the present invention is based on a further development and improvement of the processes for preparing a Plectranthus amboinicus extract, which, unexpectedly, exhibited high anti-arthritis activities.
  • a Plectranthus amboinicus extract is prepared by eluting the crude extract of Plectranthus amboinicus (Lour.) Spreng by 50% ⁇ 95% alcoholic solution, or by a normal-phase chromatography.
  • the present invention provides an extract of Plectranthus amboinicus enriching anti-arthritis activity, which is characterized by having an absorption peak of Cirsmaritin at retention time of about 45 min in a HPLC pattern detected at a wavelength of about 320 nm.
  • the extract of Plectranthus amboinicus enriching anti-arthritis activity is characterized by that: said extract was obtained by eluting the crude extract of Plectranthus amboinicus using 50% ⁇ 95% alcoholic solution, preferably using 70% ⁇ 95% alcoholic solution, and more preferably using about 95% alcoholic solution.
  • the alcoholic solution is ethanol.
  • the extract of Plectranthus amboinicus enriching anti-arthritis activity is obtained by eluting the crude extract of Plectranthus amboinicus in a normal phase chromatography using hexane as a first solvent and hexane/ethyl acetate as a second solvent.
  • the present invention provides a method for preparing an extract of Plectranthus amboinicus enriching anti-arthritis activity comprising extracting the plant or dried powder of Plectranthus amboinicus in a solvent to obtain a crude extract, and eluting the resulted crude extract by a chromatography using 50% ⁇ 95% alcoholic solution to isolate an extract enriching anti-arthritis activity.
  • the present invention provides a method for preparing an extract of Plectranthus amboinicus enriching anti-arthritis activity comprising extracting the plant or dried powder of Plectranthus amboinicus in a solvent to obtain a crude extract, and eluting the resulted crude extract by a normal phase chromatography using hexane as a first solvent and hexane/ethyl acetate as a second solvent to isolate an extract enriching anti-arthritis activity.
  • the resulted extract enriching anti-arthritis activity can be further eluted by solvents such as ethyl acetate, ethyl acetate/methanol, and/or methanol.
  • the present invention provides a method for preventing or treating arthritis comprising administrating to a subject in need thereof an effective amount of the extract of Plectranthus amboinicus enriching anti-arthritis activity as described herein.
  • the present invention provides a pharmaceutical composition for preventing or treating arthritis comprising a therapeutically effective amount of an extract of Plectranthus amboinicus enriching anti-arthritis activity as described herein, and a pharmaceutically acceptable carrier.
  • FIG. 1 is a HPLC pattern of ON-024 detected at a wavelength of 200 nm, in which there are six absorption peaks at retention times of 13, 20, 21, 27, 37 and 39 min.
  • FIG. 2 is a HPLC pattern of ON-024 detected at a wavelength of 320 nm, in which there is an absorption peak of Caffeic acid at retention time of 13 min, an absorption peak of Rosmarinic acid at 27 min, and an absorption peak at 35 min.
  • FIG. 3 is a HPLC pattern of ON-025 detected at a wavelength of 200 nm, in which there is an absorption peak at retention time of 45 min, and an absorption peak of Carvacrol at 55 min.
  • FIG. 4 is a HPLC pattern of ON-025 detected at a wavelength of 320 nm, in which there are eight absorption peaks at retention time of 39-43 min and an absorption peak of Cirsmaritin at 45 min.
  • FIG. 5 is a HPLC pattern of ON-066 detected at a wavelength of 200 nm, in which there is an absorption peak of Cirsmaritin at retention time of 45 min and an absorption peak of Carvacrol at 55 min.
  • FIG. 6 is a HPLC pattern of ON-066 detected at a wavelength of 320 nm, in which there is an absorption peak of Cirsmaritin at retention time of 45 min and an absorption peak of Salvigenin at 62 min.
  • FIG. 7 is a HPLC pattern of ON-080 detected at a wavelength of 200 nm, in which there is an absorption peak of Carvacrol at retention time of 55 min.
  • FIG. 8 is a HPLC pattern of ON-080 detected at a wavelength of 320 nm, in which there are three absorption peaks at retention time of 18-21 min, three absorption peaks at 37-41 min, two absorption peaks at 43-45 min, an absorption peak of Cirsmaritin at 45 min, four absorption peaks at 50-54 min, and an absorption peak of Salvigenin at 62 min.
