WO2022157810A1 - A synergistic herbal immunomodulatory formulation - Google Patents

A synergistic herbal immunomodulatory formulation Download PDF

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Publication number
WO2022157810A1
WO2022157810A1 PCT/IN2022/050058 IN2022050058W WO2022157810A1 WO 2022157810 A1 WO2022157810 A1 WO 2022157810A1 IN 2022050058 W IN2022050058 W IN 2022050058W WO 2022157810 A1 WO2022157810 A1 WO 2022157810A1
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Prior art keywords
formulation
cell lines
herbal formulation
powder
present
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PCT/IN2022/050058
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French (fr)
Inventor
Girish Kr Gupta
Suresh Garg
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Zeon Lifesciences Limited
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Publication of WO2022157810A1 publication Critical patent/WO2022157810A1/en

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    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • 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
    • A61K36/13—Coniferophyta (gymnosperms)
    • A61K36/15—Pinaceae (Pine family), e.g. pine or cedar
    • 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)
    • 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)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger

Definitions

  • the present invention relates to a synergistic herbal immunomodulatory formulation. More specifically the present invention relates to the synergistic efficacy of the combination of herbs fermented sea buckthorn pulp powder, nanosolubilized curcuminoid powder(20%), and pine bark extract (95%) in the desired amount characterized for a multi-component approach indicated for immunomodulatory activity.
  • SARS-CoV-2 The novel coronavirus SARS-CoV-2, commonly known as covid-19 has had devastating medical, social, and economic impacts worldwide. Patients with COVID-19 can present with acute symptoms of fever, dyspnea, pneumonia, acute respiratory distress syndrome (ARDS), and multi-system failure due to cytokine release syndrome (CRS).
  • ARDS acute respiratory distress syndrome
  • CRS cytokine release syndrome
  • hydroxychloroquine and/or antiviral drugs is being used to cure the patient suffering from covid-19.
  • treatment of SARS-CoV-2 using pharmaceutical agents is often limited by reduced bioavailability, lack of tissue specificity, and tissue toxicity of the therapeutic agent. Many of the researchers are rigorously involved in the development of a vaccine for the same.
  • Curcumin is a yellow polyphenol extracted from the rhizome of turmeric, which has shown good activity as adjuvant therapy in the treatment of patients with COVID-19. Curcumin has potential antiviral effects, including protein binding affinity towards SARS-CoV-2 proteins, which is comparable to that of drugs such as hydroxychloroquine that have been considered for clinical trials in the treatment of COVID-19. Curcumin exhibits very poor bioavailability, with very low or undetectable concentrations in blood and extraintestinal tissues. Therefore, to enhance the bioavailability of curcumin, several approaches have been taken including curcumin nanoparticles.
  • Seabuckthorn is a homologous plant of medicine and food. Seabuckthorn fruit is rich in nutrients and biologically active substances. Seabuckthorn fruit is used as a medicine to relieve cough and phlegm, strengthen the stomach and digest food, and promote blood circulation and disperse phlegm. In modern medical research, sea buckthorn can lower cholesterol, relieve angina pectoris, and prevent coronary atherosclerotic heart disease.
  • Pine bark (Pinus species) is a herb, rich in several bioflavonoids having both antiinflammatory and antioxidant effects. Research has shown that pine bark extract may result in the improvement in the symptoms of osteoarthritis and can be used in pharmaceutical and cosmetic products. Today, pine bark extracts and other OPCs products (especially grape seed extracts) are widely consumed as food ingredients or dietary supplements. Extensive research conducted with several formulations of pine bark extracts has established its safety and tolerability for human consumption.
  • US6187297B1 discloses a composition that includes sea buckthorn oil that is used a s a cosmetic product, a pharmaceutical product or a food product.’
  • EP2517716A1 discloses oral compositions having at least two botanical active ingredients derived from plants.
  • the composition contains at least two herbal active ingredients out of which one is pine.
  • US8017147B2 discloses a synergistic mixture (which may be utilized as a food or a drink or a supplement or a drug or a cosmetic or a hygienic product) that is formulated and is capable of improving a person's well being, lowering the risks of cardiovascular and/or Alzheimer's diseases and/or lowering blood sugar using natural and synthetic ingredients. Numerous ratios may be formulated for aroma, color, flavor, flow (viscosity), taste, and uniformity. Moreover, ingredients for sugar substitutes, natural preservatives, nano-dispersion, nano-emulsion, nano- encapsulation of ingredients, and apparatus for personalized nutrition are also described herein.
