Claims:WE CLAIM:
1. A polyherbal formulation comprising;
(a) an aqueous extract of Ocimum sanctum in an amount in the range of 20 wt.% to 30 wt.% with respect to the total weight of the formulation, wherein said aqueous extract of Ocimum sanctum has at least 2% of polyphenols;
(b) an aqueous extract of Tinospora cordifolia in an amount in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, wherein said aqueous extract of Tinospora cordifolia has polyphenols is in the range of 0.1 to 1%;
(c) a hydroalcoholic extract of Glycyrrhiza glabra in an amount in the range of 5 wt.% to 15 wt.% with respect to the total weight of the formulation, wherein said hydroalcoholic extract of Glycyrrhiza glabra has at least 25% of glycyrrhizinic acid and has glabridin is in the range of 0.2% to 1%;
(d) a hydroalcoholic extract of Withania somnifera in an amount in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, wherein said hydroalcoholic extract of Withania somnifera has at least 1% Withanolide-IV of and at least 0.2% of Withanolide A;
(e) a hydroalcoholic extract of Trigonella foenum in an amount in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, wherein said hydroalcoholic extract of Trigonella foenum has at least 5% of polyphenols; and
(f) an alcoholic extract of Andrographis paniculata in an amount in the range of 15 wt.% to 25 wt.% with respect to the total weight of the formulation, wherein said alcoholic extract of Andrographis paniculata has at least 20% of andrographolide.

2. The polyherbal formulation as claimed in claim 1, wherein said hydroalcoholic extract is selected from the methanolic extract and ethanolic extract.

3. The polyherbal formulation as claimed in claim 1, wherein said hydroalcoholic extract of Glycyrrhiza glabra is 30% methanolic extract, wherein said hydroalcoholic extract of Trigonella foenum is 75% ethanolic extract, wherein said hydroalcoholic extract of Withania somnifera is 70% ethanolic extract and wherein said alcoholic extract of Andrographis paniculata is 95% ethanolic extract.
4. The polyherbal formulation as claimed in claim 1, said formulation comprises:
(a) an aqueous extract of Ocimum sanctum in an amount in the range of 22 wt.% to 28 wt.% with respect to the total weight of the formulation, wherein said aqueous extract of Ocimum sanctum has at least 2% of polyphenols;
(b) an aqueous extract of Tinospora cordifolia in an amount in the range of 12 wt.% to 18 wt.% with respect to the total weight of the formulation, wherein said aqueous extract of Tinospora cordifolia has polyphenols is in the range of 0.1 to 1;
(c) a hydroalcoholic extract of Glycyrrhiza glabra in an amount in the range of 7 wt.% to 13 wt.% with respect to the total weight of the formulation, wherein said hydroalcoholic extract of Glycyrrhiza glabra has at least 25% of glycyrrhizin and has glabridin is in the range of 0.2% to 1%;
(d) a hydroalcoholic extract of Withania somnifera in an amount in the range of 12 wt.% to 18 wt.% with respect to the total weight of the formulation, wherein said hydroalcoholic extract of Withania somnifera has at least 1% of Withanoside IV and at least 0.2% of Withanolide A;
(e) a hydroalcoholic extract of Trigonella foenum in an amount in the range of 12 wt.% to 18 wt.% with respect to the total weight of the formulation, wherein said hydroalcoholic extract of Trigonella foenum has at least 5% of polyphenols; and
(f) an alcoholic extract of Andrographis paniculata in an amount in the range of 16 wt.% to 23 wt.% with respect to the total weight of the formulation, wherein said alcoholic extract of Andrographis paniculata has at least 20% of andrographolide.

5. The polyherbal formulation as claimed in claim 1, wherein said formulation is in a form selected from a tablet, an uncoated tablet, a film-coated tablet, a sugar-coated tablet, and an enteric-coated tablet.

6. The polyherbal formulation as claimed in claim 1, wherein an amount of the polyphenol present in
i. said aqueous extract of Ocimum sanctum is at least 2% with respect to the total weight of said extract;
ii. said aqueous extract of Tinospora cordifolia is in the range of 0.1 to 1% with respect to the total weight of said extract;
iii. said hydroalcoholic extract of Glycyrrhiza glabra is in the range of 1.5 to 3.5% with respect to the total weight of said extract;
iv. said hydroalcoholic extract of Withania somnifera is in the range of 0.5 to 2.5% with respect to the total weight of said extract;
v. said hydroalcoholic extract of Trigonella foenum is at least 5% with respect to the total weight of said extract; and
vi. said hydroalcoholic extract of Andrographis paniculata is in the range of 0.1 to 1% with respect to the total weight of said extract.

7. A process for the preparation of a polyherbal formulation, wherein said polyherbal formulation is in the form of a tablet, said process comprising the following steps:
a) sifting individually and then mixing, predetermined amounts of sieved powder extracts of Ocimum sanctum, Tinospora cordifolia, Glycyrrhiza glabra, Withania somnifera, Trigonella foenum, and Andrographis paniculata in a mixer for a first predetermined time period to under stirring to obtain a first mixture;
b) kneading said first mixture under stirring for a second predetermined time period followed by granulation for a third predetermined time period to obtain granules;
c) drying said granules at a predetermined temperature to obtain dried granules;
d) milling said dried granules to obtain milled granules; and
e) blending said milled granules for a fourth predetermined time period at a predetermined speed to obtain a blend followed by compressing said blend to obtain the polyherbal formulation in the form of a tablet.
8. The process as claimed in claim 7, wherein said predetermined amount of said aqueous extract of Ocimum sanctum is in the range of 20 wt.% to 30 wt.% with respect to the total weight of the formulation, said predetermined amount of said aqueous extract of Tinospora cordifolia is in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, said predetermined amount of said hydroalcoholic extract of Glycyrrhiza glabra is in the range of 5 wt.% to 15 wt.% with respect to the total weight of the formulation, said predetermined amount of said hydroalcoholic extract of Withania somnifera is in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, said predetermined amount of said hydroalcoholic extract of Trigonella foenum is in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, and said predetermined amount of said alcoholic extract of Andrographis paniculata is in the range of 15 wt.% to 25 wt.% with respect to the total weight of the formulation.

9. The process as claimed in claim 7, wherein said first predetermined time period is in the range of 8 to 15 min, said second predetermined time period is in the range of 1 to 5 min, said third predetermined time period is in the range of 2 to 10 min and said fourth predetermined time period is in the range of 3 to 10 min.

10. The process as claimed in claim 7, wherein said predetermined temperature is in the range of 40 to 70ºC and wherein said predetermined speed is in the range of 10 to 20 RPM.

