BACKGROUND OF THE INVENTION

The present invention relates to flower extracts from Leonotis leonurus var. albiflora white flower for use in methods of treating or preventing cellular damage of healthy cells caused by a cancer therapy. The flower extracts from L. leonurus var. albiflora white flower are particularly unique and useful as a chemoprotectant, as a post-chemotherapeutic and antioxidant supplement, boosting cell recovery following chemotherapy. The invention also relates to methods of treating or preventing cellular damage of healthy cells caused by a cancer therapy, comprising administering crude or purified flower extracts from L. leonurus var. albiflora white flower to a subject or administering compositions comprising said flower extracts to the subject.

Medicinal plants have been utilized by humans for thousands of years, providing the foundation of traditional medicine (TM) practices throughout history. The advent of modern conventional medicine (CM) came as a result of isolating active phytochemicals from known TM plants, and subsequently creating pure compounds that have characterized therapeutic roles. Since the isolation of therapeutic phytochemicals from plants, CM has focused on synthetic and semi-synthetic drug development, largely distributed by pharmaceutical companies. This is primarily due to the ‘single-drug/single-target’ nature of chemically synthesized compounds, as opposed to the often-uncertain pleiotropic nature of phytochemicals. However, CM drugs have proven inadequate against the increasing prevalence of complex, chronic, and other oxidative stress-related illnesses, such as diabetes, autoimmune disorders, cardiovascular diseases, and cancer, which require multiple targets of therapy. Furthermore, improved methods of isolating and characterizing therapeutic phytochemicals have also contributed to the growing acceptance and use of TM as a primary source of healthcare.

The growing interest in the use of TMs has spurred efforts at uncovering therapeutic compounds within medicinal plants, with the hope of identifying compounds which may aid in the treatment of chronic diseases. Among the many therapeutic phytochemicals found in common TM plants, phytocannabinoids (pCBs) of Cannabis sativa L. are of pharmacological interest, owing to their interaction with the human endocannabinoid system (ECS) and their role in disease treatment and prevention. Since the discovery of pCBs in C. sativa, pCB-like compounds capable of interacting with the human ECS have been identified in a number of other plant species. These atypical ligands of the human ECS, termed cannabimimetics, elicit similar pharmacological effects to pCBs of C. sativa, making them ideal candidates in the search for novel therapeutic phytochemicals to combat chronic illnesses.

South Africa boasts a rich diversity of plants used in TM, with over 3000 plants documented to have medicinal capacities. Some well-known South African TM herbs, including aloe (Aloe ferox), buchu (Agathosma spp.), cancer bush (Sutherlandia frutescens), rooibos (Aspalanthus linearis), and lion’s tail (Leonotis leonurus), have been investigated to uncover valuable therapeutic phytochemicals. Among these medicinal plants, the L. leonurus herb (commonly known as lion’s tail or wilde dagga) is of particular interest.

The Lamiaceae (mint) plant family are frequently defined by distinctive aromatic flowers. Herbs of the Lamiaceae family have been extensively utilized in TM practices across the world, with notable examples including plants of the genera Mentha, Salvia, Thymus, and Marrubium. Essential oils common across genera of the Lamiaceae family have shown promising therapeutic capabilities, often demonstrating a significant capacity for combating cellular oxidative stress, a primary contributor to the development of complex illnesses. The observed antioxidant capacity of the essential oils has been linked to the significant occurrence of phenolic compounds (phenolics) and terpenes that are distinctive of the Lamiaceae family. Phenolics are broadly defined as molecules that comprise at least one aromatic ring with one or more hydroxyl groups attached. The primary classes of phenolics include phenolic acids, stilbenes, lignans, and flavonoids classified by the number and arrangement of aromatic rings. Whereas terpenes are structurally defined by the presence of an isoprene unit and grouped according to the number of isoprene units within the chemical structure. The occurrence of antioxidant phenolics and terpenes is however not limited to plants of the Lamiaceae family. Notably, C. sativa of the Cannabaceae family is in close chemotaxonomic proximity to plants of Lamiaceae, sharing highly similar clusters of phenolics and terpenes.