  • CN1568945 A discloses the invention discloses a novel use of curcumin, i.e. the pharmaceutical use of curcumin in preparing medicament for treating and preventing severe acute respiratory syndrome (SARS).
  • SARS severe acute respiratory syndrome
  • the main objective of the present invention is to provide a synergistic herbal formulation used as an immunomodulatory agent.
  • Yet another objective of the present invention is to elucidate the synergistic action using the combination of herbs, fermented sea buckthorn pulp powder, nanosolubilized curcuminoid powder(20%), and pine bark extract (95%)(95%).
  • Yet another objective of the present invention is to provide a nanosolubilized Curcuminoid(20%) formulation with increase bioavailability compared to compare to Curcumin Extract.
  • Yet another objective of the present invention is to provide an anti-viral herbal formulation.
  • Yet another objective of the present invention is to provide a herbal formulation that will affect to the infective mechanism of the SARS-COV2 Virus.
  • the present invention describes a herbal formulation for the management of immunomodulatory activity acting synergistically in vitro and in-vivo.
  • the herbal formulation comprising of a fermented sea buckthorn pulp powder in the concentration range of 25 mg to 100 mg, a nano-solubilized curcuminoid powder(20%) in the concentration range of 125 mg to 500 mg, a pine bark extract (95%) in the concentration range of 25 to lOOmg, and a pharmaceutically accepted excipient.
  • the nano-solubilized curcuminoid powder (20%) is developed using solid dispersion which is followed by spray drying to get a homogeneous powder.
  • the herbal formulation has been evaluated in vitro using cell lines, wherein the cell lines are mammalian cell lines.
  • the cell types for the desired cell lines include, but are not limited to, dendritic cells, macrophages, and B cells.
  • the pharmaceutically accepted excipient includes, but is not limited to microcrystalline cellulose, magnesium stearate, colloidal silicon dioxide, talc, and stearic acid.
  • the present invention relates to a herbal formulation, wherein, the formulation is useful as an immunomodulatory agent having the fermented sea buckthorn pulp powder, the nano-solubilized curcuminoid powder(20%), and the pine bark extract (95%), all are mixed by either slugging and/or dry blending along with the pharmaceutically accepted excipient and nano solubilization method.
  • the formulation provides a synergistic effect to modulate immunity markers and phagocytic index on the selected cell lines.
  • Various in-vivo and in- vitro studies are also conducted to get the desired results as claimed in the present invention.
  • the herbal formulation in the present invention is administered through an oral administration means that includes, but is not limited to, a pill, tablet, capsule, concentrated syrup, food additive, suspension, emulsion, novel drug delivery system, nanoemulsion and a chewable solid.
  • the main advantage of the present invention is to provide a synergistic herbal formulation used as an immunomodulatory agent.
  • Yet another advantage of the present invention is to elucidate the synergistic action using the combination of herbs, fermented sea buckthorn pulp powder, nano-solubilized curcuminoid powder(20%), and pine bark extract (95%).
  • Yet another advantage of the present invention is to provide a nanosolubilized Curcuminoid(20%) formulation with increase bioavailability compared to compare to Curcumin Extract.
  • Yet another advantage of the present invention is to provide a simple and cost- effective solution for a variety of immunomodulatory caused ailments and conditions.
  • Yet another advantage of the present invention is to provide an anti-viral herbal formulation.
  • Yet another advantage of the present invention is to provide a herbal formulation that will affect to the infective mechanism of the SARS-COV2 Virus
  • the term “a” or “an”, as used herein, is defined as one.
  • the term “plurality”, as used herein, is defined as two as or more than one.
  • the term “another”, as used herein, is defined as at least a second or more.
  • the terms “including” and/or “having”, as used herein, are defined as comprising (i.e., open language).
  • the present invention describes a herbal formulation for the management of immunomodulatory activity acting synergistically in vitro.
  • the herbal formulation comprising of a fermented sea buckthorn pulp powder in the concentration range of 25 mg to 100 mg, a nano-solubilized curcuminoid powder(20%) in the concentration range of 125 mg to 500 mg, a pine bark extract (95%) in the concentration range of 25 to lOOmg, and an at least one pharmaceutically accepted excipient.
  • the nano-solubilized curcuminoid powder (20%) is developed using solid dispersion which is followed by spray drying to get a homogeneous free flow powder.
  • the herbal formulation has been evaluated in vitro using cell lines, wherein the cell lines are mammalian cell lines.
  • the cell types for the desired cell lines include, but are not limited to dendritic cells, macrophages, and B cells.