, Description:FIELD
The present disclosure relates to a polyherbal formulation and a process for its preparation. Particularly, the present disclosure relates to a polyherbal formulation that has an immunomodulatory activity.
DEFINITIONS
As used in the present disclosure, the following terms are generally intended to have the meaning as set forth below, except to the extent that the context in which they are used indicates otherwise.
Cell-mediated immunity: Cell-mediated immunity is an immune response that does not involve antibodies. Rather, cell-mediated immunity is the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen.
Humoral immunity: Humoral immunity is the aspect of immunity that is mediated by macromolecules found in extracellular fluids such as secreted antibodies, complement proteins, and certain antimicrobial peptides. Humoral immunity is named so because it involves substances found in the humors, or body fluids. It contrasts with cell-mediated immunity. Humoral immunity is also referred to as antibody-mediated immunity.
Delayed-type hypersensitivity (DTH): Delayed-type hypersensitivity is a unique type of cell-mediated immunity. An inflammatory response that develops 24 to 72 hours after exposure to an antigen that the immune system recognizes as foreign. This type of immune response involves mainly T cells rather than antibodies (which are made by B cells). Also called DTH.
Buffy coat: The buffy coat is the fraction of an anticoagulated blood sample that contains most of the white blood cells and platelets following density gradient centrifugation.
Hemagglutination: Hemagglutination is a reaction that causes clumping of red blood cells in presence of some enveloped viruses, such as the influenza virus.
BACKGROUND
The background information herein below relates to the present disclosure but is not necessarily prior art.
A viral infection is a proliferation of a harmful virus inside the body. Viruses cannot reproduce without the assistance of a host. Viruses infect the host by introducing their genetic material into the cells and hijacking the cell's internal machinery to multiply into many viruses. The virus can enter the body by different ways which include the respiratory tract, gastrointestinal tract, genital tract, congenital infections as well as via skin or underlying subcutaneous tissue. The presence of the virus load or severity of the infection depends on the transmission and the phase of infection. The body of the host stimulates multiple immune defense functions such as humoral immunity and cell-mediated immunity to eliminate the virus and/or viral disease. In response to the virus invasion into the body, the humoral immunity stimulates B lymphocytes to produce antibodies, IgG, IgM, and IgA that are specific for viral antigens and exerts antiviral activity. IgG antibodies are responsible for most antiviral activity in serum, while IgA is the most important antibody when viruses infect mucosal surfaces. Cell-mediated immunity refers to the recognition and/or killing of virus and virus-infected cells by leukocytes and the production of different soluble factors (cytokines) which are stimulated in response to virus entry. The cytotoxic T lymphocytes, natural killer (NK) cells and antiviral macrophages recognize and kill the virus-infected cells and the Helper T cells recognize virus-infected cells and produce several important cytokines. Cytokines produced by monocytes (monokines), T cells, and NK cells (lymphokines) play important roles in regulating immune functions and developing antiviral immune functions.
However, in most cases, the body’s defense mechanism does not work properly due to the severity of infection, polypharmacy and the presence of associated diseases in the subjects, which further weakens the immunity.
Recently, the formulations that provide immunity has gained more importance due to the emergence of a viral infection such as COVID-19. Currently, there are no drugs available to combat the disease COVID-19. While several companies around the world are focusing on developing a vaccine for COVID-19, it is clear that the vaccine for any viral disease isn’t a boost to the whole immune system. A flu shot cannot make one more resistant to colds and other illnesses, rather it boosts the immune response of the infected patient to only the strain of the flu that the vaccine is designed to protect. Moreover, commercially available treatments for COVID-19/viral infections are life-threatening and have serious side effects such as immunosuppressant (immune suppression), imbalances in liver enzymes, muscle fatigue, hyperglycemia and the like.
Therefore, there is felt a need for a polyherbal formulation that mitigates the drawbacks mentioned hereinabove.
OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.
An object of the present disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
An object of the present disclosure is to provide a polyherbal formulation that is safe.
Another object of the present disclosure is to provide a polyherbal formulation that can be administered orally.
Still another object of the present disclosure is to provide a polyherbal formulation in the form tablet.
Yet another object of the present disclosure is to provide a polyherbal formulation that has immunomodulatory activity.
Yet another object of the present disclosure is to provide a process for the preparation of a polyherbal formulation.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY
The present disclosure relates to a polyherbal formulation. The formulation comprises: a) an aqueous extract of Ocimum sanctum in an amount in the range of 20 wt.% to 30 wt.% with respect to the total weight of the formulation, wherein the aqueous extract of Ocimum sanctum has at least 2% of polyphenols; b) an aqueous extract of Tinospora cordifolia in an amount in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, wherein the aqueous extract of Tinospora cordifolia has polyphenols is in the range of 0.1 to 1%; c) a hydroalcoholic extract of Glycyrrhiza glabra in an amount in the range of 5 wt.% to 15 wt.% with respect to the total weight of the formulation, wherein the hydroalcoholic extract of Glycyrrhiza glabra has at least 25% of glycyrrhizinic acid and has glabridin is in the range of 0.2% to 1% d) a hydroalcoholic extract of Withania somnifera in an amount in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, wherein the hydroalcoholic extract of Withania somnifera has at least 1% of withanoside IV and at least 0.2% of withanolide A; e) a hydroalcoholic extract of Trigonella foenum in an amount in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, wherein the hydroalcoholic extract of Trigonella foenum has at least 5% of polyphenols; and f) an alcoholic extract of Andrographis paniculata in an amount in the range of 15 wt.% to 25 wt.% with respect to the total weight of the formulation, wherein the alcoholic extract of Andrographis paniculata has at least 20% of andrographolide.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING
The present disclosure will now be described with the help of the accompanying drawing, in which:
Figure 1 illustrates a UV-Visible chromatogram of a diluent (methanol) at 227 nm;
Figure 2 illustrates a UV-Visible chromatogram of a diluent (methanol) at 254 nm;
Figure 3 illustrates a UV-Visible chromatogram of a standard solution of Withanolide A, Withanoside-IV, and Andrographolide at 227 nm;
Figure 4 illustrates a UV-Visible chromatogram of a standard solution of Glycyrrhizinic acid at 254 nm;
Figure 5 illustrates a UV-Visible chromatogram of a sample solution of the polyherbal formulation at 227 nm for estimation of Withanolide A, Withanoside-IV and, Andrographolide;
Figure 6 illustrates a UV-Visible chromatogram of a sample solution of the polyherbal formulation at 254 nm for estimation of Glycyrrhizinic acid;
Figure 7A illustrates the effects of the polyherbal formulation of the present disclosure on a primary antibody titer (humoral immunity) in mice in accordance with an embodiment of the present disclosure;
Figure 7B illustrates the effects of the polyherbal formulation of the present disclosure on a secondary antibody titer (humoral immunity) in mice in accordance with an embodiment of the present disclosure;
Figure 7C illustrates the effects of the polyherbal formulation of the present disclosure on SRBC-induced delayed-type hypersensitivity (humoral immunity) in mice in accordance with an embodiment of the present disclosure; and
Figure 8 illustrates the effects of the polyherbal formulation of the present disclosure on Peritoneal macrophage count (cell-mediated immunity) in mice in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION
Embodiments, of the present disclosure, will now be described with reference to the accompanying drawing.
Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms “a,” “an,” and “the” may be intended to include the plural forms as well, unless the context clearly suggests otherwise. The terms "comprises," "comprising," “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the method and process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
Recently, the formulations that provide immunity has gained more importance due to the emergence of a viral infection such as COVID-19. Currently, there are no drugs to combat the disease COVID-19. While several companies around the world are focusing on developing a vaccine for COVID-19, it is clear that a vaccine for any viral disease isn’t a boost to our whole immune system. A flu shot will not make one more resistant to colds and other illnesses, it boosts the immune response of the infected patient to only the strain of the flu that the vaccine is designed to protect. Moreover, a conventional approach such as chemical treatments via drugs and traditional knowledge does provide options for immunomodulation, but they are not efficacious due to their serious adverse effects such as immunosuppressant (immune suppression), imbalances in liver enzymes, muscle fatigue, hyperglycemia and the like and poor compliance. There is a definite need for a polyherbal formulation that effectively enhances immunity without having side effects.
The formulation of the present disclosure fills this exact technical gap in the marketplace.
The present disclosure provides a polyherbal formulation and a process for its preparation.
In an aspect, the present disclosure provides a polyherbal formulation. The formulation comprises: a) an aqueous extract of Ocimum sanctum in an amount in the range of 20 wt.% to 30 wt.% with respect to the total weight of the formulation, wherein the aqueous extract of Ocimum sanctum has at least 2% of polyphenols; b) an aqueous extract of Tinospora cordifolia in an amount in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, wherein the aqueous extract of Tinospora cordifolia has polyphenols is in the range of 0.1 to 1%; c) a hydroalcoholic extract of Glycyrrhiza glabra in an amount in the range of 5 wt.% to 15 wt.% with respect to the total weight of the formulation, wherein the hydroalcoholic extract of Glycyrrhiza glabra has at least 25% of glycyrrhizinic acid and has glabridin is in range of 0.2% to 1% ; d) a hydroalcoholic extract of Withania somnifera in an amount in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, wherein the hydroalcoholic extract of Withania somnifera has at least 1% of Withanoside IV and at least 0.2% of Withanolide A; e) a hydroalcoholic extract of Trigonella foenum in an amount in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, wherein the hydroalcoholic extract of Trigonella foenum has at least 5% of polyphenols; and f) an alcoholic extract of Andrographis paniculata in an amount in the range of 15 wt.% to 25 wt.% with respect to the total weight of the formulation, wherein the alcoholic extract of Andrographis paniculata has at least 20% of andrographolide.
In an embodiment, the plant extract is derived from barks, roots, tubers, stolons, rhizomes, leaves, seeds, fruits, stems and flowers.
In an embodiment, the extracts of Ocimum sanctum, Tinospora cordifolia, Glycyrrhiza glabra, Withania somnifera, Trigonella foenum, and Andrographis paniculata are taken in the form of a powder obtained by the direct micronization of the plant material. Alternatively, the extracts may be in the form of a solid or a semi-solid. Typically, the extracts are selected from the group consisting of alcoholic, hydroalcoholic, aqueous, ether, ester, ethyl acetate, acetone and hexane extract. Typically, the extracts are prepared by using techniques selected from percolation, decoction, maceration, Soxhlet extraction and supercritical fluid extraction.
Ocimum sanctum or Ocimum tenuiflorum is also known as the Tulsi or holy basil. It is an aromatic perennial plant in the family Lamiaceae. It is native to the Indian subcontinent and widespread as a cultivated plant throughout the Southeast Asian tropics. Tulsi is recommended for treatment of cough, asthma, fever, and dysentery, and the like. It modulates both humoral and cell-mediated immune responses.