Leonotis leonurus (L.) R. Br. is an easily cultivated, woody shrub endemic to South Africa, with an extensive background in South African TM. L. leonurus belongs to the Lamiaceae (mint) herb family and is mostly identified by its brightly coloured orange flowers that bloom biannually. L. leonurus are used in a variety of administrations to relieve common ailments ranging from common skin conditions to fever and pain. Recent investigations into the extracts L. leonurus leaves have revealed significant anti-diabetic properties of the whole extract, as well as anticonvulsant properties. However, most existing research on the TM therapeutic applications of L. leonurus extracts included extracts of the whole plant (leaves, stems, and aerial parts), and did not investigate the chemical composition of the flowers.

Only one relevant study can be found on the phytochemicals within the orange flowering tops of L. leonurus (Agnihotri et aL, 2009). This study revealed only six metabolites (dihydroxyphytyl palmitate, succinic acid, uracil, luteolin-7-O-glucoside, acteoside, and geniposidic acid) within the orange flowers of L. leonurus, none of which have known roles as cannabimimetics. However, a scarcer variety exhibiting entirely white flowers (Leonotis leonurus (L.) R. Br. var. albiflora) can also be found. While preliminary phytochemical studies were published by the inventors on identification of a phytocannabinoid-like compound in flowers from Leonotis lenourus var. albiflora Benth. (Hunter et al. BMC Res Notes (2020) 13:522), the phytochemical composition of this albinistic flower variety has not been thoroughly investigated. Chemotherapeutic agents are designed to target cancerous cells and suppress tumour progression in cancer patients, often through cytotoxic mechanisms. However, their cytotoxic effects are often non-specific to cancerous cells only, leading to significant side effects when undergoing chemotherapy. Clinical data has suggested that administration of dietary antioxidants, parallel to, or following, chemotherapy treatment, may significantly alleviate the chemotoxin sideeffects on healthy cells and aid in the recovery of cells following chemotherapy.

Accordingly, the inventors investigated the phytochemical makeup of Leonotis leonurus (L.) R. Br white (var. albiflora) flower extracts for the potential occurrence of therapeutic compounds, including pCB-like compounds, and further assessed the therapeutic potential of the flower extract to aid in oxidative stress-related human diseases and combat the adverse effects of chemotherapy on healthy cells.

SUMMARY OF THE INVENTION

The present invention broadly relates to flower extracts from L. leonurus var. albiflora white flower for use as a chemoprotectant and for boosting cell recovery following chemotherapy. The invention further relates to the flower extracts for use in methods of treating or preventing cellular damage of healthy cells caused by a cancer therapy, the methods comprising administering the flower extract to a subject. Also provided are methods of treating or preventing cellular damage of healthy cells caused by cancer therapy comprising administering a crude or purified flower extract from L. leonurus var. albiflora white flower to the subject or administering compositions comprising said flower extracts to the subject.

According to a first aspect of the invention there is provided for a crude or purified flower extract from L. leonurus var. albiflora white flower, for use in a method of treating or preventing cellular damage of healthy cells caused by a cancer therapy in a subject in need thereof, the method comprising administering the flower extract to the subject.

In a first embodiment of the flower extract for use, the flower extract may be administered to the subject before, during or after the cancer therapy, for example as an adjuvant therapy or a post-chemotherapeutic agent.

According to a second embodiment of the flower extract for use, the flower extract increases cell viability of healthy cells or increases cell proliferation.

In a third embodiment of the flower extract for use, the cancer therapy may be a chemotherapeutic agent, including a cis-platinum compound.

In an alternative embodiment of the flower extract for use, the cancer therapy may be radiation therapy.

According to a further embodiment of the flower extract for use, the flower extract may be obtained using an organic solvent. Several organic solvents suitable for extracting phytochemical compounds from plants are known in the art, including acetonitrile, ethanol, methanol, butanol, and mixtures thereof. Preferably, the organic solvent is acetonitrile. In another embodiment of the flower extract for use, the subject may be a mammal, in particular a human subject.

According to yet a further embodiment of the flower extract for use, the flower extract may be provided together with a pharmaceutically acceptable carrier and may be formulated in a pharmaceutical composition.

In another embodiment of the flower extract for use, the flower extract may be administered to the subject by topical, parenteral, or oral administration.