  • the term “to prevent” or “preventing” shall generally refer to different degrees of stoppage of action or progress. To prevent is to lessen the degree or to stop something effectually, or both.
  • treatment or “treating,” as used herein, refers to improving conditions.
  • therapeutically effective amount refers to the amount of components of the composition or the pharmaceutical composition of the invention alone or in combination with other medicaments that could provide therapeutical benefits in treatment.
  • the present invention provides an extract of Plectranthus amboinicus enriching anti-arthritis activity, which is characterized by having an absorption peak of Cirsmaritin at retention time of about 45 min in a HPLC pattern detected at a wavelength of about 320 nm. This can be obtained by eluting the crude extract using 50% ⁇ 95% alcoholic solution or by a normal phase chromatography. The resulted eluate is different to the known extracts of Plectranthus amboinicus in that it is enriched with anti-arthritis activity and thus capable of treating or preventing arthritis.
  • the extract of Plectranthus amboinicus enriching anti-arthritis activity is isolated by eluting the crude extract as described here using 50% ⁇ 95% alcoholic solution, there is an absorption peak at retention time of about 45 min and an absorption peak of Carvacrol at about 55 min in a HPLC pattern detected at a wavelength of about 200 nm, as shown in FIG. 3 ; there are also 8 absorption peaks at retention time of 39-43 min and an absorption peak of Cirsmaritin at about 45 min in a HPLC pattern detected at a wavelength of about 320 nm, as shown in FIG. 4 .
  • the extract of Plectranthus amboinicus enriching anti-arthritis activity is isolated by eluting the crude extract as described herein using 70% ⁇ 95% alcoholic solution, there is an absorption peak of Cirsmaritin at retention time of about 45 min and an absorption peak of Carvacrol at about 55 min in a HPLC pattern detected at a wavelength of about 200 nm, as shown in FIG. 5 ; there are also one absorption peak of Cirsmaritin at retention time of about 45 min and one absorption peak of Salvigenin at about 62 min in a HPLC pattern detected at a wavelength of about 320 nm, as shown in FIG. 6 .
  • the extract of Plectranthus amboinicus enriching anti-arthritis activity can be isolated by a chromatography, which comprises steps of: extracting the plant or dried powder of Plectranthus amboinicus in a solvent to obtain a crude extract, and eluting the resulted crude extract by a chromatography using 50% ⁇ 95% alcoholic solution to isolate an extract enriching anti-arthritis activity.
  • the crude extract as described herein it is preferable to elute the crude extract as described herein by 70% ⁇ 95% alcoholic solution, or more preferably by about 95% alcoholic solution.
  • the alcoholic solution for elution may be methanol or ethanol. In a preferred embodiment of the instant invention, ethanol is used.
  • a method for preparing an extract of Plectranthus amboinicus enriching anti-arthritis activity comprising extracting the plant or dried powder of Plectranthus amboinicus in a solvent to obtain a crude extract, and eluting the resulted crude extract by a normal phase chromatography using hexane as a first solvent and hexane/ethyl acetate as a second solvent to isolate an extract enriching anti-arthritis activity.
  • the volume ratio of hexane to ethyl acetate in the solvent “hexane/ethyl acetate” is 1:1.
  • Plectranthus amboinicus to be extracted can be in any forms as desired, including but not limited to, a fresh or dried plant. It is preferably to extract the plant in the form of powder.
  • the crude extract of the plant can be obtained by any of the known processes, for example, extracting in a solvent, including but not limited to, alcoholic solution. According to one embodiment of the present invention, about 95% ethanol solution is used.
  • about 95% ethanol solution can be used to obtain a crude extract, and 70%—95% ethanol solution can be further used in a chromatography to elute and isolate an extract enriching anti-arthritis activity.
  • about 95% ethanol solution can be used to obtain a crude extract, and hexane can be further used in a normal phase chromatography to elute and isolate an extract enriching anti-arthritis activity.
  • the extraction process as described herein can be carried out with other appropriate condensation or purification procedures, for example, dry condensation, decompression condensation and freeze-drying.
  • the present invention further provides a method for preventing or treating arthritis comprising administrating to a subject in need thereof an effective amount of the extract as described herein.
  • the present invention also provides a pharmaceutical composition for preventing or treating arthritis comprising a therapeutically effective amount of the extract enriching anti-arthritis activity as described herein and a pharmaceutically acceptable carrier.