  • the pharmaceutically accepted excipient includes, but is not limited to microcrystalline cellulose, magnesium stearate, colloidal silicon dioxide, talc, and stearic acid.
  • the present invention relates to a herbal formulation, wherein, the formulation is useful as an immunomodulatory agent having the fermented sea buckthorn pulp powder, the nano-solubilized curcuminoid powder(20%), and the pine bark extract (95%), all are mixed by either slugging and/or dry blending along with the pharmaceutically accepted excipient.
  • the formulation provides a synergistic effect to modulate immunity markers and phagocytic index on the selected cell lines.
  • the ingredients of the formulation can be processed into forms having varying delivery systems.
  • the ingredients can be processed and included in capsules, tablets, gel tabs, novel drug delivery systems, lozenges, strips, granules, powders, concentrates, solutions, lotions, creams, or suspensions.
  • the ingredients can be formulated, individually or in combination, with other foods to provide premeasured nutritional supplements, supplemented foods, for example, singleserving bars.
  • the formulation is administered in capsule form.
  • the method for the manufacturing of the formulation includes mixing of all the ingredients by either slugging and/or dry blending and then filled in the capsule formulation.
  • the dosages of the formulation may be administered in increments determined by the disease or condition being treated.
  • the combination of all the three natural ingredients having different therapeutic properties with implying nanotechnology improve the bioavailability/efficacy that opens up the solution for prophylaxis as well as an improved therapeutic regime for SARS virus.
  • the present invention is used to evaluate the immune-boosting properties using in vitro techniques.
  • the in vitro cytotoxicity is performed for the test substances on respective cell lines in order to find toxic concentrations of the test substance and to evaluate the immune-boosting properties through checking the modulatory effect of test products on Immune markers and phagocytic index.
  • the test system for the present invention is Dendritic Cell lines
  • Cell lines will be cultured in DMEM media supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (lOOIU/ml), streptomycin (lOOg/ml), and amphotericin B (5g/ml) in a humidified atmosphere of 5% CO2 at 37 DC until confluent.
  • the cells will be dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS).
  • the stock cultures will be grown in 25 cm2 culture flasks and all experiments will be carried out in 96 micro-titer plates.
  • Each weighed test drug will be separately dissolved and volume will be made up with DMEM supplemented with 2% inactivated FBS to obtain a stock solution of 1 mg/ml concentration and sterilized by filtration. Serial two-fold dilutions will be done from this to perform cytotoxic studies.
  • Dendritic cells from C57 mice will be prepared using a standard protocol.
  • Cells will be cultured in 90-mm culture Petri dishes (2* 106 cells/10 ml) with growth medium [RPMI 1640 medium supplemented with 10% FBS, lOOU/ml penicillin, 100 pg/ml streptomycin, and 20 ng/ml rm GMCSF] for 6 days at 37 °C in a humidified 5% CO2 atmosphere and Viable cells to be counted using trypan blue exclusion method.
  • cytokines TNF-a, IL-l-p, and MIP-l-a
  • DCs 1.6 x 104 cells/well in 24-well culture plate
  • test formulations at selected non-cytotoxic concentrations in triplicates.
  • DCs treated with 0.25% DMSO will be included as control cells.
  • cell-free culture supernatants will be analyzed for secreted levels of TNF-a, IL-l-P, and MIP-l-a by ELISA.
  • LPS E. coli.
  • 10 ng/ml will be included as a positive control.
  • the modulatory effect of the test substance on selected immune markers and phagocytic index in treated cells over the control group will be determined and expressed as a respective modulatory effect over control.
  • the observed results will be presented in suitable tabular and graphical formats.
  • a bacterial Reverse Mutation Test is conducted to assess the potential of the test substance to induce point gene mutations, viz., substitution, addition or deletion of one or a few DNA base pairs in the Salmonella typhimurium.
  • the 4 strains of Salmonella typhimurium are used:TA 98,TA100,TA102,TA1535 and TAI 537.
  • the study was conducted with and without metabolic activator (S-9 fraction). Two methods were carried out - direct plate incorporation method and pre incubation method.
  • the test product was tested at the concentration of 100, 266, 707, 1880 and 5000 pg/plate by direct plate incorporation method and 50, 158, 500, 1581 and 5000 pg/plate by preincubation method using DMSO as solvent.