According to a second aspect of the present invention there is provided for a method of treating or preventing cellular damage of healthy cells caused by a cancer therapy in a subject in need thereof, comprising administering a crude or purified flower extract from L. leonurus var. albiflora white flower to the subject.

In a first embodiment of the method, the method may comprise administering the flower extract to the subject before, during or after the cancer therapy, for example as an adjuvant therapy or a post-chemotherapeutic agent.

According to a second embodiment of the method, the method comprising administering the flower extract increases cell viability of healthy cells or increases cell proliferation.

In a third embodiment of the method, the cancer therapy may be a chemotherapeutic agent, including a cis-platinum compound.

In an alternative embodiment of the method, the cancer therapy may be radiation therapy.

According to a further embodiment of the method, the flower extract may be obtained using an organic solvent. Several organic solvents suitable for extracting phytochemical compounds from plants are known in the art, including acetonitrile, ethanol, methanol, butanol, and mixtures thereof. Preferably, the organic solvent is acetonitrile.

In another embodiment of the method, the subject may be a mammal, in particular a human subject.

According to yet a further embodiment of the method, the flower extract may be comprised in a pharmaceutical composition and may further comprise a pharmaceutically acceptable carrier.

In another embodiment of the method, the flower extract may be administered to the subject by topical, parenteral, or oral administration.

According to a third aspect of the present invention there is provided for the use of a crude or purified flower extract of leonurus var. albiflora white flower in the manufacture of a medicament for use in a method of treating or preventing cellular damage of healthy cells caused by a cancer therapy in a subject in need thereof, the method comprising administering the medicament to the subject. BRIEF DESCRIPTION OF THE FIGURES

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

Figure 1 : Preliminary HPTLC cannabimimetic screening of L. leonurus and L. leonurus var. albiflora. Phytochemical thin-layer chromatogram profiles of L. leonurus crude extracts (75% acetonitrile). WF: L. leonurus var. albiflora white flower extract. WL: L. leonurus var. albiflora leaf extract. OF: L. leonurus orange flower extract. OL: L. leonurus leaf extract. Mobile phase: chloroform, 10% MeOH. Loading volume: 6 pL. pCB-specific compounds were derivatized with FBBS (0.1%, 1 mM NaOH) under dark conditions. HPTLC plates were captured under visible light following derivatization. The respective observed track Rf values and visualized track hues are indicated in Table 1 of Example 1.

Figure 2: UV-HPTLC phytochemical profile of L. leonurus and L. leonurus var. albiflora flower extracts. Phytochemical thin-layer chromatogram profiles of L. leonurus var. albiflora white flower (WF) and L. leonurus orange flower extracts (OF) (75% acetonitrile, freeze- dried, resuspended in ddH ₂ O and filter sterilized). Mobile phase: chloroform, 10% MeOH. Loading volume: 12 pL. Compounds containing aromatic rings were visualized under UV254 light. The respective observed track R _f values and visualized track hues are indicated in Table 3 of Example 1.

Figure 3: Optimized HPTLC cannabimimetic screening of L. leonurus and L. leonurus var. albiflora flower extracts. Phytochemical thin-layer chromatogram profiles of L. leonurus var. albiflora white flower (WF) and L. leonurus orange flower extracts (OF) (75% acetonitrile, freeze-dried, resuspended in ddH ₂ O and filter sterilized). Mobile phase: chloroform, 10% MeOH. Loading volume: 12 pL. pCB-specific compounds were derivatized with FBBS (0.1 %, 1 mM NaOH) under dark conditions. HPTLC plates were captured under visible light following derivatization. The respective observed track R _f values and visualized track hues are indicated in Table 2 of Example 1 .

Figure 4: LC-MS/MS phytochemical profile of L. leonurus var. albiflora white flower acetonitrile extract. Total ion chromatogram (positive [M+H] ⁺ and negative [M-H]- ionization overlay in red and blue, respectively) of the predicted metabolites within the white flower extract of L. leonurus var. albiflora (75% v/v acetonitrile extract, freeze-dried and reconstituted in ddH ₂ O). Samples were prepared for LC-MS/MS analysis with HPLC-grade MeOH (50% v/v). Compound peaks were analyzed based on their ion adduct m/z and screened using the Metabolomics Workbench metabolite repository. Peak height represents relative intensity of the compound. The x-axis represents compound retention time. The respective predicted compounds are indicated in Table 4.