In accordance with the embodiments of the present disclosure, the amount of the aqueous extract of Ocimum sanctum is in the range of 20 wt.% to 30 wt.% of the total weight of the formulation wherein the aqueous extract of Ocimum sanctum has at least 2% of polyphenols. In an exemplary embodiment, the amount of aqueous extract of Ocimum sanctum is 25 wt.% wherein the aqueous extract of Ocimum sanctum has 2% of polyphenols.
Tinospora cordifolia is an herbaceous vine of the family Menispermaceae indigenous to tropical regions of the Indian subcontinent. It is also known as Giloy. It has anti-diabetic, antioxidant, antihepatotoxic, and immunomodulatory properties. Tinospora cordifolia led to the activation of macrophages. The macrophages are the first line of defense in our body and play a crucial role in innate (nonspecific) and adaptive (specific) immunity.
In accordance with the embodiments of the present disclosure, the amount of the aqueous extract of Tinospora cordifolia is in the range of 10 wt.% to 20 wt.% of the total weight of the formulation wherein the aqueous extract of Tinospora cordifolia has polyphenol is in the range of 0.1 to 1%. In an exemplary embodiment, the amount of aqueous extract of Tinospora cordifolia is 15 wt.% wherein the aqueous extract of Tinospora cordifolia extract has 0.46% of polyphenols.
Glycyrrhiza glabra is also known as Liquorice or licorice or Mulethi. It is a flowering plant of the bean family Fabaceae, from the root of which a sweet, aromatic flavouring can be extracted. The liquorice plant is an herbaceous perennial legume native to Western Asia, North Africa and southern Europe. It is known for its antiviral, antimicrobial, anti-inflammatory and antitumor activities. Glycyrrhizin is known to inhibit hepatitis C virus, coxsackievirus B3, influenza virus and COVID-19 virus and has excellent immunostimulant properties.
In accordance with the embodiments of the present disclosure, the amount of the hydroalcoholic extract of Glycyrrhiza glabra is in the range of 5 wt.% to 15 wt.% of the total weight of the formulation wherein the hydroalcoholic extract of Glycyrrhiza glabra has at least 25% of glycyrrhizinic acid. In an exemplary embodiment, the amount of the hydroalcoholic extract of Glycyrrhiza glabra is 9 wt.% wherein the hydroalcoholic extract of Glycyrrhiza glabra has 25% of glycyrrhizinic acid and has glabridin is in range of 0.2% to 1%.
Withania somnifera, commonly known as ashwagandha, Indian ginseng, poison gooseberry, and winter cherry, is a plant in the Solanaceae or nightshade family. Several other species in the genus Withania are morphologically similar. It has immunostimulant properties.
In accordance with the embodiments of the present disclosure, the amount of the hydroalcoholic extract of Withania somnifera is in the range of 10 wt. % to 20 wt. % of the total weight of the formulation wherein, the hydroalcoholic extract of Withania somnifera has at least 1% of withanoside IV and at least 0.2% of withanolide A. In an exemplary embodiment, the amount of the hydroalcoholic extract of Withania somnifera is 15 wt.% wherein the hydroalcoholic extract of Withania somnifera has 1 % of Withanoside IV and 0.2% of Withanolide A.
Trigonella foenum or Fenugreek is an annual plant in the family Fabaceae, with leaves consisting of three small obovate to oblong leaflets. It is cultivated worldwide as a semiarid crop. Its seeds and leaves are common ingredients in dishes from the Indian subcontinent, and have been used as a culinary ingredient since ancient times.
In accordance with the embodiments of the present disclosure, the amount of the hydroalcoholic extract of Trigonella foenum is in the range of 10 wt.% to 20 wt.% of the total weight of the formulation wherein the hydroalcoholic extract of Trigonella foenum has at least 5% of polyphenols. In an exemplary embodiment, the amount of the hydroalcoholic extract of Trigonella foenum is 15 wt.% wherein the hydroalcoholic extract of Trigonella foenum has 5% of polyphenols.
Andrographis paniculata, commonly known as creat or green chiretta, is an annual herbaceous plant in the family Acanthaceae, native to India and Sri Lanka. It acts as a natural immune booster and is most widely used to treat cold and flu symptoms and is also used to treat other diseases like HIV, upper respiratory tract infections and ulcerative colitis.
In accordance with the embodiments of the present disclosure, the amount of the hydroalcoholic extract of Andrographis paniculata is in the range of 15 wt.% to 25 wt.% of the total weight of the formulation wherein the alcoholic extract of Andrographis paniculata has at least 20% of Andrographolide. In an exemplary embodiment, the amount of the alcoholic extract of Andrographis paniculata is 18 wt.% of the total weight of the formulation wherein the alcoholic extract of Andrographis paniculata has 20% of Andrographolide.
In accordance with the embodiments of the present disclosure, the hydroalcoholic extract of Glycyrrhiza glabra 30% is methanolic extract, the hydroalcoholic extract of Trigonella foenum is 75% ethanolic extract, the hydroalcoholic extract of Withania somnifera is 70% ethanolic extract and the alcoholic extract of Andrographis paniculata is 95% ethanolic extract.
In accordance with the embodiments of the present disclosure, the aqueous extract of Ocimum sanctum comprises at least 2% of polyphenols with respect to the total weight of the extract, the aqueous extract of Tinospora cordifolia comprises polyphenols in an amount in the range of 0.1 to 1% with respect to the total weight of the extract, the hydroalcoholic extract of Glycyrrhiza glabra comprises polyphenols in an amount is in the range of 1.5 to 3.5% with respect to the total weight of the extract, the hydroalcoholic extract of Withania somnifera comprises polyphenols in an amount in the range of 0.5 to 2.5% with respect to the total weight of the extract, the hydroalcoholic extract of Trigonella foenum comprises polyphenols at least 5% with respect to the total weight of the extract, and the alcoholic extract of Andrographis paniculata comprises polyphenols in an amount in the range of 0.1 to 1% with respect to the total weight of the extract.
In accordance with the embodiments of the present disclosure, the polyherbal formulation comprises a) an aqueous extract of Ocimum sanctum in an amount in the range of 22 wt.% to 28 wt.% with respect to the total weight of the formulation, wherein the aqueous extract of Ocimum sanctum has at least 2% of polyphenols, b) an aqueous extract of Tinospora cordifolia in an amount in the range of 12 wt.% to 18 wt.% with respect to the total weight of the formulation, wherein the aqueous extract of Tinospora cordifolia has polyphenols is in the range of 0.1 to 1%, c) a hydroalcoholic extract of Glycyrrhiza glabra in an amount in the range of 7 wt.% to 13 wt.% with respect to the total weight of the formulation, wherein the hydroalcoholic extract of Glycyrrhiza glabra has at least 25% of glycyrrhizinic acid and has glabridin is in the range of 0.2% to 1%, a hydroalcoholic extract of Withania somnifera in an amount in the range of 12 wt.% to 18 wt.% with respect to the total weight of the formulation, wherein the hydroalcoholic extract of Withania somnifera has at least 1% of Withanoside IV and at least 0.2% of Withanolide A, a hydroalcoholic extract of Trigonella foenum in an amount in the range of 12 wt.% to 18 wt.% with respect to the total weight of the formulation, wherein the hydroalcoholic extract of Trigonella foenum has at least 5% of polyphenols, and an alcoholic extract of Andrographis paniculata in an amount in the range of 16 wt.% to 23 wt.% with respect to the total weight of the formulation, wherein the alcoholic extract of Andrographis paniculata has at least 20% of andrographolide.
In an exemplary embodiment, the formulation comprises 25 wt.% aqueous extract of Ocimum sanctum, 15 wt.% aqueous extract of Tinospora cordifolia, 9 wt.% hydroalcoholic extract of Glycyrrhiza glabra, 15 wt.% hydroalcoholic extract of Withania somnifera, 15 wt.% hydroalcoholic extract of Trigonella foenum, and 18 wt.% alcoholic extract of Andrographis paniculata.
In an embodiment of the present disclosure, the formulation is in the form of a tablet selected from an uncoated tablet, a film-coated tablet, a sugar-coated tablet, and an enteric-coated tablet.
In an exemplary embodiment, the formulation is a film coated tablet.
In another aspect, there is provided with a process for the preparation of the polyherbal formulation in the form of a tablet. The process is described in detail as:
In a first step, sifting individually powder extracts of Ocimum sanctum, Tinospora cordifolia, Glycyrrhiza glabra, Withania somnifera, Trigonella foenum, and Andrographis paniculata through a sieve no. 20 to obtain a sieved extract. The predetermined amounts of sieved extracts of Ocimum sanctum, Tinospora cordifolia, Glycyrrhiza glabra, Withania somnifera, Trigonella foenum, and Andrographis paniculata are then mixed in the rapid mixer granulator (RMG) for a first predetermined time period under stirring to obtain a first mixture.
In an embodiment of the present disclosure, the predetermined amount of the aqueous extract of Ocimum sanctum is in the range of 20 wt.% to 30 wt.% with respect to the total weight of the formulation, the predetermined amount of the aqueous extract of Tinospora cordifolia is in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, the predetermined amount of the hydroalcoholic extract of Glycyrrhiza glabra is in the range of 5 wt.% to 15 wt.% with respect to the total weight of the formulation, the predetermined amount of the hydroalcoholic extract of Withania somnifera is in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, the predetermined amount of the hydroalcoholic extract of Trigonella foenum is in the range of 10 wt.% to 20 wt.% with respect to the total weight of the formulation, and the predetermined amount of the alcoholic extract of Andrographis paniculata is in the range of 15 wt.% to 25 wt.% with respect to the total weight of the formulation.
In accordance with an embodiment of the present disclosure, the first predetermined time period is in the range of 8 to 15 min. In an exemplary embodiment, the first predetermined time period is 10 min.
In a second step, the so obtained first mixture is kneaded under stirring for a second predetermined time period followed by granulation for a third predetermined time period to obtain granules.
In accordance with an embodiment of the present disclosure, the second predetermined time period is in the range of 1 to 5 min. In an exemplary embodiment, the second predetermined time period is 2 min.
In accordance with an embodiment of the present disclosure, the third predetermined time period is in the range of 2 to 10 min. In an exemplary embodiment, the third predetermined time period is 4 min.
In a third step, the granules are dried at a predetermined temperature to obtain a dried granules.
In accordance with an embodiment of the present disclosure, the predetermined temperature is in the range of 40 to 70ºC. In an exemplary embodiment, the predetermined temperature is 55 ºC.
In a fourth step, the dried granules are milled to obtain a milled granules.
In a fifth step, the so obtained milled granules are blended for a fourth predetermined time period at a predetermined speed to obtain a blend followed by compressing the blend to obtain the polyherbal formulation in the form of tablet.
In accordance with an embodiment of the present disclosure, the fourth predetermined time period is in the range of 3 to 10 min. In an exemplary embodiment, the fourth predetermined time period is 5 min.
In accordance with an embodiment of the present disclosure, the predetermined speed is in the range of 10 to 20 RPM. In an exemplary embodiment, the predetermined speed is 15 RPM.
The polyherbal composition of the present disclosure is an immunomodulatory activity. Moreover, the polyherbal composition of the present disclosure is safe and effective.
The foregoing description of the embodiments has been provided for purposes of illustration and not intended to limit the scope of the present disclosure. Individual components of a particular embodiment are generally not limited to that particular embodiment, but, are interchangeable. Such variations are not to be regarded as a departure from the present disclosure, and all such modifications are considered to be within the scope of the present disclosure.
The present disclosure is further described in light of the following experiments which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure. The following experiments can be tested to scale up to industrial/commercial scale and the results obtained can be extrapolated to the industrial scale.
EXPERIMENTAL DETAILS:
EXPERIMENT 1: Preparation of the polyherbal formulation in the form of uncoated tablet:
The polyherbal formulation in the form of tablet was prepared by blending the ingredients in appropriate quantities as given in Table-1
Table 1 Polyherbal formulation
Sr. No. Ingredients Experiment No.1 (in mg per two tablets)
1. Ocimum sanctum extract 400
2. Tinospora cordifolia extract 250
3. Glycyrrhiza glabra extract 150
4. Withania somnifera extract 250
5. Trigonella foenum extract 250
6. Andrographis paniculata extract 300
Total weight of two tablets-1600 mg