  • RESULTS Mean numbers of revertant colonies counted in the above mentioned concentrations did not increase significantly over the solvent control in both the methods in the presence and absence of metabolic activator. The revertant colonies observed were within the limits of established number of spontaneous revertants. The number of revertant colonies/plate in the positive controls/mutagens increased by 4.5 to 34.8 and 4.4 to 36.4 fold in method 1 and method 2 respectively under test conditions. The test substance is not mutagenic in Bacterial reverse mutation test at the tested concentrations under the test conditions.
  • test substance is not mutagenic in Bacterial reverse mutation test at the tested concentrations under the test conditions
  • Rats were administered a single dose of 2000 mg/kg orally and then observed individually for the first four hours, then over a period of 24 hours and once daily for 14 days.
  • the median lethal dose of test substance in female rats after single oral treatment is above 2000 mg/kg body weight and is classified as Category 5 and Safe.
  • mice which were divided into six groups.
  • test product showed non-significant phagocytic activity in a dosedependent manner when compared to the positive control.
  • the high Dose of 500 mg/kg of the test product showed better enhancing of carbon clearance than reference standard group.

Abstract

The present invention describes a herbal formulation for the management of immunomodulatory activity acting synergistically in vitro. The herbal formulation comprising of a fermented sea buckthorn pulp powder in the concentration range of 25 mg to 100 mg, a nano-solubilized curcuminoid powder(20%) in the concentration range of 125 mg to 500 mg, a pine bark extract (95%) in the concentration range of 25 to 100mg, and an at least one pharmaceutically accepted excipient. In an embodiment of the present invention, the herbal formulation has been evaluated in vitro using cell lines, wherein the cell lines are mammalian cell lines. In another aspect of the invention, the cell types for the desired cell lines are selected from, but not limited to dendritic cells, macrophages, and B cells.

Description

A SYNERGISTIC HERBAL IMMUNOMODULATORY FORMULATION
FIELD OF THE INVENTION
The present invention relates to a synergistic herbal immunomodulatory formulation. More specifically the present invention relates to the synergistic efficacy of the combination of herbs fermented sea buckthorn pulp powder, nanosolubilized curcuminoid powder(20%), and pine bark extract (95%) in the desired amount characterized for a multi-component approach indicated for immunomodulatory activity.
BACKGROUND OF THE INVENTION
The novel coronavirus SARS-CoV-2, commonly known as covid-19 has had devastating medical, social, and economic impacts worldwide. Patients with COVID-19 can present with acute symptoms of fever, dyspnea, pneumonia, acute respiratory distress syndrome (ARDS), and multi-system failure due to cytokine release syndrome (CRS). The use of either hydroxychloroquine and/or antiviral drugs is being used to cure the patient suffering from covid-19. Unfortunately, treatment of SARS-CoV-2 using pharmaceutical agents is often limited by reduced bioavailability, lack of tissue specificity, and tissue toxicity of the therapeutic agent. Many of the researchers are rigorously involved in the development of a vaccine for the same. Many subjects in the trials seem to have a limited benefit from vaccination, which is active in developing immunity, particularly in young subjects because their immune response is very effective whereas, in elderly subjects, the immune response seems to be very limited, and therefore, vaccination in these subjects seems to have a minor effect and benefit
Curcumin is a yellow polyphenol extracted from the rhizome of turmeric, which has shown good activity as adjuvant therapy in the treatment of patients with COVID-19. Curcumin has potential antiviral effects, including protein binding affinity towards SARS-CoV-2 proteins, which is comparable to that of drugs such as hydroxychloroquine that have been considered for clinical trials in the treatment of COVID-19. Curcumin exhibits very poor bioavailability, with very low or undetectable concentrations in blood and extraintestinal tissues. Therefore, to enhance the bioavailability of curcumin, several approaches have been taken including curcumin nanoparticles.
Seabuckthorn is a homologous plant of medicine and food. Seabuckthorn fruit is rich in nutrients and biologically active substances. Seabuckthorn fruit is used as a medicine to relieve cough and phlegm, strengthen the stomach and digest food, and promote blood circulation and disperse phlegm. In modern medical research, sea buckthorn can lower cholesterol, relieve angina pectoris, and prevent coronary atherosclerotic heart disease.
Pine bark (Pinus species) is a herb, rich in several bioflavonoids having both antiinflammatory and antioxidant effects. Research has shown that pine bark extract may result in the improvement in the symptoms of osteoarthritis and can be used in pharmaceutical and cosmetic products. Today, pine bark extracts and other OPCs products (especially grape seed extracts) are widely consumed as food ingredients or dietary supplements. Extensive research conducted with several formulations of pine bark extracts has established its safety and tolerability for human consumption.
