Extracts of sanicula elata for use in treating pulmonary tuberculosis

Abstract

This disclosure relates to a pharmaceutical composition comprising a pharmaceutically effective amount of a crude or purified ethanol extract of the plant Sanicula elata. Also provided is a plant extract preparation of Sanicula elata for use in treating respiratory conditions by macrophage immunomodulation. Also described are methods of treating respiratory conditions using the extracts or compositions containing them.

Description

[0001] EXTRACTS OF SANICULA ELATA FOR USE IN TREATING PULMONARY TUBERCULOSIS

[0002] BACKGROUND OF THE INVENTION

[0003] This invention relates to a plant extract preparation of Sanicula elata, or a herbal composition comprising the plant extract, for use in a method of treating a respiratory condition, including pulmonary tuberculosis. Also described herein are methods of treating a respiratory condition, such as pulmonary tuberculosis by administering the plant extract preparation of Sanicula elata or the composition to a subject as well as uses of the extract in such methods.

[0004] Tuberculosis (TB) is a disease that causes major morbidity and mortality around the globe, which disproportionally affects middle and low-income countries. Until 2019 TB was the biggest infectious killer of humans from a single pathogen. TB is also a major burden to healthcare systems globally. In South Africa, 450 000 patients get infected with TB each year, with 270 000 co-infected with HIV and 89 000 deaths annually. Further, 1.8% of new cases are multi-drug resistant. Thus, alternative treatment interventions are much needed to help reduce the disease burden.

[0005] Mycobacterium tuberculosis (Mtb) is the causal agent of TB, a slow-growing acid-fast bacillus that primarily manifests as pulmonary disease. Mtb can also develop into extrapulmonary TB by systemic infection. It is further capable of penetrating the blood-brain barrier. Furthermore, its peculiar physiology enables it to thwart attempts to create effective drugs against this bacterium.

[0006] Conventional antitubercular chemotherapeutic drugs presently used worldwide primarily comprise synthetic antibiotics, e.g., isoniazid (i.e., isonicotinic acid hydrazide), sodium PAS (sodium para amino salicylate), fluoroquinolones (e.g. moxifloxacin), calcium methanesulfonate of isoniazid, cyclocerine, morphazinamide hydrochloride, rifampicin, ethambutol, sparfloxicin, pyrazinamide and streptomycin, used singly or in combination.

[0007] While use of the above-mentioned drugs, preferably along with the BCG vaccination, provide improved patient outcomes, several drawbacks are noticed, especially in terms of side effects of such drugs and the requirement of prolonged intake / duration of therapy. Another problem observed is that even after treatment, some mycobacteria continue to reside in the subject. It is therefore imperative to develop alternatives to the above classes of drugs so that either the dosage or the intake duration is reduced or the problem of resistance to the drugs is obviated, and to provide increased access by patients.

[0008] Macrophages are most common host cells of Mtb in pulmonary TB (PTB). Autophagy is an intracellular catabolic process that assists in the removal of invading pathogens through a lysosomal degradation mechanism. The activation of autophagy in infected host cells, such as macrophages, is a promising treatment strategy against Mtb infection, which may also work against drug-resistant strains with or without other drugs. However, in order for the Mtb to be destroyed when it enters the lung tissue, immune alveolar macrophages must maintain the pro-inflammatory phenotype.

[0009] Macrophages derived from monocyte precursors undergo specific differentiation depending on the local tissue environment. The various macrophage functions are linked to the type of receptor interaction on the macrophage and the presence of cytokines. Similar to the T helper type 1 and T helper type 2 (TH1 -TH2) polarization, two distinct states of polarized activation for macrophages have been defined: the classically activated (M1 ) macrophage phenotype and the alternatively activated (M2) macrophage phenotype.

[0010] Granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) are involved in the differentiation of monocytes to macrophages. Human GM-CSF can polarize monocytes towards the M1 macrophage subtype with a “pro- inflammatory” cytokine profile (e.g., TNF-alpha, IL-1 beta, IL-6, IL-12 and IL-23), and treatment with M-CSF produces an “anti-inflammatory” cytokine (e.g., IL-10, TGF-beta and IL-1 ra) profile similar to M2 macrophages. Classically activated (M1 ) macrophages have the role of effector cells in TH1 cellular immune responses. The alternatively activated (M2) macrophages appear to be involved in immunosuppression and tissue repair. LPS and the TH1 cytokine IFN-gamma polarize macrophages towards the M1 phenotype which induces the macrophage to produce large amounts of IL-1 beta, TNF, IL-12, and IL-23. This helps to drive antigen specific TH1 and TH 17 cell inflammatory responses forward and thus participates to the clearance of invading microorganisms.

[0011] The antimicrobial functions of M1 macrophages are linked to up-regulation of enzymes, such as inducible nitric oxide synthase (iNOS) that generates nitric oxide from L-arginine. The secretion of IL-6, IL-23, and IL-1 beta are important factors in the induction and maintenance of Thl7 cells. In some cases, inflammatory responses can trigger tissue damage (toxic activity or reactive oxygen), resulting in an uncontrolled macrophage inflammatory response which could become pathogenic. For example, uncontrolled macrophage inflammatory response participates in the pathogenesis of inflammatory bowel disease (IBD). In contrast, exposure of macrophages to the TH2 cytokine IL-4 produces a M2 phenotype which induces the production of high levels of IL-10 and IL-IRA and low expression of IL-12. These cells reduce inflammation, are immunoregulators, promote tissue remodeling and tumor progression. It has recently been demonstrated in vitro that macrophages are capable of complete repolarization from M2 to M1 and change again in response to fluctuations in the cytokine environment.

[0012] There has been much recent interest globally in the use of immunomodulatory drugs as an alternative approach to combat TB, due to the key role played by immune cells, and in particular macrophages, in TB infection. Natural products obtained from plants are gaining great attention as potential candidates for consideration in drug discovery and can serve as future templates for new drug development. For example, aflatoxins are produced as secondary metabolites by fungi, and are known for their toxic and carcinogenic effects and food contaminants in humans. Synergistic effects of plant-based compounds (baicalein, celastrol and nobiletin) with synthetic anti-fungus drug (strobilurins) were found to have strong efficacy against the aflatoxins produced by fungus. In the case of Mtb, four plant derived compounds (4-hydroxybenzaldehyde, benzoic acid, bergapten, psoralen and p- hydroxybenzaldehyde acid) have exhibited potential as future anti-TB drug candidates that could be considered for TB treatment. One study has found that 4-hydroxybenaldehyde and bergapten have an anti-TB effect against the MTB-RNA polymerase enzyme as well as the InhA protein, displaying better results than rifampicin and isoniazid which are the first line drugs for the same targets. Further, these first line TB drugs have adverse effects on TB patients; rifampicin and INH are known to induce hepatotoxicity.