Figure 5: LC-MS/MS phytochemical profile of L. leonurus orange flower acetonitrile extract. Total ion chromatogram (positive [M+H] ⁺ and negative mode [M-H]- ionization overlay in red and blue, respectively) of the predicted metabolites within the orange flower extract of L. leonurus (75% v/v acetonitrile extract, freeze-dried and reconstituted in ddH ₂ O). Samples were prepared for LC-MS/MS analysis with HPLC-grade MeOH (50% v/v). Compound peaks were analyzed based on their ion adduct m/z and screened using the Metabolomics Workbench metabolite repository. Peak height represents relative intensity of the compound. The x-axis represents compound retention time. The respective predicted compounds are indicated in Table 5.

Figure 6: LC-MS/MS based putative identification of the cannabimimetic compound adrenoyl- EA in L. leonurus var. albiflora whole white flower extract. A and B: L. leonurus var. albiflora whole white flower extract TIC spectra in negative [M-H]- and positive [M+H] ⁺ mode ionization, respectively (as reported in Figure 3). C and D: TIC spectra (in negative [M-H]- and positive [M+H] ⁺ mode ionization, respectively) exhibiting the isolated tentative cannabimimetic compound (as obtained from the HPTLC plate scrape; track e, Figure 3). The isolated peak (peak x) was tentatively identified as the cannabimimetic compound adrenoyl-EA, based off the observed maximum relative intensity and the corresponding ion adduct m/z and retention time to the respective whole flower extract spectra (m/z = 374.26, RT: 30.78 min for negative and m/z = 376.26, RT: 30.77 min for positive [M+H] ⁺ mode ionization, respectively).

Figure 7: The total antioxidant capacities of L. leonurus var. albiflora white flower and L. leonurus orange flower extracts. The total antioxidant capacity (TAC) of the L. leonurus var. albiflora white flower (WF) and L. leonurus orange flower (OF) acetonitrile extracts, as expressed in nmole/pL Trolox equivalents (0.5 mg/mL whole flower extract). Each sample was analysed in triplicate (n=3) and the mean ± SEM graphically indicated. Statistical significance between the TAC of two flower varieties was determined using a two-tailed t-test (****p<0.0001 ).

Figure 8: The human gDNA protecting potential of L. leonurus var. albiflora white flower and L. leonurus orange flower extracts. Control sample lanes: U (undamaged gDNA), D (Fenton’s oxidative reagent damaged gDNA), P (Trolox antioxidant protected gDNA). Treatment samples: Lanes 1 -5: 0.5, 0.25, 0.125, 0.0625, and 0.03125 mg/mL, respectively for the whole white flower (WF) and orange flower (OF) extracts. The red line serves as the index protection potential based off the protected gDNA sample (P).

Figure 9: Cytotoxic effect of cisplatin on human breast epithelial (MCF-12A) cells. The dose-dependent cytotoxic effect of cisplatin on MCF-12A human breast epithelial cells. Cells were exposed to a range of concentrations of cisplatin (10, 20, and 30 pg/mL; A, B, C, and D, respectively) and assessed for cellular viability by comparing cell morphology changes and in an MTT assay, following 48 h of cisplatin treatment and relative to an untreated control (UTC). All samples were assessed in experimental replicates (n=7) and the mean ± SEM graphically indicated. The IC ₅ o value of cisplatin was determined as the concentration of cisplatin exhibiting 50% relative cell viability (16.43 pg/mL). Figure 10: Assessment of L. leonurus var. albiflora white flower and L. leonurus orange flower extracts on MCF-12A cell viability. Cell viability of MCF-12A cells was determined through an MTT assay 48 h post-treatment with a range of concentrations of the var. albiflora white flower (WF) and orange flower (OF) extracts of L. leonurus (1 , 0.5, 0.25, and 0.125 mg/mL whole flower extract). Percentage viability was determined relative to an untreated control cell line (UTC), with cisplatin as a cytotoxic control (Cis IC50: 16.43 pg/mL). Each sample was analysed in replicate (n=7) and the mean ± SEM graphically indicated. Statistical significance of increased cellular viability was determined using a two-tailed t-test, relative to the untreated vehicle control (***p = 0.0001 , ****p < 0.0001 ).