US6187297B1 discloses a composition that includes sea buckthorn oil that is used a s a cosmetic product, a pharmaceutical product or a food product.’
EP2517716A1 discloses oral compositions having at least two botanical active ingredients derived from plants. The composition contains at least two herbal active ingredients out of which one is pine.
US8017147B2 discloses a synergistic mixture (which may be utilized as a food or a drink or a supplement or a drug or a cosmetic or a hygienic product) that is formulated and is capable of improving a person's well being, lowering the risks of cardiovascular and/or Alzheimer's diseases and/or lowering blood sugar using natural and synthetic ingredients. Numerous ratios may be formulated for aroma, color, flavor, flow (viscosity), taste, and uniformity. Moreover, ingredients for sugar substitutes, natural preservatives, nano-dispersion, nano-emulsion, nano- encapsulation of ingredients, and apparatus for personalized nutrition are also described herein.
CN1568945 A discloses the invention discloses a novel use of curcumin, i.e. the pharmaceutical use of curcumin in preparing medicament for treating and preventing severe acute respiratory syndrome (SARS).
The above mention prior art applications teach a composition including one or more of the constituent ingredients of the present invention. However, none of them teaches the entire combination of constituent ingredients did not set forth herein nor do any of them teach the features, aspects and purposes of the present invention. Thus, The need for the present invention arises as none of the existing inventions have been characterized for the multi-component approach related to a variety of immunomodulatory based conditions, symptoms, and diseases.
OBJECTIVE OF THE INVENTION
The main objective of the present invention is to provide a synergistic herbal formulation used as an immunomodulatory agent.
Yet another objective of the present invention is to elucidate the synergistic action using the combination of herbs, fermented sea buckthorn pulp powder, nanosolubilized curcuminoid powder(20%), and pine bark extract (95%)(95%).
Yet another objective of the present invention is to provide a nanosolubilized Curcuminoid(20%) formulation with increase bioavailability compared to compare to Curcumin Extract.
Yet another objective of the present invention is to provide a simple and cost- effective solution for a variety of immunomodulatory caused ailments and conditions. Yet another objective of the present invention is to provide a relatively stable, and palatable formulation that is able to be used as a multipurpose nutritional supplement and a food product.
Yet another objective of the present invention is to provide an anti-viral herbal formulation.
Yet another objective of the present invention is to provide a herbal formulation that will affect to the infective mechanism of the SARS-COV2 Virus.
Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided hereinbelow, in which various embodiments of the disclosed invention are illustrated by way of example.
SUMMARY OF THE INVENTION
The present invention describes a herbal formulation for the management of immunomodulatory activity acting synergistically in vitro and in-vivo. The herbal formulation comprising of a fermented sea buckthorn pulp powder in the concentration range of 25 mg to 100 mg, a nano-solubilized curcuminoid powder(20%) in the concentration range of 125 mg to 500 mg, a pine bark extract (95%) in the concentration range of 25 to lOOmg, and a pharmaceutically accepted excipient. Herein, the nano-solubilized curcuminoid powder (20%) is developed using solid dispersion which is followed by spray drying to get a homogeneous powder. In an embodiment of the present invention, the herbal formulation has been evaluated in vitro using cell lines, wherein the cell lines are mammalian cell lines. In another aspect of the invention, the cell types for the desired cell lines include, but are not limited to, dendritic cells, macrophages, and B cells. In an embodiment of the present invention, the pharmaceutically accepted excipient includes, but is not limited to microcrystalline cellulose, magnesium stearate, colloidal silicon dioxide, talc, and stearic acid. The present invention relates to a herbal formulation, wherein, the formulation is useful as an immunomodulatory agent having the fermented sea buckthorn pulp powder, the nano-solubilized curcuminoid powder(20%), and the pine bark extract (95%), all are mixed by either slugging and/or dry blending along with the pharmaceutically accepted excipient and nano solubilization method. In the preferred embodiment of the invention, the formulation provides a synergistic effect to modulate immunity markers and phagocytic index on the selected cell lines. Various in-vivo and in- vitro studies are also conducted to get the desired results as claimed in the present invention. The herbal formulation in the present invention is administered through an oral administration means that includes, but is not limited to, a pill, tablet, capsule, concentrated syrup, food additive, suspension, emulsion, novel drug delivery system, nanoemulsion and a chewable solid.
The main advantage of the present invention is to provide a synergistic herbal formulation used as an immunomodulatory agent.
Yet another advantage of the present invention is to elucidate the synergistic action using the combination of herbs, fermented sea buckthorn pulp powder, nano-solubilized curcuminoid powder(20%), and pine bark extract (95%).