[0013] Accordingly, the inventors of the present invention aimed to investigate the potential in vitro immunomodulatory effects of extracts of various medicinal plants, including an extract of Sanicula elata, for use in treatment of PTB and other respiratory conditions and to further provide active compounds from extracts of Sanicula elata based on evidence that extracts of this plant may have immunomodulatory effects.

[0014] SUMMARY OF THE INVENTION

[0015] The present invention relates to a plant extract preparation of Sanicula elata for use in treating respiratory conditions, such as pulmonary tuberculosis, by macrophage immunomodulation. Also described herein are methods of treating respiratory conditions using the extracts or compositions containing them.

[0016] According to a first aspect of the present invention there is provided for a pharmaceutical composition comprising a crude or purified ethanol extract of the plant Sanicula elata.

[0017] In one embodiment, the pharmaceutical composition may further comprise an additional pharmaceutically active ingredient, such as one or more selected from the group consisting of isoniazid, sodium para amino salicylate, a fluoroquinolone, calcium methanesulfonate of isoniazid, cyclocerine, morphazinamide hydrochloride, rifampicin, ethambutol, sparfloxicin, pyrazinamide and streptomycin.

[0018] According to a second aspect of the present invention there is provided for a crude or purified extract from the plant Sanicula elata for use in a method of treating a respiratory condition, the method comprising administering the extract to a subject in need thereof.

[0019] In a first embodiment of the crude or purified extract for use, the extract may exhibit an immunomodulatory effect.

[0020] According to a second embodiment of the crude or purified extract for use, the treatment of the respiratory condition may be as a result of immunomodulation of macrophages. In one embodiment, the immunomodulation of macrophages modulates the macrophages towards the killing (M1) phenotype.

[0021] In a third embodiment of the crude or purified extract for use, the respiratory condition may be selected from the group consisting of coughing, chest pain, runny nose, fever, asthma, chronic obstructive pulmonary disease, allergic nasitis, phlegm, acute and chronic bronchitis, bronchiolitis, sore throat, tonsillitis, laryngitis and pulmonary tuberculosis. Preferably, the respiratory condition is pulmonary tuberculosis.

[0022] According to a fourth embodiment of the crude or purified extract for use, the extract may be obtained from a plant part selected from the group consisting of a whole plant, a leaf, a stem, a root, and a seed. Preferably, the extract is a whole plant extract.

[0023] In a fifth embodiment of the crude or purified extract for use, the extract may be an organic solvent-derived extract. In one embodiment, the organic solvent may be selected from the group consisting of ethanol, methanol and butanol or mixtures thereof. In a preferred embodiment, the solvent may be ethanol, preferably 70% ethanol.

[0024] According to a further embodiment of the crude or purified extract for use, the method may comprise administering the extract in combination with an additional pharmaceutically active ingredient, such as one or more selected from the group consisting of isoniazid, sodium para amino salicylate, a fluoroquinolone, calcium methanesulfonate of isoniazid, cyclocerine, morphazinamide hydrochloride, rifampicin, ethambutol, sparfloxicin, pyrazinamide and streptomycin.

[0025] In another embodiment of the crude or purified extract for use, the extract may be formulated as a pharmaceutical composition comprising a pharmaceutically effective amount of the crude or purified extract and one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0026] According to a third aspect of the present invention there is provided for a method of treating a respiratory condition in a subject, the method comprising administering a crude or purified extract from the plant Sanicula elata to a subject in need thereof.

[0027] In a first embodiment of the method of treating a respiratory condition, the extract may exhibit an immunomodulatory effect.

[0028] According to a second embodiment of the method of treating a respiratory condition, the treatment of the respiratory condition may be as a result of immunomodulation of macrophages. In one embodiment, the immunomodulation of macrophages modulates the macrophages towards the killing (M1 ) phenotype.

[0029] In a third embodiment of the method of treating a respiratory condition, the respiratory condition may be selected from the group consisting of coughing, chest pain, runny nose, fever, asthma, chronic obstructive pulmonary disease, allergic nasitis, phlegm, acute and chronic bronchitis, bronchiolitis, sore throat, tonsillitis, laryngitis and pulmonary tuberculosis. Preferably, the respiratory condition is pulmonary tuberculosis.

[0030] According to a fourth embodiment of the method of treating a respiratory condition, the extract may be obtained from a plant part selected from the group consisting of a whole plant, a leaf, a stem, a root, and a seed. Preferably, the extract is a whole plant extract.

[0031] In a fifth embodiment of the method of treating a respiratory condition, the extract may be an organic solvent-derived extract. In one embodiment, the organic solvent may be selected from the group consisting of ethanol, methanol and butanol or mixtures thereof. Preferably, the solvent is ethanol, more preferably 70% ethanol.

[0032] According to a further embodiment of the method of treating a respiratory condition, the method may comprise administering the extract in combination with an additional pharmaceutically active ingredient, such as one or more selected from the group consisting of isoniazid, sodium para amino salicylate, a fluoroquinolone, calcium methanesulfonate of isoniazid, cyclocerine, morphazinamide hydrochloride, rifampicin, ethambutol, sparfloxicin, pyrazinamide and streptomycin.

[0033] In another embodiment of the method of treating a respiratory condition, the extract may be formulated as a pharmaceutical composition comprising a pharmaceutically effective amount of the crude or purified extract and one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0034] According to a further aspect of the present invention there is provided for the use of a crude or purified extract of Sanicula elata in the preparation of a medicament for use in a method of treatment of a respiratory condition, the method comprising administering the medicament to a subject.

[0035] In one embodiment of the use of the crude or purified extract of Sanicula elata, the respiratory-related condition may be selected from the group consisting of coughing, chest pains, runny nose, fever, asthma, chronic obstructive pulmonary disease, allergic nasitis, phlegm, acute and chronic bronchitis, bronchiolitis, sore throat, tonsillitis, laryngitis and pulmonary tuberculosis. Preferably, the respiratory condition is pulmonary tuberculosis.

[0036] BRIEF DESCRIPTION OF THE FIGURES

[0037] Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

[0038] Figure"! : A graph showing the results of the toxicity testing using

[0039] Hoechst33342 / PI dual Staining. Sanicula elata. Buch-Ham Ex.D.Don (SE) showed > 80% cell viability. Figure 2: A graph showing nitric oxide production by macrophages treated with Sanicula elata. (SE), which was shown to have pro-inflammatory activity compared to LPS activated cells (LPS control).

[0040] Figure 3: A graph showing percentage cell viability using the MTT bio-assay.

[0041] Figure 4: A graph showing the inflammatory results on five fractions obtained from the crude extract.

[0042] Figure 5: Flow Chart for the Phytochemical Investigation leading to Compound Isolation.