Figure 11 : Assessment of the potential MCF12-A cellular protection capacities of L. leonurus var. albiflora white flower and L. leonurus orange flower extracts against cisplatin-induced cytotoxicity. MCF-12A cell viability was determined using an MTT assay and by visually comparing cell morphology changes following respective sample and control treatments (UTC: untreated control; Cis: IC ₅ o 16.43 pg/mL for 48 h; W and O: 0.5 mg/mL white and orange flower extract for 48 h, respectively; W:Cis and O:Cis: 0.5 mg/mL white and orange flower extract prior treatment for 24 h, and subsequent treatment with 16.43 pg/mL for 48 h). Each sample was analysed in replicate (n=7) and the mean ± SEM graphically indicated. Statistical significance of increased/decreased cellular viability was determined using a two-tailed t-test, relative to the untreated vehicle control (****p < 0.0001 )

DETAILED DESCRIPTION OF THE INVENTION

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.

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.

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.

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.

The present invention relates to the use of crude or purified extracts from L. leonurus var. albiflora white flower as a chemoprotectant and post-chemotherapy cell proliferation aid. The invention further relates to adjuvant and post-chemotherapeutic compositions comprising the extract, wherein the extract is capable of increasing cell proliferation following chemotherapy administration, as well as protecting healthy cells from apoptosis during chemotherapy administration, thereby increasing cell viability during chemotherapy administration.

The inventors of the present invention have found that, when comparing the HPTLC profiles of white and orange flower varieties of L. leonurus, it is evident that the flower extract profiles of both varieties of L. leonurus exhibited unique compounds to those of observed in the corresponding leaf extract profile. Based on the ubiquitous occurrence of aromatic moieties (which nominally absorb light at 254nm) within the molecular structure of antioxidant phytochemicals (i.e. phenolics and terpenes), the inventors tentatively identified a number of unknown phenolics and/or terpenes within both the white and orange flower extracts. Further, the evident difference in the HPTLC profiles of the white and orange flower extracts suggests a significant level of diversity within the phytochemical makeup of the two flower varieties, and particularly within the cannabinoid-related group of compounds. LC-MS/MS data of L. leonurus var. albiflora white flower acetonitrile extract exhibited a range of 30 tentatively identified compounds within the groups of terpenes and terpene derivatives, iridoid glucosides, flavonoids, flavonoid glycosides, fatty acids, and endocannabinoids. Individual compounds of the groups coumarins (Scopoletin), furans (Methanofuran), ethanolamines (1 -Eicosyl-glycero-3- phosphoethanolamine), and diacylglycerophosphates (1 -Dodecanoyl-2- (4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoyl)-glycero-3-phosphate) were also predicted to occur.

A great majority of phytochemical research has largely focused on the identification of compounds that exhibit ROS scavenging capacities, in an effort characterize dietary antioxidants capable of combating oxidative stress-related diseases. To this extent, it has been widely observed that phenolics and terpenes exhibit significant antioxidant capacities, providing a promising strategy in limiting oxidative stress. Among the tentatively identified compounds within the L. leonurus var. albiflora white flower extract, the inventors found a range of subclasses of phenolics. Inclusive were the flavonoids naringenin 7-O-glucoside, naringenin 7-[3-acetyl-6-p- coumaroylglucoside], apigenin 8-C-glucoside, apigenin, and luteolin 7-4’-dimethyl ether 6-C- glucoside. Flavonoids are a well-studied class of phenolics known to protect DNA from oxidative stress. Naringenin, apigenin, and luteolin have all presented antioxidant activities primarily linked to mechanisms of cytokine suppression. Interestingly, none of the tentatively identified flavonoids within the leonurus var. albiflora white flower extract was predicted to occur in the orange flower extracts of the L. leonurus. The majority of predicted flavonoids of the orange flower extract included luteolin derivatives. Along with the identified luteolin derivatives, the flavonoids epigallocatechin 3-O-(3,5-dimethylgallate), chlorogenic acid, and methyl cinnamic acid were also predicted, all of which are capable of ROS scavenging activities.