The temperature and humidity of the manufacturing area was not more than 25°C and 40°C respectively.
EXPERIMENT 2: Assay determination of active constituents of polyherbal formulation by using HPLC
Active constituents such as glycyrrhizinic acid, Withanolide-A, Withanolide-IV, and Andrographolide were assayed by using chromatograms wherein the peaks are measured for each constituent.
Chromatographic conditions:
Buffer solution Preparation: 1.36 gm of Potassium dihydrogen phosphate was dissolved in 900mL of water and pH was adjusted by using 0.5 ml of phosphoric acid and final volume was made up to 1000 mL with water. The buffer solution was mixed well, degassed and filtered through a 0.45µ filter.
Mobile Phase A: Buffer solution,
Mobile Phase B: Acetonitrile, and
Diluent: Methanol
Column : Inertsil ODS, 3V, 250 x 4.6 mm and 5µm.
Column Temperature: 35°C, Sampler Temperature: 10°C
Wave length: Glycyrrhizin – 254 nm, Withanolide-A, Withanolide-IV, and Andrographolide 227 nm.
Flow rate: 1.0 mL/min
Injection Volume: 20 ?L
Run time: 40 minutes
Retention Time: Glycyrrhizin - 20.7 min, Withanolide-A- 14 min, Withanolide-IV-25.1 min, and Andrographolide 15.8 min. .
Elution : Gradient
Table 2: Gradient program for the HPLC runs employed for detection and resolution of all the active constituents of polyherbal formulation.
Time (minutes) % Mobile phase :A (buffer solution) % Mobile phase :B (methanol)
0.0 90 10
10.0 65 35
14.0 65 35
15.0 55 45
19.0 55 45
20.0 50 50
25.0 50 50
27.0 20 80
31.0 20 80
35.0 90 10
40.0 90 10