[0043] Figure 6: Molecular Structure of Isolated Compound S7 (Sucrose).

[0044] Figure 7: Nitric oxide production of macrophages treated with four compounds (S1 , S7, S9, S10). Bar graph represents quadruplicate values of one experiment. Error bars represent the standard deviation of the mean.

[0045] Figure 8: Cytotoxicity of macrophages treated with four compounds (S1 , S7, S9, S10). Bar graph represents quadruplicate values of one experiment. Error bars represent the standard deviation of the mean.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

[0048] The invention as described should not be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0049] As used throughout this specification and in the claims which follow, the singular forms “a”, “an” and “the” include the plural form, unless the context clearly indicates otherwise.

[0050] The terminology and phraseology used herein is for the purpose of description and should not be regarded as limiting. The use of the terms “comprising”, “containing”, “having” and “including” and variations thereof used herein, are meant to encompass the items listed thereafter and equivalents thereof as well as additional items. It is, however, contemplated as a specific embodiment of the present disclosure that the term “comprising” encompasses the possibility of no further members being present, i.e., for the purpose of such an embodiment “comprising” is to be understood as having the meaning of “consisting of”.

[0051] In its broadest sense, the invention relates to the use of an extract of Sanicula elata or a composition comprising the extract for biomedical applications, especially for the modulation of macrophages for the treatment of respiratory-related diseases or conditions, such as pulmonary tuberculosis. The inventors of the present invention have prepared a plant extract composition, which was studied for various biomedical applications including its potential immunomodulatory effect on macrophages with the aim of developing an alternative treatment against pulmonary tuberculosis.

[0052] Macrophages, as the first cells to encounter the Mtb organism were treated with ten medicinal plants, in the presence of the bacterial protein lipopolysaccharide (LPS), that scientifically mimics the Mtb organisms, and only one plant was able to meet the intentions of the current work, Sanicula elata, which was to modulate the macrophages. The present study was able to pick the biological marker that characterized the exact phenotype (M1 cells) that the human body requires to kill the organism during infection. This is to say that this plant modulated the cells towards the fighting and killing phenotype, while at the same time displaying no toxic effect upon investigation. Further, cytotoxicity testing proved the plant nontoxic while it positively drove the switching of macrophages in the correct direction i.e. M1 phenotype.

[0053] Medicinal plants are rich in phytochemicals, and they remain untapped sources of new interventions, that could be explored and scientifically investigated for their biological activities for management of many diseases including pulmonary tuberculosis. Compounds derived from medicinal plants have shown inhibitory activity against Mtb based on various mechanisms. Thus, the present study seeks to investigate medicinal plants and compounds therefrom for the immunomodulation of macrophages. Current synthetic TB drugs are based on the different structures and pathways of Mtb, and, for immunomodulation, the focus is on macrophages as the therapeutic target cell to combat TB. S. elata ethanolic extract (105 g) was suspended in 400 mL of distilled water and successively partitioned with organic solvents, n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (BuOH). This afforded four organic fractions and an aqueous residue. Each fraction was concentrated by drying in vacuo on a rotary evaporator to yield dry fractions. Proinflammatory and cytotoxicity screening of all fractions was conducted using the Griess method and MTT technique respectively. The aqueous residue and the DCM fraction was found to immunomodulate raw 264.7 macrophages. Based on the biological results, the aqueous residue was subjected to chromatography analysis and NMR-Spectroscopy analysis. Four compounds (S1 , S5, S7 and S10) were characterized, and one compound elucidated (S7). The four compounds were further screened for immunomodulatory properties using the same cell line. No nitric oxide was produced by macrophages treated with the four compounds, showing that they are biologically active when used in combination. S7 was elucidated in this study as sucrose, a disaccharide (having two sugar units), with one unit each of D-glucose (pyranose sugar, six-member ring) and L-fructose (furanose sugar, five-member ring) in a p-1 ,2 linkage.

[0054] Accordingly, the present invention provides for the use of a crude or purified extract from Sanicula elata for immunomodulation of macrophages to treat respiratory conditions or diseases. Also provided are pharmaceutical compositions comprising the extract from Sanicula elata for immunomodulation of macrophages, and treatment of respiratory conditions.

[0055] As used herein, the term “treatment” or “treat” refers to any administration of the extract or compositions herein to partially or completely alleviate, ameliorate, relieve, improve, delay onset of, reduce severity of, and / or reduce frequency, incidence or severity of one or more symptoms, features, and / or causes of a particular disorder, and / or condition. Such treatment may be of a subject who does not exhibit signs of the relevant condition or of a subject who exhibits only early signs of the condition. Alternatively, or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant condition. For example, a subject may be suffering from respiratory-related conditions such as coughing, chest pains, runny nose, fever, asthma, chronic obstructive pulmonary disease, allergic nasitis, phlegm, acute and chronic bronchitis, bronchiolitis, sore throat (pharyngitis), tonsillitis, laryngitis, pulmonary tuberculosis, and the like.

[0056] As used herein, the terms “improve”, “ameliorate”, and “heal” are used interchangeably and refer to when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) and / or frequency.

[0057] According to the present invention, the crude or purified extract from Sanicula elata has application as a bioactive for immunomodulation of macrophages.

[0058] As used herein, the phrase “bioactive” refers to a substance that has activity in a biological system (e.g., in a cell, such as an isolated cell, in a culture of cells, in a tissue, in an organism etc.). For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. As used herein the term “immunomodulation” refers to a change in the body's immune system, caused by agents that activate or suppress its function. In one embodiment, the immunomodulation is of macrophages and results in the pro-inflammatory M1 phenotype of macrophages.

[0059] As used herein the term “crude extract” refers to a concentrated preparation of a plant extract obtained by removing secondary metabolites from the crude plant material with the aid of a suitable solvent. This may be done, for example, by submerging the crude plant material in the suitable solvent, removing the solvent and consequently evaporating all or nearly all of the solvent. As used herein the term “purified extract” refers to an extract obtained by separating the constituent parts of the crude extract from each other. By way of a non-limiting example, the constituent parts of the crude extract may be separated from one another by separating the polar constituents from the non-polar constituents. In so doing the active polar and / or non-polar constituents may thus be concentrated.

[0060] Those skilled in the art will appreciate that there are a number of methods for obtaining extracts from crude plant material. These methods include, among others, cutting, chopping, macerating and / or grinding raw plant material in at least one solvent in order to obtain a plant extract. It will also be appreciated that the plant material may be fresh material or dry plant material.

[0061] The solvent may be an organic solvent. Organic solvents typically used in the preparation of plant extracts include ethanol, methanol, butanol dichloromethane, chloroform, acetone and / or mixtures thereof. In one embodiment, the organic solvent is ethanol. Preferably, the organic solvent is 70% ethanol.