Yet another advantage of the present invention is to provide a nanosolubilized Curcuminoid(20%) formulation with increase bioavailability compared to compare to Curcumin Extract.
Yet another advantage of the present invention is to provide a simple and cost- effective solution for a variety of immunomodulatory caused ailments and conditions.
Yet another advantage of the present invention is to provide a relatively stable and palatable formulation that can be used as a multipurpose nutritional supplement
Yet another advantage of the present invention is to provide an anti-viral herbal formulation.
Yet another advantage of the present invention is to provide a herbal formulation that will affect to the infective mechanism of the SARS-COV2 Virus Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided hereinbelow, in which various embodiments of the disclosed invention are illustrated by way of example
DETAILED DESCRIPTION OF THE INVENTION
Definition
The term “a” or “an”, as used herein, is defined as one. The term “plurality”, as used herein, is defined as two as or more than one. The term “another”, as used herein, is defined as at least a second or more. The terms “including” and/or “having”, as used herein, are defined as comprising (i.e., open language).
The term “comprising” is not intended to limit the present invention with such terminology rather is used in a wider sense. Any invention using the term comprising could be separated into one or more claims using “consisting” or “consisting of’. The term “comprising” may be used interchangeably with the terms “having” or “containing”.
Reference in this document to “one embodiment”, “certain embodiments”, “an embodiment”, “another embodiment”, and “yet another embodiment” or similar terms, throughout the document means that a specific feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases in various places, this specification throughout are not necessarily all referring to the same embodiment. Furthermore, the specific features, structures, or characteristics are combined in any suitable manner in one or more embodiments without limitation.
The term “or” as used herein is to be interpreted as inclusive or meaning any one or more combinations. Therefore, “A, B or C” means any of the following: “A; B; C; A and B; A and C; B and C; A, B and C”. An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in mutually exclusive, inherently. As used herein, the term "one or more" generally refers to, but is not limited to, singular as well as the plural form of the term.
The drawings featured in the figures are to illustrate certain convenient embodiments of the present invention and are not to be considered as a limitation to that. Term "means" preceding a present participle of operation indicates the desired function for which there is one or more embodiments, i.e., one or more methods, for achieving the desired function and that one skilled in the art could select from these or their equivalent in view of the disclosure herein and use of the term "means" is not intended to be limiting.
The present invention describes a herbal formulation for the management of immunomodulatory activity acting synergistically in vitro. The herbal formulation comprising of a fermented sea buckthorn pulp powder in the concentration range of 25 mg to 100 mg, a nano-solubilized curcuminoid powder(20%) in the concentration range of 125 mg to 500 mg, a pine bark extract (95%) in the concentration range of 25 to lOOmg, and an at least one pharmaceutically accepted excipient. Herein, the nano-solubilized curcuminoid powder (20%) is developed using solid dispersion which is followed by spray drying to get a homogeneous free flow powder.
In an embodiment of the present invention, the herbal formulation has been evaluated in vitro using cell lines, wherein the cell lines are mammalian cell lines. In another aspect of the invention, the cell types for the desired cell lines include, but are not limited to dendritic cells, macrophages, and B cells.
In an embodiment of the present invention, the pharmaceutically accepted excipient includes, but is not limited to microcrystalline cellulose, magnesium stearate, colloidal silicon dioxide, talc, and stearic acid.
The present invention relates to a herbal formulation, wherein, the formulation is useful as an immunomodulatory agent having the fermented sea buckthorn pulp powder, the nano-solubilized curcuminoid powder(20%), and the pine bark extract (95%), all are mixed by either slugging and/or dry blending along with the pharmaceutically accepted excipient.
In the preferred embodiment of the invention, the formulation provides a synergistic effect to modulate immunity markers and phagocytic index on the selected cell lines.
In another preferred embodiment of the invention, the ingredients of the formulation can be processed into forms having varying delivery systems. For example, the ingredients can be processed and included in capsules, tablets, gel tabs, novel drug delivery systems, lozenges, strips, granules, powders, concentrates, solutions, lotions, creams, or suspensions. The ingredients can be formulated, individually or in combination, with other foods to provide premeasured nutritional supplements, supplemented foods, for example, singleserving bars. In one example, the formulation is administered in capsule form.
In an embodiment of the present invention, the method for the manufacturing of the formulation includes mixing of all the ingredients by either slugging and/or dry blending and then filled in the capsule formulation. In another aspect of the invention, the dosages of the formulation may be administered in increments determined by the disease or condition being treated.