Inference: It is evident from table 2 that all the active ingredients of the polyherbal formulation is properly separated by using HPLC.
Preparation of individual Standard stocks:
Glycyrrhizin Standard stock: (100µg / mL)
2.6 mg of the Glycyrrhizin acid reference standard (Natural remedies) was transferred to 25 mL volumetric flask. 15 mL of 100% methanol was added and sonicated to dissolve the Glycyrrhizin. The final volume was made up to the mark with 100% methanol and mixed well to obtain a standard stock solution of Glycyrrhizin.
Withanolide-A Standard stock: (100µg / mL)
2.5 mg of the Withanolide-A reference standard (TCI) was transferred into 25 mL volumetric flask. 15 mL of 100% methanol was added and sonicated to dissolve the Withanolide-A. The final volume was made up to the mark with 100% methanol and mixed well to obtain a standard stock solution of Withanolide-A.
Withanolide-IV Standard stock: (100µg / mL)
2.5 mg of the Withanolide-IV reference standard (TCI) was transferred into 25 mL volumetric flask. 15 mL of 100% methanol was added and sonicated to dissolve the Withanolide-IV. The final volume was made up to the mark with 100% methanol and mixed well to obtain a standard stock solution of Withanolide-IV.
Andrographolide Standard stock: (each 100µg / mL)
2.5 mg of the Andrographolide reference standard (TCI) was transferred into 25 mL volumetric flask. 15 mL of 100% methanol was added and sonicated to dissolve the Andrographolide. The final volume was made up to the mark with 100% methanol and mixed well to obtain a standard stock solution of Andrographolide.
Mix Standard preparation (10µg / mL)
1 mL standard stocks of each Glycyrrhizin, Withanolide-A, Withanolide-IV, and Andrographolide were taken into 10mL volumetric flask and diluted up to the mark with 100% methanol and mixed well to obtain a mixed standard solution of Glycyrrhizin, Withanolide-A, Withanolide-IV, and Andrographolide.
Sample Preparation:
At least 20 tablets were taken randomly and an average weight of tablets was calculated. The tables were crushed into fine powder. 400 mg of tablet powder was transferred to 100 mL volumetric flask and 60 mL of 100% methanol was added and sonicated for 30 minutes with intermediate shaking. Made the final volume with 100% methanol and mixed well. The solution was filtered through a 0.45 ? nylon filter and the filtrate was collected by discarding the first 3 mL of the filtrate.
System Suitability:
The chromatographs were conducted for standard preparations and the multiple peak responses were recorded which was illustrated in figures 1 to 6. Retention time, peak type, and area of each active constituent were recorded which is mentioned below in table 3:
Table 3:
Peak No. Peak name Retention time Area Peak type (baseline-mean -baseline- BMB)
1. Glycyrrhizin 20.682 448151 BMB
2. Withanolide-A 25.097 70051 BMB
3. Withanolide-IV 14.083 47635 BMB
4. Andrographolide 15.773 5840094 BMB