[0062] It will be understood that the crude or purified extract from Sanicula elata of the invention may be in the form of a pharmaceutical composition, to be administered to a subject, for immunomodulation of macrophages to treat respiratory conditions.

[0063] As described herein the extract or pharmaceutical compositions of the invention are extracts or pharmaceutical compositions suitable for oral, intravenous, cutaneous or subcutaneous use on a subject. The subject may include a living animal, preferably a mammal and most preferably a human.

[0064] The extract can be prepared in any desired delivery form known in the art of pharmaceuticals for example, the extract may be prepared as a tablet, capsule, tincture, power, inhalant and / or liquid. Other conventional formulations, including known carriers and additives, will be readily apparent to those skilled in the art.

[0065] The extract may be formulated as a pharmaceutical composition, by methods know to those skilled in the art. Pharmaceutically acceptable ingredients may be used. The term "pharmaceutically acceptable" refers to properties and / or substances which are acceptable for administration to a subject from a pharmacological or toxicological point of view. Further “pharmaceutically acceptable” refers to factors such as formulation, stability, patient acceptance and bioavailability which will be known to a manufacturing pharmaceutical chemist from a physical / chemical point of view.

[0066] The pharmaceutical composition of the invention containing the extract may be in a form suitable for oral administration use. Suitable forms of the pharmaceutical composition include, for example, tablets, capsules, tinctures, powers, inhalants and / or liquids.

[0067] The “suitable forms” of the pharmaceutical composition may be combined with “pharmaceutically acceptable carriers” and other elements known in the art to produce tablets, capsules, tinctures, powers, inhalants and / or liquids. The pharmaceutical composition may further be combined with other ingredients which promote the absorption of the extract into the body. By “pharmaceutically acceptable carrier” is meant a solid or liquid filler, diluent or encapsulating substance which may be safely used for the administration of the pharmaceutical composition to a subject. The compositions of the invention may further include additives which enhance the properties of the composition. Examples of such additives include, but are not limited to rheology modifiers, suspending agents, thickeners, denaturants, humectants, solvents, emollients, emulsifiers and preservatives.

[0068] The use of the compositions entails administration of an effective amount of the extract or a pharmaceutical composition containing the extract to a subject in order to treat a respiratory condition, such as pulmonary tuberculosis, by immunomodulation of macrophages. The term “effective amount” in the context of treating a respiratory condition refers to the administration of an amount of the extract or the composition comprising the active plant extract to an individual in need of treatment, either as a single dose or as several doses of the extract or the pharmaceutical composition containing the extract.

[0069] It will be appreciated by those of skill in the art that suitable dosages of the pharmaceutical composition, as well as the frequency of administration of an effective amount of the composition will be dependent on several factors. These factors include the individual components used, the formulation of the pharmaceutical composition, the condition being treated, the severity of the condition, the age, weight, health and general physical condition of the subject being treated, and other medication that the subject may be taking, and other factors as are known to those skilled in the art. It is expected that the effective amount will fall within a relatively broad range that can be determined through routine trials.

[0070] In the present invention, the extract was non-toxic to the subject. Further, the pro- inflammatory behaviour of S. elata is proven by the biological marker measured, nitric oxide, which gave a positive signal that S. elata can maintain the correct macrophage phenotype that could kill the Mtb microorganism.

[0071] The following examples are offered by way of illustration and not by way of limitation.

[0072] EXAMPLE 1

[0073] Preparation of Plant Extracts

[0074] Whole plants including the leaves, the roots, and the bark were collected from medicinal plants (Table 1 ) and washed in clean running water. These were allowed to dry for 7 - 10 days at temperature ranges of 25 - 30 °C. After drying, all samples were milled up by mortar into powder and stored in dry capped plastic bottles ready for extracting. Table 1 : Details of the plants used in this study.

[0075] Extracts of ten reported medicinal plants (Table 2) were prepared according to the following method. To measure the quantity that is sufficient for the Soxhlet extractor jar, the standard weighing boat was used to measure powders and different weights were taken for the medicinal plant. The weight of powdered material per individual plant is shown in Table 2, with the weights of all medicinal plants in the weight range of 20 g - 70 g. Ethanol was considered for the hydro-alcoholic ratio based on its property of extracting both the non-polar and polar phytochemicals in plants. The plant material was extracted using the Soxhlet plant extraction method. Briefly, plant material was soaked in 70% ethanol to initiate the extraction process and allowed to stand overnight for complete extraction. The extracts were subsequently dried in the oven at 40 °C.

[0076] All dried material was divided into 50 mg aliquots and labelled for experimental work on cytotoxicity investigations and biological studies. Table 2: The weight of powdered material used for extractions per individual plant

[0077] EXAMPLE 2

[0078] Preliminary Cytotoxicity Testing

[0079] Sample preparation and cell line maintenance

[0080] The ethanol extracts were reconstituted in dimethyl sulfoxide (DMSO) to give the final concentration of 100 mg / ml (50 mg dried extract was dissolved in 500 pl DMSO). The test samples were stored at 4 °C until required for further testing. The human hepatocarcinoma cell line C3A was used to test the toxicity of the 10 extracts. Cells were maintained in 10 cm culture dishes in 10 ml complete medium (EMEM-LG, NEAA +10% FBS + 1x penicillin / streptomycin) and incubated at 37 °C in 5% CO2, and 100% humidity until needed.

[0081] Treatment of cells with extracts for cytotoxicity testing

[0082] Cells were seeded in 96 well plates at 4000 cells / well (100 pl aliquots) and left overnight to attach and subsequently treated. Dilutions of each extract (400 pg / ml) were prepared in a complete medium. Three-point dilution series were tested, with dilutions prepared to yield 3 concentrations of 400 pg / ml, 200 pg / ml and 100 pg / ml. 100 pl aliquots of each dilution were added to 100 pl of attached cells in the 96 well plates, yielding final concentrations of 200 pg / ml, 100 pg / ml and 50 pg / ml. Cells were then incubated at 37 °C, in 5% CO2. Melphalan (100 Mm) was used as a positive control at a concentration of 40 pM.

[0083] Hoechst 33342 / PI dual staining

[0084] Hoechst 33342 and Propidium Iodide (PI) are nuclear dyes that are used to distinguish between three population groups (live-, apoptotic-, and dead cells). The Hoechst 33342 dye binds to the minor grooves of double-stranded DNA and because it is a membrane-permeant dye, it stains all nucleic acids. PI intercalates between DNA base pairs with little or no sequence preferences. The permeability of PI is dependent on the integrity of the cell membrane and the correct functioning of the efflux pumps. Therefore, dead cells stain more easily and enable non-viable populations to be distinguished. Live cells give a less intense blue, fluorescent signal while dead cells would have a more pronounced red fluorescent signal.