In yet another aspect of the invention, the combination of all the three natural ingredients having different therapeutic properties with implying nanotechnology improve the bioavailability/efficacy that opens up the solution for prophylaxis as well as an improved therapeutic regime for SARS virus.
There are two types of studies that were conducted to get the desired results as claimed in the present invention. The two types of studies are :
1. In vitro Studies
2. In- vivo Studies The present invention is used to evaluate the immune-boosting properties using in vitro techniques.
Experiment No.l
The in vitro cytotoxicity is performed for the test substances on respective cell lines in order to find toxic concentrations of the test substance and to evaluate the immune-boosting properties through checking the modulatory effect of test products on Immune markers and phagocytic index. The test system for the present invention is Dendritic Cell lines
Test culture preparation
Cell lines will be cultured in DMEM media supplemented with 10% inactivated Fetal Bovine Serum (FBS), penicillin (lOOIU/ml), streptomycin (lOOg/ml), and amphotericin B (5g/ml) in a humidified atmosphere of 5% CO2 at 37 DC until confluent. The cells will be dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS). The stock cultures will be grown in 25 cm2 culture flasks and all experiments will be carried out in 96 micro-titer plates.
Test solution Preparation
Each weighed test drug will be separately dissolved and volume will be made up with DMEM supplemented with 2% inactivated FBS to obtain a stock solution of 1 mg/ml concentration and sterilized by filtration. Serial two-fold dilutions will be done from this to perform cytotoxic studies.
IMMUNOMODULATORY ACTIVITY IN DENDRITIC CELLS
Dendritic cells from C57 mice will be prepared using a standard protocol. Cells will be cultured in 90-mm culture Petri dishes (2* 106 cells/10 ml) with growth medium [RPMI 1640 medium supplemented with 10% FBS, lOOU/ml penicillin, 100 pg/ml streptomycin, and 20 ng/ml rm GMCSF] for 6 days at 37 °C in a humidified 5% CO2 atmosphere and Viable cells to be counted using trypan blue exclusion method. Estimation of cytokines (TNF-a, IL-l-p, and MIP-l-a) in DCs
For estimation of cytokines activity, DCs (1.6 x 104 cells/well in 24-well culture plate) will be treated with test formulations at selected non-cytotoxic concentrations in triplicates. DCs treated with 0.25% DMSO will be included as control cells. After 24 h of incubation, cell-free culture supernatants will be analyzed for secreted levels of TNF-a, IL-l-P, and MIP-l-a by ELISA. LPS (E. coli.) (10 ng/ml) will be included as a positive control.
OBSERVATIONS
The modulatory effect of the test substance on selected immune markers and phagocytic index in treated cells over the control group will be determined and expressed as a respective modulatory effect over control. The observed results will be presented in suitable tabular and graphical formats.
Experiment No.2
A bacterial Reverse Mutation Test is conducted to assess the potential of the test substance to induce point gene mutations, viz., substitution, addition or deletion of one or a few DNA base pairs in the Salmonella typhimurium.
PROCEDURE
The 4 strains of Salmonella typhimurium are used:TA 98,TA100,TA102,TA1535 and TAI 537. The study was conducted with and without metabolic activator (S-9 fraction). Two methods were carried out - direct plate incorporation method and pre incubation method. The test product was tested at the concentration of 100, 266, 707, 1880 and 5000 pg/plate by direct plate incorporation method and 50, 158, 500, 1581 and 5000 pg/plate by preincubation method using DMSO as solvent.
RESULTS Mean numbers of revertant colonies counted in the above mentioned concentrations did not increase significantly over the solvent control in both the methods in the presence and absence of metabolic activator. The revertant colonies observed were within the limits of established number of spontaneous revertants. The number of revertant colonies/plate in the positive controls/mutagens increased by 4.5 to 34.8 and 4.4 to 36.4 fold in method 1 and method 2 respectively under test conditions. The test substance is not mutagenic in Bacterial reverse mutation test at the tested concentrations under the test conditions.
CONCLUSION
The test substance is not mutagenic in Bacterial reverse mutation test at the tested concentrations under the test conditions
Summary table of mean ± SD number of spontaneous revertant colony counts for Solvent control & test Substance (5000 pg) group in both the methods employed.
Figure imgf000012_0001
Experiment No.3
In the in-vivo studies, an Acute Oral Toxicity Study in Wistar Rats is conducted to assess the toxicity/safety of the test substance by acute oral administration in adult healthy female wistar albino rats.