Similar to flavonoids, terpenes and their derivatives also exhibit therapeutic benefits attributed to their role in limiting oxidative stress. LC-MS/MS analysis of the white flower extract of L. leonurus var. albiflora exhibited numerous terpene compounds, as predicted from the metabolite screening experiments. The predicted terpenes included p-cymene, geniposidic acid, geniposide, loganic acid, harpagide, marrubiin, and leoleolorin A. The antioxidant effects of p- cymene, a monoterpene present in numerous medicinal plants, have been observed in a variety of studies. Iridoid glucosides, including the tentatively identified geniposidic acid, geniposide, loganic acid, and harpagide, are monoterpene derivatives commonly found in plants of Lamiaceae and are among the numerous antioxidant-capable compounds of focus in pharmacology. Marrubiin is a labdane diterpene that has previously been investigated as a constituent of Leonotis leonurus (L.) R. Br. leaf extracts capable of significant anti-diabetic properties. Beyond its potential in relieving diabetic symptoms, marrubiin has recently been investigated for its antifungal and antioxidant capacities.

Herein, the inventors show that MCF-12A cell treatment with varying concentrations of L. leonurus (L.) R. Br. var. albiflora white flower extracts exhibited a proportionate increase in percentage cell viability, relative to the untreated control cell line. Independent controls included MCF-12A cell treatments with cisplatin only, white flower extract only, and orange flower extract only, with both the white flower and orange flower extract controls showing significantly increased cellular viability, when compared to the untreated control cell line. Both the white and orange flower extracts showed significant levels of cellular protection against cisplatin, when compared to the cisplatin only treatment control. The percentage protection of each flower extract relative to the cisplatin only control was also discerned, indicating the level of cellular proliferation. The chemoprotective effect of both flower extracts was further observed by the significant level of MCF-12A cell viability even in the presence of cisplatin, as discerned by adhered and transparent viable cells in the respective combined flower extract and cisplatin treatments, when compared to the rounded and darkened cells present in the cisplatin only control treatment. The ability of both the white and orange flower extracts to significantly increase cellular viability and provide significant levels of cellular protection against cisplatin was particularly surprising to the inventors, given the differences between the phytochemical composition of the flower extracts.

The degree of cell adherence and the level of observed translucence has been directly related to cell viability of MCF-12A cells. From these results, both flower extracts not only exhibited the lack of cytotoxic effects but presented a significant increase in cell viability (even at low extract concentrations), relative to untreated cells. Following the observed MCF-12A increased cellular viability as a result of exposure to the respective flower extracts, the inventors treated MCF-12A cells with the white and orange flower extracts and subsequently exposed the cells to cisplatin. The observed results of the MTT assay following the combined treatment suggested a significant ability of both the white (var. albiflora) and orange L. leonurus flower extracts to protect healthy MCF-12A cells against the cytotoxic action of cisplatin. This suggestion was supported by the observed cellular morphologies following the combined treatment, wherein cells that were initially treated with the flower extracts prior to cisplatin treatment showed a greater number of viable cells, when compared to the evident widespread MCF-12A cell death consequent of cisplatin treatment alone. It is surprising that, although the white and orange flower varieties of L. leonurus flower extracts have completely different phytochemical profiles, extracts of both varieties provide a chemoprotective effect and increase cell viability and proliferation either during or following treatment of cells with a chemotherapeutic.

Thus, the present invention relates to flower extracts from L. leonurus var. albiflora white flower or pharmaceutical compositions comprising the flower extracts, for use in methods of treating or preventing cellular damage of healthy cells caused by a cancer therapy by administering the flower extracts or compositions to the subject.

As used herein, the term “treatment”, “treating” or “treat” refers to any administration of the extract or compositions herein to partially or completely alleviate, ameliorate, and/or reduce severity of cellular damage of healthy cells caused by a cancer therapy and/or increase healthy cell proliferation. Accordingly, the treatment may be to reduce, allay or mitigate the side-effects of the cancer therapy.