Inference: It is evident from table 3 and figures 1 to 6 that, the extracts and the polyherbal formulation of the present disclosure show the presence of the active constitutes at a specific wavelength.
Procedure:
Separately injected the diluent, standard preparation and sample preparation into the chromatograph. The response for the analyte peaks was measured and recorded into the chromatograms.
The quantity in mg per tablet and % assay was calculated of each content using the following formula:
Calculation:

Glycyrrhizin
(mg/tablet) = At x Wt.std x 1 x 100 x P x Avg.Wt
As 25 10 Wt.spl 100

Withanolide-A (mg/tablet) = At x Wt.std x 1 x 100 x P x Avg.Wt
As 25 10 Wt.spl 100
Withanolide-IV (mg/tablet) = At x Wt.std x 5 x 1 x 100 x P x Avg.Wt
As 25 10 10 Wt.spl 100

Andrographolide (mg/tablet) = At x Wt.std x 1 x 1 x 100 x P x Avg.Wt
As 25 10 10 Wt.spl 100
where,
At: Average area of analyte peak in Sample Solution
As: Average area of analyte peak in Standard Solution
Wt. std. : Weight of standard in mg
Wt. spl: Weight of sample in mg
Avg. Wt.: Average weight in mg
P: Potency of Standard in % (as such basis)
L.C.: Label Claim in mg
EXPERIMENT 3: Determination of the total polyphenol content by UV-VIS spectroscopy:
The amount of the total polyphenol content present in each active constituents were measured by using the Folin Ciocalteu method.
Reagents required:
Diluent: Milli Q water; Standard: Gallic acid; Folin Ciocalteu Reagent: Dilute Folin Ciocalteu reagent by taking 10 mL to 20 mL with diluent, and 7% Na2CO3 Reagent (7 gm Na2CO3 in 100 mL water).
Preparation of standard stock solution:
50 mg of gallic acid was dissolved in 100 mL of diluent. Further, 2 mL of the above solution was diluted with 25 mL of diluent to obtain a standard stock solution.
Preparation of sample solution:
20 tablets were powdered. 150 mg of the powdered tablet was transferred into 100 mL volumetric flask followed by adding 80 mL of diluent and heated the solution on Water bath at 80°C for 60 minutes. Mixed the above solution followed by cooling and dilute 5ml of the above solution to 25ml with diluent to obtain the sample solution.
Method:
1 mL standard solution and 2 mL sample solution were taken in a separate 25 mL volumetric flask. Added 2 mL of Folin ciocalteu reagent in each flask and mixed well. Further, added 11 mL of 7% Na2CO3 solution in each flask and made up the volume with diluent to obtain a solution. The solution was mixed and allowed to stand in a dark place for 30 min. Absorbance was measured after 30 min at 760nm.
Similarly, the blank solution was prepared without adding sample and % of total polyphenol content in the portion of a sample taken and calculated by using the following formulae.
Calculation:
Total Polyphenol (%w/w) = At x Wt.std x 2 x 1 x 100 x 25 x 25 x P x 100
As 100 25 25 Wt.spl 5 2 100
where,
At : Average absorbance of Sample Solution.
As : Average absorbance of standards Solution.
Wt. std. : Weight of standard in mg.
Wt. spl. : Weight of sample in mg.
P : Potency of working Standard.
EXPERIMENT 4: Evaluation of the immunomodulatory effect of the polyherbal formulation in animal models of immunity: (as per the IAEC and CPCSEA guidelines)
Objectives:
• To sensitize the mice with Sheep RBC (SRBC) solution;
• To determine the effect of treatment of polyherbal formulation of the present disclosure on SRBC-induced antibody (HA) titer and delayed-type hypersensitivity (DTH) response; and
• To determine the cell-mediated immunity using peritoneal macrophage count.
Materials:
Cyclophosphamide, 1% neutral red solution and Alsever’s solution were procured from Sigma Aldrich. Other chemicals and reagents used for the study were of analytical grade from approved vendors. Swiss albino mice weighing between 20-25 g were procured from the National Institute of Biosciences, Pune. The animals were maintained under standard environmental conditions and were fed with a standard pellet diet and water ad libitum.
Methods:
The mice were divided into five groups containing six animals each group.
Group-I- Vehicle control and received vehicle;
Group-II- Cyclophosphamide (25 mg/kg, p.o.) for 5 days;
Group-III- SRBC (sheep RBC) group receive SRBC solution
Group-IV- Polyherbal formulation (329 mg/kg) of the present disclosure for 8 days after sensitization with SRBCs; and
Group-V- Cyclophosphamide (25 mg/kg, p.o.) for 5 days and the polyherbal formulation (329 mg/kg) of the present disclosure.
The respective treatment was administered for 8 days (from day 0 to day 7) after sensitization with SRBCs. Cyclophosphamide was administered for 5 days as follows: one day before sensitization (day -1), on the day of sensitization (day 0) and day 1, 2 and 3 after sensitization with SRBCs (1, 2 and 3 days).

Sheep Red blood cells (SRBC) Preparation:
Sheep blood (56 ml) was collected in Alsever’s solution, and cells were isolated by centrifugation at 1000 rpm for 15 min. Plasma and the buffy coat were removed followed by washing of cells with five volumes of 0.9% NaCl thrice. The so formed pellet was suspended in two volumes of 0.05 M Tris-HCl with 0.1 mM EDTA (pH 7.6) and mixed thoroughly and again centrifuged at 25,000 rpm for 30 min. The process was repeated till the supernatant becomes clear.

SRBC preserves the native antigenicity of the erythrocytes (RBCs) while rendering the cells generally resistant to lysis by osmotic shock, freeze-thawing or immune hemolysis. This reagent (SRBC) can be used directly in hemagglutination procedures, or may be coupled with various proteins for hemagglutination testing.

Sheep RBC-induced antibody (HA) titer:
The groups of six mice were immunized with 20 µL of SRBC suspension (5?×?109 SRBC/mL) subcutaneously into their right hind footpad to precisely evaluate the effects on antibody production. This immunization day was referred to as Day 0. Seven days later (Day + 7), each mouse was injected intradermally into the left hind footpad with 20 µL of SRBC suspension (5?×?109 SRBC/mL). Blood samples were collected from each mouse on Day + 7 (post-challenge) for the evaluation of primary antibody titer by retro-orbital puncture under light ether anesthesia and again on Day + 14 (post-challenge) for secondary antibody titer. The levels of antibodies were calculated. The serum samples were obtained.25 µL of serum sample was added into one well of 96-well microtiter plate. Serial two-fold dilutions of the serum samples were done by using 25?µL of normal saline each time of transfer across the plate. After that, 25 µL of 1 percent (v/v) SRBC suspension in normal saline was added to 25 µL of diluted serum in each well. The plate was kept for 1 h at room temperature and then the well contents were tested for hemagglutination, i.e. until unmistakably negative patterns were shown by control wells. For the given mice, the value of the highest serum dilution demonstrating hemagglutination was identified as the antibody titer.