[0085] From the stock solution of 5 mg / ml Hoechst 33342, 10 pil / ml was added to 10 ml PBS. From the stock solution of 100 pg / ml PI, 20 pl PI was added to 1 ml PBS in the presence of Ca2+and Mg2+to ensure adherence of the cells to the plate. Media and treatment were aspirated from the plates and 100 pl staining solution (Hoechst dye) was added to each well. Plates were incubated at 37 °C for 15 - 20 minutes. After incubation, 10 pl PI was added to each well and the plates were acquired immediately using DAPI filters for Hoechst and Texas Red filters for PI.

[0086] Data acquisition and analysis

[0087] Quantification of live and dead cells was performed using the ImageXpress Micro XLS Widefield Microscope (Molecular Devices) using a 10x Plan Fluor objective and DAPI and Texas Red filter cubes. Nine image sites were acquired per well which is representative of roughly 75% of the surface area of each well. Acquired images were analysed by the MetaXpress software and Multi-Wavelength Cell Scoring Application Module. Acquired data were transferred to an Excel spreadsheet and analysed and processed.

[0088] Eighty percent cell viability was considered as the cut-off mark to determine the cytotoxicity of the leaf, root, buck, and bulb extracts against the C3A cells, which implied that the extracts were considered toxic when cell viability falls below 80%. Sanicula elata. Buch- Ham Ex. D. Don (SE) showed > 80% cell viability (Figure 1 ), suggesting that S. elata has pro- inflammatory activity compared to LPS activated cells (LPS control).

[0089] EXAMPLE 3

[0090] Pro / Anti-Inflammatory Screening of the Plant Extracts

[0091] Preparation of RA W 264.7 macrophages

[0092] One millilitre vial of frozen cells was thawed at 37 °C in a water bath. The seeding and sub-culturing was performed under sterile conditions in the safety cabinet. The cells were seeded in RPMI1640 culture medium supplemented with 10% FBS (RPMI complete medium) into 96-well plates at a density of 1 x 105cells per well and allowed to attach overnight.

[0093] Subsequently, the spent culture medium was aspirated from the wells. RPMI medium (100 pl) was then added to the cells to give final concentrations of 50 pg / ml, 100 pg / ml, and 200 pg / ml. To assess the anti-inflammatory / pro-inflammatory activity of the 10 extracts, 100 pl of LPS (final concentration 500 ng / ml) containing medium was added to the corresponding wells, and amino guanidine (100 pM) was used as the positive control to indicate antiinflammatory activity while LPS was used as a positive control to indicate pro-inflammatory activity of plant samples. The cells were incubated for further 24 hrs. Nitric oxide production

[0094] To quantify Nitric Oxide (NO) production, 50 pl of spent culture medium was transferred to a new 96-well plate after 24 hrs incubation, and 50 pl Griess reagent added. Absorbance was measured using a Bio Tek® Power Wave XS spectrophotometer (Winooski, VT, USA) at 540 nm. To determine the concentration of NO in each sample, a standard curve of sodium nitrite dissolved in culture media was used.

[0095] Cytotoxicity screening on all test samples

[0096] To confirm the absence of toxicity as a contributing factor across all samples, cell viability was assessed using tetrazolium salts assay (MTT). The medium and treatments remaining in each well were removed and replaced with 100 pl medium containing 0.5 mg / ml MTT and further incubation was allowed for 30 minutes at 37 °C. Thereafter, MTT was removed and 100 pl DMSO was added to each well to solubilize the formazan crystals. Absorbance was then measured using a BioTek® Power Wave XS spectrophotometer (Winooski, VT, USA) at 540 nm. All test samples were quadrupled in both the inflammatory and MTT bio-assays.

[0097] Pro- / Anti-inflammatory assay

[0098] The ethanoic extracts were assessed for potential pro / anti-inflammatory activity using RAW 264.7 macrophages. Macrophages were cultured in multi-well plates and activated by exposure to 500 ng / mL lipopolysaccharides (LPS), which induces the expression of iNOS with concomitant production of nitrate, along with other inflammatory mediators. LPS is a strong activator of immune cells including B cells, monocytes, macrophages, and other LPS reactive cells. Such immune cells require an immunogenic stimulator such as LPS to produce cytokines. The LPS activated macrophages were treated with either 50 pg / ml or 100 pg / ml of the plant extracts. The treated samples were centrifuged to collect the cell culture supernatant and cytokine ELISA was performed on the supernatant. Simultaneously, evaluation of cell viability against RAW 264.7 macrophages using the MTT bio-assay was performed. Nitric oxide production by macrophages treated with Sanicula elata. is shown in Figure 2. Sanicula elata was shown to have pro-inflammatory activity compared to LPS activated cells (LPS control) (Figure 3).

[0099] EXAMPLE 4

[0100] In Vitro Anti-Inflammatory Screening of 5 Selected Fractions (F1-F5)

[0101] Reagents

[0102] RAW 264.7 mouse macrophages were purchased from Cellonex (South Africa). Sulfanilamide Solution and NED Solution were made as per manufacturer’s instructions and products purchased from Promega. Lipopolysaccharide (LPS) and aminoguanidine were purchased from Sigma-Aldrich (St. Louise, MO, USA). RPMI1640 culture medium and fetal bovine serum (FBS) were from GE Healthcare Life Sciences (Logan, UT, USA).

[0103] Sample preparation and anti-inflammatory screening

[0104] Extracts were solubilized using DMSO to make a stock of 100 mg / mL. Samples were stored at 4 °C until required. Aminoguanidine (AG) was used as a positive control to indicate anti-inflammatory activity. RAW 264.7 cells were seeded in RPMI1640 culture medium supplemented with 10% FBS (RPMI complete medium) into 96-well plates at a density of 1 x 105cells per well and allowed to attach overnight. Subsequently, day spent culture medium was removed and 50 pL sample aliquots (diluted in RPMI complete medium) added to give final concentrations of 16.125 pg / mL, 31 .25 pg / mL, 62.5 pg / mL, 125 pg / mL and 250 pg / mL for all fractions.

[0105] To assess the anti-inflammatory activity, 50 pL of LPS (final concentration of 500 ng / mL) containing medium was added to the corresponding wells. Aminoguanidine (AG) was used as the positive control at 100 pM and 200 pM. Cells were incubated for a further 24 hr. To quantify NO production, 50 pL of the spent culture medium was transferred to a new 96- well plate. Sulfanilamide solution and NED solution was prepared as per manufacturer’s instructions. Initially, 50 pL Sulfanilamide solution was added to the spent culture medium and incubated for 10 minutes in the dark at room temperature. Thereafter, 50 pL NED solution was added to each well and further incubated for 5 - 10 minutes in the dark at room temperature. Absorbance was measured at 540 nm (BioTek® PowerWave XS spectrophotometer). A standard curve using sodium nitrite dissolved in culture medium was used to determine the concentration of NO in each sample. The inflammatory results on five fractions obtained from the crude extract are shown in Figure 4.