The term “prevention”, “preventing” or “prevent”, when used in relation to cellular damage of healthy cells caused by a cancer therapy, includes administration of the flower extracts or compositions herein to increase cell viability during treatment of a subject with a cancer therapy relative to a subject which does not receive the flower extract or composition. Administration for treatment or prevention may be before, during or after the cancer therapy.

It will be understood that the extract of the invention may be in the form of a crude flower extract, a purified flower extract or a pharmaceutical composition comprising the flower extract.

As used herein the term “crude extract” or “crude flower extract” refers to a concentrated preparation of a plant flower extract obtained by removing secondary metabolites from the crude plant flower material with the aid of a suitable solvent. This may be done, for example, by submerging the crude plant material in a suitable solvent, removing the solvent and consequently evaporating all or nearly all of the solvent. As used herein the term “purified extract” or “purified flower extract” refers to a flower extract obtained by separating the constituent parts of the crude flower extract from each other. By way of a non-limiting example, the constituent parts of the crude flower 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.

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

The solvent may be an organic solvent. Organic solvents typically used in the preparation of plant extracts include but are not limited to acetonitrile, ethanol, methanol, butanol dichloromethane, chloroform, acetone and/or mixtures thereof. Any appropriate route of administration of the extracts or pharmaceutical compositions comprising the extracts may be employed, such as, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracistemal, intraperitoneal, intranasal, aerosol, topical, or oral administration.

As used herein the term “subject” includes a mammal, preferably a human or animal subject, but most preferably the subject is a human subject.

“Suitable forms” of the pharmaceutical composition for may include, for example, tablets, capsules, tinctures, powers, inhalants and/or liquids.

Other pharmaceutically acceptable ingredients may be used with the extracts or pharmaceutical compositions of the invention. The term “pharmaceutically acceptable” refers to properties and/or substances which are acceptable for administration, such as parenteral, or oral 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.

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 extract, mixture, pharmaceutical composition and/or medicament to a subject.

It will be appreciated that the crude or purified flower extract and/or pharmaceutical composition comprising the crude or purified flower extract may also be used in applications for animal and veterinary products.

The pharmaceutical compositions, flower extracts, and compounds of the invention can be provided either alone or in combination with other active compounds (for example, small molecules, nucleic acid molecules, peptides, or peptide analogues).

The use of the flower extracts or pharmaceutical compositions or methods of treatment and/or prevention using the flower extracts or pharmaceutical compositions entails administration of an effective amount of the flower extract or a pharmaceutical composition to a subject in order to provide a chemotherapeutic protective effect or to increase cell viability or cell proliferation during or following administration of a chemotherapeutic substance or ingredient. The term “effective amount” in the context of providing a chemotherapeutic protective effect or increasing cell viability or cell proliferation during or following administration of a chemotherapeutic active ingredient refers to the administration of an amount of the active flower extract or the pharmaceutical composition containing the flower extract to an individual, either a single dose or several doses of the flower extract or pharmaceutical composition containing the flower extract, to achieve the desired therapeutic result.

Any of the compositions of the invention may be administered in a single dose or in multiple doses. It will be appreciated that the exact dosage and frequency of administration of the effective amount will be dependent on several factors. These factors include the individual components used, the formulation of the flower extract or pharmaceutical composition containing the flower extract, the nature and 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, the dosage of chemotherapeutic 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.

Dosage values may vary and be adjusted over time according to the individual need and the judgment of the person administering or supervising the administration of the flower extracts or pharmaceutical compositions of the invention.

The examples provided below are offered by way of illustration and not by way of limitation.

All statistical analyses were performed using the GraphPad Prism software (GraphPad Prism v 7.0, GraphPad Software, California, USA, www.graphpad.com). All observed experimental values were analyzed based the mean ± the standard error of the mean (SEM) across the experimental replicates. Statistical significance was determined using a two-tailed t test, with significance being determined based off a resultant p value < 0.05.