Table 4: Primary antibody titer in CP treated animals
Animal No. Vehicle Control Cyclophosphamide (CP) (25 mg/Kg) CP + polyherbal formulation
(329 mg/Kg) SRBC SRBC + polyherbal formulation (329 mg/Kg)
1 3 1 4 7 6
2 5 2 5 8 7
3 4 1 6 9 8
4 3 2 7 11 9
5 3 1 8 9 11
6 5 2 5 8 7

Inference: It is evident from table 4 that there is a significant decrease (p<0.01) in the secondary antibody titer in CP group when compared to vehicle control group mice, as CP is an immunosuppressant drug. There is a significant (p<0.001) increase in the secondary antibody titer in SRBC group of mice when compared to the vehicle control group. There was no significant change in the primary and the secondary antibody titer in case of the treatment of mice by the polyherbal formulation of disclosure when compared to SRBC mice.
Table 5: Secondary antibody titer in CP treated animals
Animal No. Vehicle Control Cyclophosphamide (25 mg/Kg) CP + polyherbal formulation
(329 mg/Kg) SRBC SRBC + polyherbal formulation (329 mg/Kg)
1 3 1 6 9 10
2 4 1 7 10 9
3 5 2 9 11 9
4 3 2 8 12 8
5 4 2 9 10 12
6 5 2 7 10 9

Inference: It is evident from table 5 that there is a significant decrease (p<0.01) in the secondary antibody titre in CP group when compared to vehicle control group mice, as CP is an immunosuppressant drug. There is a significant (p<0.001) increase in the secondary antibody titre in SRBC group of mice when compared to vehicle control group. There was no significant change in the primary and the secondary antibody titre in case of treatment of mice by using the polyherbal formulation of the present disclosure when compared to SRBC mice.
Table 6: Effect of polyherbal formulation on primary and secondary antibody titer in mice:
Parameters Vehicle Control Cyclophosphamide
(25 mg/Kg) Cyclophosphamide + polyherbal formulation
(329 mg/Kg) SRBC
(sheep RBC)
SRBC + polyherbal formulation
(329 mg/Kg)
Primary antibody titer 3.82 ±
0.40
1.50 ±
0.22
5.82 ±
0.60
8.66±
0.56 8.00 ±
0.72
Secondary antibody titer 4.00 ±
0.36
1.66 ±
0.21
7.66±
0.48 10.33 ±
0.42 9.50 ±
0.56

Where, Values are expressed as mean ± SEM, n = 6 and measured using one-way ANOVA followed by Newmans-Keul multiple comparison test.
Inference:
It is evident from table 6 and figures 7A and 7B that there was a significant decrease (p<0.01) in the primary antibody titer and the secondary antibody titer in CP group when compared to the vehicle control group mice, as CP is an immunosuppressant drug. There was a significant (p<0.001) increase in the primary and the secondary antibody titer in SRBC group of mice when compared to the vehicle control group. Hence, it was observed that the treatment of CP administered mice with the polyherbal formulation of the present disclosure significantly (p<0.001) increased the primary and the secondary antibody titer when compared to CP group. There was no significant change in the primary and the secondary antibody titer in the polyherbal formulation of the present disclosure treated mice when compared to SRBC mice. Therefore, from the above explanation, it is evident that the immunosuppressant effect caused by CP is reversed by the treatment of mice with the polyherbal formulation (329 mg/kg) of the present disclosure. It means that the polyherbal formulation of the present disclosure has an immunostimulant role in humoral immunity.
SRBC-induced delayed-type hypersensitivity (DTH) response:
Male Swiss albino mice (20-25 g) were divided into five groups containing 6 animals in each group as follows-
Group I –Vehicle control and received vehicle;
Group II –Cyclophosphamide (25 mg/kg, p.o.);
Group III- Cyclophosphamide (25 mg/kg, p.o.)+ polyherbal formulation (329 mg/kg);
Group IV- SRBC; and
Group V-SRBC +polyherbal formulation of the present disclosure (329 mg/kg).

Daily drug treatment was started 14 days prior to the challenge, i.e. on the same day as SRBC immunization. Vehicle control and cyclophosphamide-treated mice received the vehicle or cyclophosphamide, respectively every day. Both group of mice were immunized on Day 0 with 20 µL of SRBC solution (5?×?109 SRBC/mL) was subcutaneously injected into their right hind footpad. After 14 days of gavage treatments, the thickness of each mice left footpad was measured by using a plethysmometer just before an SRBC challenge. Then the mice were challenged by injecting 20 µL SRBC solution (5?×?109 SRBC/mL) subcutaneously into their left hind footpad (designated time 0). After 24 h, the footpad thickness was re-measured. The difference was taken as an indicator of DTH between the paw volumes of the left footpad just before and 24 hours after the challenge (in ml).

Paw volume at 0 hour in delayed-type of immunity
Table 7:
Animal No. Vehicle Control CP (25 mg/kg) CP + polyherbal formulation (329 mg/kg) SRBC (Control) SRBC + polyherbal formulation (329 mg/kg)
1 1.23 1.17 1.45 2.02 2.07
2 1.17 1.35 1.80 1.98 2.00
3 1.19 1.36 1.73 2.17 2.28
4 1.28 1.25 1.50 1.99 1.97
5 1.28 1.36 1.87 2.30 2.17
6 1.34 1.38 1.48 2.16 2.20

Inference: It is evident from table 7 that the paw volume (ml) of CP treated mice was found to be increased when compared to the vehicle control group. It is indicated from table 7 that when the polyherbal formulation is administered with cyclophosphamide (CP) in mice the difference in paw volume is significantly (p<0.001) increased in comparison with individually cyclophosphamide that signifies the immunostimulant effect of the present formulation.
Table 8: Paw volume at 24 hours in delayed-type of immunity
Animal No. Vehicle Control CP (25 mg/kg) CP + polyherbal formulation (329 mg/kg) SRBC (Control) SRBC + polyherbal formulation (329 mg/kg)
1 0.01 0.01 0.27 0.85 0.89
2 0.01 0.07 0.66 0.79 0.84
3 0.01 0.01 0.42 0.92 0.96
4 0.00 0.03 0.14 0.72 0.75
5 0.01 0.05 0.63 0.98 1.03
6 0.00 0.12 0.10 0.88 0.92

Inference: It is evident from table 8 that the polyherbal formulation (329 mg/kg) of the present disclosure significantly improved the delayed-type immunity.
Table 9: The difference in paw volume (ml)
Animal No. Vehicle Control CP (25 mg/kg) CP + polyherbal formulation (329 mg/kg) SRBC (Control) SRBC + polyherbal formulation (329 mg/kg)
1 0.01 0.01 0.27 0.85 0.89
2 0.01 0.07 0.66 0.79 0.84
3 0.01 0.01 0.42 0.92 0.96
4 0 0.03 0.14 0.72 0.75
5 0.01 0.05 0.63 0.98 1.03
6 0 0.12 0.1 0.88 0.92

Inference: It is evident from the results of table 9 that the polyherbal formulation (329 mg/Kg) of the present disclosure shows the immunomodulatory effect in humoral (both immediate and delayed) and cell-mediated models of immunity.
Table 10: Effect of polyherbal formulation on SRBC-induced DTH response in mice
Parameters Vehicle Control Cyclophosphamide
(25 mg/Kg) Cyclophosphamide + polyherbal formulation
(329 mg/Kg) SRBC
(sheep RBC)
SRBC + polyherbal formulation
(329 mg/Kg)
Paw volume (ml) 0.006±0.004 0.048±
0.422 0.370±
0.098*** 0.856± 0.038### 0.898±
0.040

Where,
Values are expressed as mean ± SEM, n = 6 and measured using one-way ANOVA followed by Newmans-Keul multiple comparison test.