  • An effective amount of the extract enriching anti-arthritis activity in a pharmaceutical composition of this invention for treating arthritis is 0.5-10 g/day.
  • Dosage will depend on the nature and states of the symptoms being treated, ages and general physical conditions of the patient being treated, administration route and any therapies practiced previously.
  • carrier or “pharmaceutically acceptable carrier,” as used herein, refers to the diluents, excipients, acceptable agents or the like that are well known by those of ordinary skill in the art and can be used in the preparation of pharmaceutical composition.
  • the composition of the invention can be delivered through any medically acceptable route, such as orally, parentally e.g. intramuscularly, intravenously, subcutaneously, interperitoneally, transdermally, by rectum or inhalation, or vaginal, ocular or nasal routes.
  • the pharmaceutical composition delivered parentally can be in any forms as desired, including but not limited to, solution, suspension, emulsion, and solid injectable composition that is able to dissolve or suspend in a solvent immediately after use.
  • the injectable solution can be prepared by dissolving, suspending, or emulsifying one or more active agents in a diluent.
  • Some examples of the said diluent are distilled water for injection, saline, mineral oils, alcohols, and a combination thereof.
  • the injectable solution can also contain stabilizers, solvents, suspending agents, emulsifying agents, smoothing agents, buffer, preservatives and others.
  • the injectable solution is prepared by sterilization in the final preparation step or by sterile procedures.
  • the pharmaceutical composition of the invention can also be prepared as sterile solid preparations by, for example, freeze-drying, and can be sterilized immediately before use or dissolved in sterile injectable water or other sterile diluents.
  • the pharmaceutical composition of the present invention can also be delivered orally, the form of which can be solid or liquid.
  • Solid compositions include tablets, pills, capsules, powders, granules and the like.
  • Oral compositions also include gargles and lozenges. Capsules include hard and soft capsules.
  • one or more active compounds can be used alone or in combination with diluents, chelating agents, disintegrating agents, lubricants, stabilizers, and cosolvents, to form preparations with known methods subsequently. When needed, such preparations can be coated with a coating agent, or two or more coating agents.
  • oral liquid compositions include pharmaceutically acceptable liquid solutions, suspensions, emulsions, syrups, medicated wine, and the like.
  • one or more active compounds can be dissolved, suspended or emulsified in universal diluent (e.g. purified water, ethanol or a combination thereof). Except for such diluent, the above composition can also contain wetting agents, suspending agent, emulsifiers, sweeteners, flavoring agents, spices, preservatives, buffer and the like.
  • universal diluent e.g. purified water, ethanol or a combination thereof.
  • the above composition can also contain wetting agents, suspending agent, emulsifiers, sweeteners, flavoring agents, spices, preservatives, buffer and the like.
  • the resulted condensed liquor was separated with a HP-20 column Briefly, the condensed liquor mixed thoroughly with RO water was applied into the column, followed by passing through the column RO water of four times of the column volume to collect a first eluate. Then, a second eluate was collected by further passing through the column with a solution composed of RO water of four times of the column volume and 95% ethanol in a ratio of 1:1 (v/v). The second eluate was then condensed under reduced pressure and freeze-dried to produce an extract of Plectranthus amboinicus , designated as ON-024, i.e. a control sample extracted in 0% ⁇ 50% ethanol solution.
  • ON-024 i.e. a control sample extracted in 0% ⁇ 50% ethanol solution.
  • a fourth eluate was collected by subsequently passing through the column with ethyl acetate, after which it was condensed under reduced pressure and freeze-dried to obtain an extract of Plectranthus amboinicus isolated by ethyl acetate.
  • Example 2 The resulted condensed liquor as prepared in Preliminary Extraction of Example 1 was separated with a HP-20 column Briefly, the condensed liquor mixed thoroughly with RO water was applied into the column, followed by passing two times of the column volume of RO water through the column to collect a first eluate. Then, a second eluate was collected by further passing through the column with a solution composed of two times of the column volume of RO water and 95% ethanol in a ratio of 1:1 (v/v). The second eluate was then condensed under reduced pressure and freeze-dried to produce an extract of Plectranthus amboinicus , designated as ON-022.
  • a fourth eluate was collected by subsequently passing through the column with two times of the column volume of ethyl acetate, after which it was condensed under reduced pressure and dried to obtain an extract of Plectranthus amboinicus isolated by ethyl acetate.