Procedure
1. Rats were administered a single dose of 2000 mg/kg orally and then observed individually for the first four hours, then over a period of 24 hours and once daily for 14 days.
2. General behavior, adverse effects and mortality were observed throughout the experimental period. Body weights were recorded on test day 0 (prior to administration), day 3, day 7 and day 14.
3. Necropsy was done for all the animals and examined macroscopically.
Results
• No abnormalities were detected for the animals necropsied at terminal sacrifice.
• The limit test did not cause any mortality or signs of toxicity in the rats tested during the observation period.
Conclusion
The median lethal dose of test substance in female rats after single oral treatment is above 2000 mg/kg body weight and is classified as Category 5 and Safe.
Experiment No.4
An Immunomodulatory Activity of the SCP Blend by Carbon Clearance Assay in Swiss Albino Mice is conducted to evaluate the immunomodulatory activity of SCP -Blend in experimental mice. Procedure
The experiment was conducted on the mice which were divided into six groups.
Each group was given treatment except normal control. At the end of last dose after the 48 hours of interval the colloidal carbon ink were injected to the mice through the tail vein. The blood samples were collected at “0”hours and “15” minutes and were analyzed at 660nm.The absorbance was recorded and the rate of carbon clearance was assessed and reported.
Results
The 3 doses of test product showed non-significant phagocytic activity in a dosedependent manner when compared to the positive control. The high Dose of 500 mg/kg of the test product showed better enhancing of carbon clearance than reference standard group.
Conclusion
Test product enhanced the rate of carbon clearance (Phagocytic activity) which was comparable to the reference standard.
Figure imgf000014_0001
Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided herein, in which various embodiments of the disclosed present invention are illustrated by way of example and appropriate reference to accompanying drawings. Those skilled in the art to which the present invention pertains may make modifications resulting in other embodiments employing principles of the present invention without departing from its spirit or characteristics, particularly upon considering the foregoing teachings. Accordingly, the described embodiments are to be considered in all respects only as illustrative, and not restrictive, and the scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description or drawings. Consequently, while the present invention has been described with reference to particular embodiments, modifications of structure, sequence, materials and the like apparent to those skilled in the art still fall within the scope of the invention as claimed by the applicant.

Claims

I/WE CLAIM
1. A herbal formulation for the management of immunomodulatory activity acting synergistically in vitro comprising of a fermented sea buckthorn pulp powder in the concentration range of 25 mg to 100 mg; a nano-solubilized curcuminoid powder(20%) in the concentration range of 125 mg to 500 mg; a pine bark extract (95%) in the concentration range of 25 to lOOmg; and an atleast one pharmaceutically accepted excipient; wherein, the nano-solubilized curcuminoid powder(20 %) is develop using solid dispersion which is followed by spray drying to get a homogeneous free flow powder.
2. The herbal formulation as claimed in claim 1, has been evaluated in-vitro using cell lines, wherein the cell lines are mammalian cell lines.
3. The cell lines as claimed in claim 2, wherein the cell types are selected from, but not limited to dendritic cells, macrophages, and B cells.
4. The herbal formulation as claimed in claim 1, wherein, atleast one pharmaceutically accepted excipient is selected from, but not limited to microcrystalline cellulose, magnesium stearate, colloidal silicon dioxide, talc, and stearic acid.
5. The herbal formulation as claimed in claim 1, wherein, the formulation is useful as an immunomodulatory agent comprising of the fermented sea buckthorn pulp powder, the nano-solubilized curcuminoid powder, and the pine bark extract (95%), all are mixed by either slugging and/or dry blending along with atleast one pharmaceutically accepted excipient.
6. The formulation as claimed in claim 1, provide synergistic effects to modulate immunity markers and phagocytic index on the treated cell lines.
7. The herbal formulation as claimed in claim 1, wherein, the formulation is administered through an oral administration that is selected from, but not limited to, a pill, tablet, capsule, concentrated syrup, food additive, suspension, emulsion, novel drug delivery system, nanoemulsion and a chewable solid. The herbal formulation as claimed in claim 1, wherein the herbal formulation is stated as a nutritional Supplement.
PCT/IN2022/050058 2021-01-25 2022-01-25 A synergistic herbal immunomodulatory formulation WO2022157810A1 (en)

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WO2009002298A1 (en) * 2007-06-22 2008-12-31 Supranaturals, Llc Antioxidant drink for dietary supplementation
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WO2009002298A1 (en) * 2007-06-22 2008-12-31 Supranaturals, Llc Antioxidant drink for dietary supplementation
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