Inference:
It is evident from table 10 and figures 7C that the paw volume (ml) of CP treated mice were found to be increased when compared to the vehicle control group. Treatment of CP administered mice with the polyherbal formulation of the present disclosure significantly (p<0.001) increased the difference in paw volume when compared to the CP treated mice indicating the immunostimulant effect of the polyherbal formulation of the present disclosure. However, there was no significant change in the paw volume difference observed in the polyherbal formulation treated mice when compared to SRBC mice.

Peritoneal macrophages count:
Male Swiss albino mice (20-25 g) were divided into two groups containing 6 animals in each group as follows-
Group I –Vehicle Control and received vehicle
Group II- SAVABOOST Tablets (329 mg/kg)

The treatment was administered per-orally (p.o.) daily for 20 days. On day 21, 5 ml of cold phosphate buffer saline (PBS) was injected intraperitoneally into each animal. The mice were killed by cervical dislocation and the abdominal cavity was gently massaged for 1 min. The skin of the abdomen was sterilized by wiping with cotton dipped in 95% alcohol. An incision was made at a lower part of the abdomen and the animal was lifted by holding the tail and peritoneal fluid was collected in a glass Petri dish. The petri dish was kept at 37°C for 1 h in an incubator. After which it was allowed to cool at 25°C. The supernatant was then discarded and 2 ml of cold 2% EDTA solution was added to it and the petri dish was stored in the refrigerator at 4°C for 30 min. After 30 min, the Petri dish was removed from the refrigerator. The contents were transferred in a centrifuge tube and centrifuged at 2000 rpm for 5 min. The supernatant was discarded and the pellet was suspended in 1 ml PBS. The macrophage cell suspension was carefully pipetted by a WBC pipette till mark 0.5 and diluted with 1% neutral red solution in PBS till mark 11 of the pipette. The cells were counted in the WBC square of Neubauer's chamber of hemocytometer under 10X of the microscope. The cell count was obtained and calculated for cells per cumm.

Table 11: Peritoneal macrophages count
Animal No. Vehicle Control Polyherbal formulation (329 mg/kg)
1 114 526
2 106 514
3 98 586
4 95 544
5 73 585
6 86 654

Inference: It is evident from table 11 that the polyherbal formulation of the present disclosure significantly increases (p<0.001) the peritoneal macrophage count in mice in comparison with the vehicle control group of mice, thereby indicating the immunostimulant effect of the polyherbal formulation of the present disclosure.
Table 12: Effect of polyherbal formulation on Peritoneal macrophage count (cell-mediated immunity) in mice:
Parameters Vehicle Control Polyherbal formulation
(329 mg/Kg)
Peritoneal macrophage count 95.30 ± 5.92
568.10± 21.02

Where,
Values are expressed as mean ± SEM, n = 6 and measured by unpaired Student’s ‘t’ test.
Inference:
It is evident from table 12 and figure 8 that the peritoneal macrophage count of the polyherbal formulation group mice was significantly (p<0.001) increased when compared to the vehicle control group of mice indicating the immunostimulant effect of the polyherbal formulation of the present disclosure.

Conclusion
• The administration of cyclophosphamide caused a significant decrease in the primary and the secondary antibody titer in mice.
• Treatment of mice with the polyherbal formulation (329 mg/kg) of the present disclosure caused improvement in cyclophosphamide induced decrease in primary and secondary antibody titer. Further, a significant improvement was also observed in delayed-type immunity upon treatment with the polyherbal formulation (329 mg/kg) of the present disclosure.
• The peritoneal macrophage count in mice was also found significantly improved with the polyherbal formulation.
• The polyherbal formulation (329 mg/Kg) of the present disclosure have significant immunomodulatory effect in humoral (both immediate and delayed) and cell-mediated models of immunity.
• No immunosuppressant effect was observed upon treatment with the polyherbal formulation of the present disclosure at 329 mg/kg dose even in presence of the antigen.

EXPERIMENT 5: Comparative examples
Table 13:
S.No Active ingredient Extract amt. (in 800mg)/tablet
Active present (mg /tablet) Present formulation 2-tablets/day (mg/person/day) Rat dose (mg/ Kg/ day) according to reference literature Human equivalent dose (mg/kg/day) as per present disclosure Human equivalent dose (mg/kg/day) as per literature
1. Withania somnifera extract(1.5%-total Withanolides) 125 1.87 3.74 30 4.86 316.22
2. Andrographis paniculata extract (20% Andrographolide ) 150 30 60 4 0.65 42.16
3. Glycyrrhiza glabra extract (25% Glycyrrhizin) 75 18.75 37.5 20 3.24 210.81
4. Ocimum sanctum extract (3% polyphenols) 200 6 12 850 137.84 8959.46
5. Trigonella foenum extract (5% total polyphenols) 125 6.25 12.5 100 16.22 1054.05
Inference: It is evident from table 13 that the formulation of the present disclosure uses in very low amounts in comparison to the conventional formulations as reported in the prior art. Surprisingly, the contents of active ingredients in the polyherbal formulation of the present disclosure are several folds less than what is reportedly used by conventional formulations indicating that the present formulation has improved properties of immunomodulatory effects as compared to individual extracts reported in the literature.

TECHNICAL ADVANCES AND ECONOMICAL SIGNIFICANCE
The present disclosure described herein above has several technical advantages including, but not limited to, the realization of a polyherbal formulation:
• has an improved immunostimulant activity and hence effective;
• has better patient compliance;
• requires a very low amount of active ingredients in order to make the formulation;
• that has no serious side effects or adverse events and hence safe; and
• that has no immunosuppressant activity.
The embodiments as described herein above, and various features and advantageous details thereof are explained with reference to the non-limiting embodiments in the following description.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results.
The foregoing description of specific embodiments so fully reveal the general nature of the embodiments herein, that others can, by applying current knowledge, readily modify and/or adapt for various applications of such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Further, it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
Having described and illustrated the principles of the present disclosure with reference to the described embodiments, it will be recognized that the described embodiments can be modified in arrangement and detail without departing from the scope of such principles.
While considerable emphasis has been placed herein on the components and component parts of the preferred embodiments, it will be appreciated that many embodiments can be made and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other changes in the preferred embodiment as well as other embodiments of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.