  • Example 2 The resulted condensed liquor as prepared in Preliminary Extraction of Example 1 was separated with a HP-20 column Briefly, the condensed liquor mixed thoroughly with RO water was applied into the column, followed by passing through the column with four times of the column volume of RO water to collect a first eluate. Then, a second eluate was collected by further passing through the column with a solution composed of four times of the column volume of RO water and 95% ethanol solution in a ratio of 3:7 (v/v). The second eluate was condensed under reduced pressure and freeze-dried to produce an extract of Plectranthus amboinicus , designated as ON-065.
  • a fourth eluate was collected by subsequently passing through the column with four times of the column volume of ethyl acetate, after which it was condensed under reduced pressure and dried to obtain an extract of Plectranthus amboinicus isolated by ethyl acetate.
  • Silica Gel in a weight equals to that of the dried Plectranthus amboinicus was used to fill the column by use of hexane, after which the filled column was further covered with the CE-gel prepared as described above. Six times of the column volume of hexane was used to elute the column to obtain a first eluate, which was then condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus isolated by hexane, designated as ON-078.
  • a third eluate was collected by subsequently passing through the column with six times of the column volume of ethyl acetate.
  • the resulted third eluate was condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus isolated by ethyl acetate, designated as ON-083.
  • a fourth eluate was collected by further passing through the column with six times of the column volume of ethyl acetate/methanol solution in a ratio of 1:1 (v/v).
  • the resulted fourth eluate was condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus isolated by ethyl acetate/methanol, designated as ON-084.
  • a final eluate was collected by subsequently passing through the column with four times of the column volume of methanol.
  • the resulted final eluate was condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus isolated by methanol, designated as ON-085.
  • a third eluate was collected by subsequently passing through the column with four times of the column volume of hexane/ethyl acetate solution in a ratio of 1:1 (v:v).
  • the resulted third eluate was condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus isolated by hexane/ethyl acetate, designated as ON-092.
  • a final eluate was collected by further passing through the column with six times of the column volume of ethyl acetate.
  • the resulted final eluate was condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus isolated by ethyl acetate, designated as ON-083.
  • a third eluate was collected by subsequently passing through the column with two times of the column volume of hexane/ethyl acetate solution in a ratio of 1:1 (v:v).
  • the resulted third eluate was condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus isolated by hexane/ethyl acetate, designated as ON-081.
  • a fourth eluate was collected by subsequently passing through the column with two times of the column volume of hexane/ethyl acetate solution in a ratio of 1:1 (v:v).
  • the resulted fourth eluate was condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus isolated by hexane/ethyl acetate, designated as ON-082.
  • a fifth eluate was collected by subsequently passing through the column with six times of the column volume of hexane.
  • the resulted fifth eluate was condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus isolated by hexane, designated as ON-083.
  • a sixth eluate was collected by subsequently passing through the column with six times of the column volume of hexane/methanol solution in a ratio of 1:1 (v:v).
  • the resulted sixth eluate was condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus isolated by hexane/methanol, designated as ON-084.
  • a final eluate was collected by further passing through the column with four times of the column volume of methanol.
  • the resulted final eluate was condensed under reduced pressure and dried to obtain an extract of Plectranthus amboinicus isolated by methanol, designated as ON-085.
  • a second eluate was collected by passing through the column with six times of the column volume of hexane/ethyl acetate solution in a ratio of 9:1 (v:v).
  • a third eluate was collected by passing through the column with six times of the column volume of hexane/ethyl acetate solution in a ratio of 1:1 (v:v).
  • the resulted third eluate was condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus isolated by hexane/ethyl acetate, designated as ON-086.
  • a final eluate was collected by subsequently passing through the column with six times of the column volume of hexane/ethyl acetate solution in a ratio of 1:1 (v:v).
  • the resulted final eluate was condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus , designated as ON-088.
  • a final eluate was collected by further passing through the column with six times of the column volume of hexane/ethyl acetate solution in a ratio of 1:1 (v:v).
  • the resulted final eluate was condensed under reduced pressure and dried to produce an extract of Plectranthus amboinicus , designated as ON-090.