[0106] The pro-inflammatory behaviour of S. elata is proven by the biological marker measured, nitric oxide, which gave a positive signal that S. elata can maintain the correct phenotype that could kill the PTB microorganism, provides promising evidence for use of S. elata as a novel PTB drug.

[0107] EXAMPLE 5

[0108] Phytochemical Extraction and Isolation of Compounds from Sanicula elata

[0109] Reagents and general experimental procedure

[0110] The organic solvents used (Shalom Laboratory Supplies, Durban, KZN, South Africa) were of analytical grade and were redistilled before use. Thin-layer chromatography (TLC) was performed on 0.20 mm pre-coated silica gel 60 with fluorescent indicator UV254, purchased from MACHEREY-NAGEL GmbH & Co. KG (Valencienner Str. 1 1 , 52355 Duren, Germany). The developed TLC plates were visualized under the ultraviolet (UV) light at 254 and 366 nm wavelengths and sprayed with 10% sulfuric acid in MeOH as a general organic compound detection (chromogenic) reagent. Compound isolation was carried out by column chromatography (CC) on silica gel 60 adsorbent (0.063-0.200 mm, 70-230 mesh ASTM, Merck, Darmstadt, Germany). The compound isolation flow chart is presented in Figure 5.

[0111] Extraction and fractionation

[0112] Sanicula elata ethanolic leaf extract (105 g) was suspended in 400 mL of distilled water and successively partitioned with organic solvents, n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (BuOH), in a 2.0 L separating funnel, according to Oriola et aL, 2022. This afforded four organic fractions and the aqueous residue (Table 3). Each fraction was concentrated to dryness in vacuo on a rotary evaporator to yield dry fractions.

[0113] Table 3: Partition Fractions from the Ethanolic Extract of S. elata plant.

[0114] Isolation of compounds by column chromatography

[0115] Silica gel Open Column Chromatography (CC)

[0116] The method described by Oriola et aL, 2022 was used, with slight modification. Here aqueous fraction (38 g) was solubilized in 40 mL of methanol-distilled water solvent mixture (MeOH-H2O, 1 :1 ) to form a suspension. This suspended fraction was adsorbed onto 60 g of silica gel and dry-packed in a 180 g of silica gel layered glass column. The column set up was eluted with solvent systems in an increasing order of polarity as follows: EtOAc-MeOH (100:0, 95:5, 90:10, 80:20, 70:30, 60:40, 40:60, 20:80, 0:100%), MeOH-H20 (100:0, 90:10, 80:20, 50:50, 0:100%). The elution process afforded 103 eluates (column fractions). Eluates 58, 59, 65 and 66 afforded solid deposits after about 48 h of elution. They were filtered and washed with 50 mL each of MeOH to afford an ash amorphous powder, coded S1 , and whitish cubical crystals, coded S2-S4, respectively. Furthermore, the 103 eluates were bulked into eleven subtractions, F1-F11 , based on TLC analysis (Table 4). Bulked column fraction F8 was filtered, and the residue was washed with 50 mL of MeOH to afford an ash amorphous powder. Upon TLC of the powdered substance in MeOH-H20 (9:1 ), it gave a single brownish spot at a retardation factor (Rf) of 0.20 and was coded compound S1 (2.54 g). The eluates numbered 59, 65 and 66 in the F8 bulked fraction were also filtered and washed with 50 mL each of MeOH to afford compounds S2 (119 mg), S3 (96 mg) and S4 (91 mg).

[0117] Table 4: Column Bulked Fractions of S. elata Aqueous plant Fraction

[0118] * Column bulked fraction afforded solid deposits after 48 h of elution, leading to isolates S1 - S4. Retardation factor (Rf) = distance moved by solute / distance by solvent system

[0119] Sephadex LH-20 Column Fractionation of Column Bulked Fraction F8

[0120] Subtraction F8 (5.1 g) was suspended in 20 mL of DCM-MeOH (70:30%). It was pipetted into a 30 g Sephadex LH-20 column that had been pre-swollen and equilibrated with 100 mL each of MeOH and DCM-MeOH (70:30%) respectively. A gradient elution profile comprising DCM-MeOH (70:30, 30:70, 0:100%) and MeOH-H20 (80:20) was used, which afforded 28 eluates (column fractions). The eluates were bulked into four subtractions, F8i - F8iv, based on TLC analysis (Table 5). Subtractions F8i and FSiii were candidates for further purification based on their characteristic TLC patterns which showed major spots, revealing them to have a major compound each with slight impurity.

[0121] Table 5: Column Bulked Fractions of Subtraction F8

[0122] * Subtraction selected for further fractionation

[0123] Silica cel Column Chromatography Fractionation of Subtraction F8i

[0124] Subtraction F8i (297 mg) was adsorbed onto 2 g of silica gel and eluted on a 5 g silica gel short column, using 50 mL each of CHCh-MeOH solvent systems (75:25, 70:30, 60:40, 50:50, 30:70, 0:100%), to afford 10 eluates. Based on TLC analysis, the eluates were bulked into four subtractions, F8i(a) - F8i(d). Subtraction F8i(c) appeared as a purple-coloured spot at a Rf of 0.45 in DCM-MeOH (1 :1 ), suggesting an isolate with steroidal or terpenoidal nucleus, coded S5 (95 mg). The gradient elution profile of subtraction F8i on silica gel column is presented in Table 6.

[0125] Table 6: Gradient Elution Profile of Subtraction F8i on silica gel Column

[0126] * Pure substance (S5) based on TLC analysis in DCM-MeOH (1 :1 ) and CHCh-MeOH (1 :1 )

[0127] Silica gel Column Chromatography Fractionation of Subtraction F8iii

[0128] Subtraction FSiii (690 mg) was adsorbed onto 5 g of silica gel and eluted on a 5 g silica gel column, using 100 mL each of CHCh-MeOH solvent systems (90:10, 80:20, 70:30, 50:50 and 0:100%), to afford 17 fractions. Eluates 10, 11 and 12 gave white crystalline substances after 24 h of elution. Each substance was washed with 50 mL of MeOH to afford compounds S6 (71 mg), S7 (60 mg), and S8 (121 mg), respectively. Upon TLC analysis with DCM-MeOH (1 :1 ), they gave blackish colouration at Rf0.55, 0.53, and 0.52 respectively, indicating that they are sugar-containing (glycosidic) compounds. The filtrate of eluate 11-12 also gave a blackish colour at a Rf of 0.52 in DCM-MeOH (1 :1 ), which also suggests compound S9 (77 mg) is glycosidic. Finally, eluate 13 appeared as a brownish single spot, based on TLC analysis at a Rf of 0.49, suggesting compound S10 (89 mg) to be phenolic in nature. The gradient elution profile of subtraction FSiii on silica gel column is presented in Table 7.