  • ON-024 an extract isolated by 0%—50% ethanol solution
  • ON-025 an extract isolated by 50%—95% ethanol solution
  • ON-066 an extract isolated by a 70%—95% ethanol solution
  • ON-080 an extract isolated by a normal phase chromatography
  • the HPLC pattern of ON-024 detected at a wavelength of 200 nm showed six absorption peaks at retention times of 13, 20, 21, 27, 37 and 39 min ( FIG. 1 ); and that detected at a wavelength of 320 nm showed an absorption peak of Caffeic acid at retention time of 13 min, an absorption peak of Rosmarinic acid at 27 min, and an absorption peak at 35 min ( FIG. 2 ).
  • the HPLC pattern of ON-025 detected at a wavelength of 200 nm showed an absorption peak at retention time of 45 min, and an absorption peak of Carvacrol at 55 min ( FIG.
  • CFA Complete Freund's Adjuvant
  • vehicle 1% carboxymethylcellulose, 10 mL/kg
  • test compounds at 500 mg/kg were administered to the animals by oral gavage (PO).
  • PO oral gavage
  • PBMCs peripheral blood mononuclear cells
  • FP Ficoll-Paque
  • FBS heat inactivated fetal bovine serum
  • PHA-induced PBMC proliferation was used as a technical platform for the assessment of anti-inflammation efficacy of various extracts of the present invention.
  • Human PBMCs were co-cultured with PHA at a final concentration of 5 ng/mL and various extracts of Plectranthus amboinicus at a final concentration of 0 ⁇ 90 ng/mL for 3 days.
  • PBMCs treated by dexamethasone (DEX) before stimulation by PHA (5 ng/mL) were also included in the experiment as positive control samples. After cells were incubated for 3 days at 37° C.
  • MTT methyl thiazolyl tetrazolium
  • the extracts of Plectranthus amboinicus provided by the instant invention were all found to be capable of inhibiting the proliferation of lymphocytes induced by PHA in a dose dependent manner.
  • the cytotoxicity of the extracts to cell (without PHA treatment) was also determined by MTT assays.
  • ON-023 40 ng/mL
  • ON-022 40 ng/mL
  • ON-022 40 ng/mL

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US20150079202A1 (en) * 2013-09-03 2015-03-19 ONENESS BIOTECH CO., Ltd Use of patchouli extract in the preparation of compositions with an anti-microorganism effect
US10674928B2 (en) 2014-07-17 2020-06-09 Medtronic, Inc. Leadless pacing system including sensing extension
US10390720B2 (en) 2014-07-17 2019-08-27 Medtronic, Inc. Leadless pacing system including sensing extension
USRE48197E1 (en) 2014-07-25 2020-09-08 Medtronic, Inc. Atrial contraction detection by a ventricular leadless pacing device for atrio-synchronous ventricular pacing
US9399140B2 (en) 2014-07-25 2016-07-26 Medtronic, Inc. Atrial contraction detection by a ventricular leadless pacing device for atrio-synchronous ventricular pacing
US10279168B2 (en) 2014-11-11 2019-05-07 Medtronic, Inc. Leadless pacing device implantation
US9724519B2 (en) 2014-11-11 2017-08-08 Medtronic, Inc. Ventricular leadless pacing device mode switching
US9808628B2 (en) 2014-11-11 2017-11-07 Medtronic, Inc. Mode switching by a ventricular leadless pacing device
US9623234B2 (en) 2014-11-11 2017-04-18 Medtronic, Inc. Leadless pacing device implantation
US9492668B2 (en) 2014-11-11 2016-11-15 Medtronic, Inc. Mode switching by a ventricular leadless pacing device
US9492669B2 (en) 2014-11-11 2016-11-15 Medtronic, Inc. Mode switching by a ventricular leadless pacing device
US9289612B1 (en) 2014-12-11 2016-03-22 Medtronic Inc. Coordination of ventricular pacing in a leadless pacing system
US11207527B2 (en) 2016-07-06 2021-12-28 Cardiac Pacemakers, Inc. Method and system for determining an atrial contraction timing fiducial in a leadless cardiac pacemaker system
WO2023274123A1 (en) * 2021-06-28 2023-01-05 Oneness Biotech Co., Ltd. Methods for preparation of plectranthus amboinicus extracts
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WO2023227087A1 (en) * 2022-05-27 2023-11-30 Oneness Biotech Co., Ltd. Plectranthus amboinicus extract for use in inhibiting immune responses

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