[0129] Table 7: Gradient Elution Profile of Subtraction FSiii on silica gel Column

[0130] * Fractions afforded solid deposits after 24 h of elution, thus afforded S6 - S10 after filtration and cleaning in MeOH

[0131] The physical and TLC characterization of the ten elicited substances, S1 - S10, are presented in Table 8.

[0132] Table 8: Physicochemical Properties of Isolates from S. elata plant EXAMPLE 6

[0133] Spectroscopic Characterisation of Isolated Compounds from Sanicula elata

[0134] Nuclear Magnetic Resonance (NMR) Spectroscopy

[0135] The one-dimensional (1H and13C) and two-dimensional NMR spectral data of the isolated compounds were acquired in deuterium oxide (D2O) and deuterated methanol (CD3OD), purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). The NMR was recorded on an Avance 600 MHz Spectrometer (Bruker Biospin GmbH-Rheinstetten, Germany) at the Central Analytical Facilities, Stellenbosch University, Stellenbosch, South Africa. Tetramethyl silane (TMS) was used as an internal standard, while chemical shifts were recorded in part per million (ppm).

[0136] High-Resolution Mass Spectrometry (HRESI-MS)

[0137] The mass spectra of isolated compounds were recorded on a Quadrupole Time-of- Flight (QToF) Synapt G2 coupled to a Waters UPLC (LC-MS)., ESI probe, ESI Pos, Cone Voltage 15 V (Waters Corporation, Milford, MA, USA). The mass was determined by electrospray ionization method (HRESI-MS). The mass-to-charge ratios were acquired in the positive ion mode, over a scan range of m / z 100-2000.

[0138] Spectral characterization of compound S7

[0139] Based on NMR analysis, compound S7 was characterized as Sucrose, C12H22O11 (342.29 g / mol):

[0140] 1H NMR (400 MHz, D2O): 5 3.39 (1 H, t, J=13.2 Hz, H-4), 3.48 (1 H, dd, J=4.0, 4.0 Hz, H-2), 3.59 (2H, s, H-T), 3.68 (1 H, t, J=9.6 Hz, H-3), 3.73 (2H, d, J=3.2 Hz, H-6), 3.74 (2H, d, J=2.8 Hz, H-6’), 3.79 (1 H, m, H-5), 3.82 (1 H, m, H-5’), 3.97 (1 H, t, J=8.8 Hz, H-4’), 4.12 (1 H, d, J=8.8 Hz, H-3’), 5.33 (1 H, d, J=3.6 Hz, H-1 ).

[0141] 13C NMR (100 MHz, D2O): 5 60.1 (C-6), 61.3 (C-T), 62.3 (C-6’), 69.2 (C-4), 71 .0 (C-2), 72.4 (C-5), 72.5 (C-3), 73.9 (C-4’), 76.4 (C-3’), 81.3 (C-5’), 92.1 (C-1 ), 103.6 (C-2’). NMR of S7 is tabulated in Table 9 in comparison with the NMR spectra of sucrose, according to Hernandez-Garcia et al. (2019).

[0142] Structure elucidation of compound S7

[0143] The1H NMR spectrum of S7 showed eleven (1 1 ) oxygenated proton signals. Based on their integration, three of the signals were elucidated as methylene protons (CH2) at 5 3.59, 3.73 and 3.74, while the remaining eight were methine (CH) protons at 5H 3.39-5.33. The13C NMR spectrum of S7 showed twelve (12) signals representing 12 carbon atoms. Based on the HSQC experiment, the methylene carbons resonated as 5c 60.1 , 61 .3 and 62.3. The 2D NMR experiments (COSY, HSQC and HMBC) assisted in the full assignment of the molecule, and by comparison to the NMR spectral data of sucrose reported from Acacia famesiana fruits (Hernandez-Garcia et aL, 2019) as presented in Table 9, compound S7 was elucidated in this study as Sucrose (Figure 6), a disaccharide (having two sugar units), with one unit each of D- glucose (pyranose sugar, six-member ring) and L-fructose (furanose sugar, five-member ring) in a p-1 ,2 linkage.

[0144] Table 9: NMR data assignment of sucrose isolated as S7 and literature compound.

[0145] Chemical shifts (6H and 5C) are expressed in part per million (ppm), coupling constant (J) are expressed in hertz (Hz), singlet (s), doublet (d), triplet (t)

[0146] EXAMPLE 7

[0147] In Vitro Macrophage Activation of Selected Compounds (S1 , S7, S9 and S10)

[0148] Reagents

[0149] RAW 264.7 mouse macrophages were purchased from Cellonex (South Africa). Sulfanilamide Solution and NED Solution were made as per manufacturer’s instructions and products purchased from Promega. Lipopolysaccharide (LPS) and aminoguanidine were purchased from Sigma-Aldrich (St. Louise, MO, USA). RPMI1640 culture medium and fetal bovine serum (FBS) were from GE Healthcare Life Sciences (Logan, UT, USA).

[0150] Sample preparation and macrophage activation

[0151] The four samples, S1 , S7, S9 and S10 were screened against RAW 264.7 cells for their potential to activate macrophages. The four compounds were solubilized using DMSO to make a 100 mg / mL stocks and stored at 4°C until used. LPS was used to as a positive control for macrophage activation. RAW 264.7 cells were seeded in RPMI 1640 culture medium supplemented with 10% FBS (complete medium) into 96-well plates at a density of 1 x 105cells per well and allowed to attach overnight. The following day spent culture medium was removed and 50 pL of fraction aliquots (diluted in RPMI complete medium) added to give final concentrations of 10, 50 and 100 pg / mL. LPS was used as the positive control at 1 pg / mL. Cells were incubated for 24 hours. To quantify NO production, nitrite was measured in spent culture medium using the Griess reagent system based on the diazotization reaction originally described by Griess (1879). Later 50pL of the spent culture medium was transferred to a new 96-well plate and 50pL of sulphanilamide solution (1% sulphanilamide in 5% phosphoric acid) was added. Plates were incubated for 10 minutes at room temperature, protected from light, after which 50pL of NED solution (0.1 % / V-1 -napthylethylenediamine dihydrochloride in water) was added. Plates were incubated for 10 minutes at room temperature, protected from light, and absorbance was measured at 540 nm using a BioTek® PowerWave XS spectrophotometer (Winooski, VT, USA). A standard curve using sodium nitrite (100 pM in complete medium) was used to determine the concentration of NO (pM) in each sample. Figure 7 shows the result of the proinflammatory experiment.

[0152] Cytotoxicity screening

[0153] To confirm the absence of toxicity as a contributory factor, cell viability was assessed using MTT. This was done by the removal of the remaining of the medium and treatments in each well and replacing it with medium containing 0.5 mg / mL MTT and further incubated for 30 minutes at 37°C. Thereafter, MTT was removed and 100 pL DMSO was added to each well to solubilise the formazan crystals. Absorbance was measured at 540nm using a BioTek® PowerWave XS spectrophotometer (Winooski, VT, USA). Figure 8 shows the cytotoxic potential of compounds against the cells.

[0154] Disaccharides are naturally occurring and have been reported to have biological activities, such as trehalulose, which is an isomer of sucrose, which showed antioxidant activity by reducing the oxidative decomposition of omega-3 fatty acids embedded in a yogurt matrix in vitro (Kowalczyk et.al.,2015). In the present study, one compound, S7 (2-(4-Hydroxy-2,5-bis-hydroxymethyl-tetrahydrofuran-3-yloxy)-6-hydroxymethyl tetrahydropyran-3,4,5-triol), which is a disaccharide (sucrose) was characterized.

[0155] Based on the structural similarities between trehalulose, (glucose-fructose, with a 1 -1 glycosidic linkage) and the present sucrose, with a 1 -3 glycosidic linkage, the inventors hypothesize that this unusual disaccharide could be responsible for the antioxidant activity observed in the crude plant extract. The four compounds characterized from Sanicula elata aqueous fraction (F5) in this study did not modulate the RAW 264.7 cells when tested individually, resulting in no nitric oxide production. However, this does not rule out the presence of synergistic mechanisms between the compounds. Thus, it is likely that the observed immunomodulation reported from the crude extract and from the water fraction, has been driven by a combination of several major compounds in this plant.

[0156] REFERENCES

[0157] Hernandez-Garcia, E., Garcia, A., Avalos-Alanis, F.G., Rivas-Galindo, V.M., Delgadillo-Puga, C., del Rayo Camacho-Corona, M. 2019. Nuclear magnetic resonance spectroscopy data of isolated compounds from Acacia farnesiana (L) Willd fruits and two esterified derivatives. Data in Brief. Vol 22, 255-268.

[0158] Oriola, A.O., Aladesanmi, A. J., Idowu, T.O., Akinkunmi, Ezekiel O. Oyedeji, A. O., Ogunsina, M. O., Arthur, G., Schweizer, F. 2022. Saponin from Massularia acuminata Inhibits the Growths of Some Microbial and Human Cancerous Cells in vitro. Chemistry Africa. Vol 5, 1959-1966.

Claims

CLAIMS1 . A pharmaceutical composition comprising a pharmaceutically effective amount of a crude or purified ethanol extract of the plant Sanicula elata and one or more pharmaceutically acceptable carriers, diluents and / or excipients.

2. The pharmaceutical composition of claim 1 , wherein the composition further comprises an additional pharmaceutically active ingredient.

3. A crude or purified extract from the plant Sanicula elata for use in a method of treating a respiratory condition, the method comprising administering the extract to a subject in need thereof.

4. The crude or purified extract for use of claim 3, wherein the extract exhibits an immunomodulatory effect.

5. The crude or purified extract for use of claim 3 or 4, wherein the treating of the respiratory condition is by immunomodulation of macrophages.

6. The crude or purified extract for use of claim 5, wherein the immunomodulation of macrophages modulates the macrophages towards the killing (M1 ) phenotype.

7. The crude or purified extract for use of any one of claims 3 to 6, wherein the respiratory condition is selected from the group consisting of coughing, chest pain, runny nose, fever, asthma, chronic obstructive pulmonary disease, allergic nasitis, phlegm, acute and chronic bronchitis, bronchiolitis, sore throat, tonsillitis, laryngitis and pulmonary tuberculosis.

8. The crude or purified extract for use of any one of claims 3 to 7, wherein the extract is obtained from a whole plant of Sanicula elata.

9. The crude or purified extract for use of any one of claims 3 to 8, wherein the extract is an organic solvent-derived extract.

10. The crude or purified extract for use of claim 9, wherein the organic solvent is ethanol.

11. The crude or purified extract for use of any one of claims 3 to 10, wherein the method comprises administering the extract in combination with an additional pharmaceutically active ingredient.

12. The crude or purified extract for use of claim 11 , wherein the additional pharmaceutically active ingredient is one or more selected from the group consisting of isoniazid, sodium para amino salicylate, a fluoroquinolone, calcium methanesulfonate of isoniazid, cyclocerine, morphazinamide hydrochloride, rifampicin, ethambutol, sparfloxicin, pyrazinamide and streptomycin13. A method of treating a respiratory condition in a subject, comprising administering a crude or purified extract from the plant Sanicula elata to a subject in need thereof.

14. The method of claim 13, wherein the extract exhibits an immunomodulatory effect.

15. The method of claim 13 or 14, wherein treating the respiratory condition is by immunomodulation of macrophages.

16. The method of claim 15, wherein the immunomodulation of macrophages modulates the macrophages towards the killing (M1 ) phenotype.

17. The method of any one of claims 13 to 16, wherein the respiratory condition is selected from the group consisting of coughing, chest pain, runny nose, fever, asthma, chronic obstructive pulmonary disease, allergic nasitis, phlegm, acute and chronic bronchitis, bronchiolitis, sore throat, tonsillitis, laryngitis and pulmonary tuberculosis.

18. The method of any one of claims 13 to 17, wherein the extract is obtained from a whole plant of Sanicula elata.

19. The method of any one of claims 13 to 18, wherein the extract is an organic solvent- derived extract.

20. The method of claim 19, wherein the organic solvent is ethanol.21 . The method of any one of claims 13 to 20, wherein the method further comprises administering the extract in combination with an additional pharmaceutically active ingredient.

22. The method of claim 21 , wherein the additional pharmaceutically active ingredient is one or more selected from the group consisting of isoniazid, sodium para amino salicylate, a fluoroquinolone, calcium methanesulfonate of isoniazid, cyclocerine, morphazinamide hydrochloride, rifampicin, ethambutol, sparfloxicin, pyrazinamide and streptomycin.

23. Use of a crude or purified extract of Sanicula elata in the preparation of a medicament for use in a method of the prevention or treatment of a respiratory condition, the method comprising administering the medicament to a subject.

24. The use according to claim 23, wherein the respiratory-related condition may be selected from the group consisting of coughing, chest pains, runny nose, fever, asthma, chronic obstructive pulmonary disease, allergic nasitis, phlegm, acute and chronic bronchitis, bronchiolitis, sore throat, tonsillitis, laryngitis and pulmonary tuberculosis.

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