EXAMPLE 1

Phytochemical profiling and pCB screening of two Leonotis leonurus flower varieties

Plant material and metabolite extraction

Flowers of both white and orange L. leonurus plants were harvested at the Bottom Road Sanctuary, Zeekoevlei, Western Cape Province, South Africa (GPS coordinates: -34.057951 , 18.499391 ). All harvested flower samples were stored at -20 °C in preparation for analysis. Harvested plant material was collected in liquid N ₂ and freeze-dried (20 h; Sentry 2.0 SP Scientific, USA). The freeze-dried plant material was then finely ground in liquid N ₂ using a pestle and mortar. The ground plant material was placed in a microfuge tube and acetonitrile subsequently added as previously described (Routaboul et aL, 2006; 50 mg/mL, 75% v/v acetonitrile). Each sample was vortexed vigorously and placed on ice (30 s). Samples were subsequently sonicated and centrifuged at room temperature (water bath sonication >200W ultrasonic power, 20 min; 13 000 x g, 5 min, RT). The supernatant of each sample was placed in a new microfuge tube and acetonitrile was added to the microfuge tube containing the remaining pellet, which were both stored overnight (1 mL 75% v/v acetonitrile, 4 °C). Subsequently, the pellet was centrifuged, and the supernatant subsequently pooled with the stored supernatant from the primary extraction (13 000 x g, 5 min, RT). Each pooled extraction was filter sterilized concentrated using a vacuum evaporator (EZ-2, GeneVac, UK; 500 pL, > 4 h). The concentrated extract was weighed and subsequently reconstituted in ddH ₂ O to create a stock solution of extract (0.05 mg/pL whole flower extract). HPTLC phytochemical screening

Each prepared sample extract was loaded onto precoated silica coated high performance thin layer chromatography (HPTLC) plates with UV254 fluorescent indicator (Macherey-Nagel, Germany; 12 pL loading V, millipore 60 silica). The developing chamber was conditioned with the mobile solvent phase and the plate was subsequently placed in the chamber and allowed to develop fully before the plate was removed and dried fully (CAMAG, Switzerland; 30 min; chloroform, 10% v/v methanol). The developed plate was visualized in a UV-viewing cabinet and the resulting profile captured (Spectroline® CM Cabinet, Sigma-Aldrich). The plate was sprayed with a reagent solution of Fast Blue B salt (FBBS) dissolved in NaOH and incubated in the dark overnight (1% w/v FBBS, 1 mM NaOH). Following development, the resulting profiles were captured using a high-resolution image scanner. Potential compounds of interest were scraped from the silica plate into a microfuge tube and dissociated from the silica with HPLC grade MeOH and placed in a microfuge tube and stored in preparation for LC-MS/MS analysis (500 pL, 50% v/v MeOH, stored at -20 °C).

Preliminary HPTLC phytochemical and cannabimimetic screening of L. leonurus orange flower and L. leonurus var. albiflora white flower extracts.

Based on the derivatisation of pCBs with FBBS and the consequent chromophore development, HPTLC profiles of L. leonurus and L. leonurus var. albiflora leaf and flower acetonitrile extracts presented a range of potential cannabimetic compounds (Figure 1 ). The identified compound tracks from each extract were evaluated based on the visualized hue and respective Rf value of the track (Table 1. The HPTLC profile of the white flower extract of L. leonurus var. albiflora presented 4 tracks positive for potential cannabimimetics (Figure 1 , Table 1 ; a: Rf 0.09; red/purple hue, b: Rf 0.63; pink/brown hue, c: Rf 0.70; red/purple hue, d: Rf 0.91 ; pink/orange hue). The corresponding L. leonurus var. albiflora leaf extract HPTLC profile showed 2 faint positive tracks (Figure 1 , Table 1 ; e: Rf 0.07; brown/pink hue, f: Rf 0.63; brown/pink hue). The HPTLC profile of L. leonurus showed 4 positive tracks in the orange flower extract, and a further 4 tracks in the corresponding leaf extract (Figure 1 , Table 1 ; flower extract: g: Rf 0.02; yellow hue, h: Rf 0.1 1 ; red/purple hue, i: Rf 0.68; pink/orange hue, j: Rf 0.72; pink/orange hue, leaf extract: k: Rf 0.02; yellow hue, I: Rf 0.11 ; brown hue, m: Rf 0.40; yellow hue, n: Rf 0.69; brown hue). When compared to the leaf extract profiles of both varieties of L. leonurus, the flower extract profiles exhibit more distinct chromophores with visualized track hues typical of pCBs (Figure 1 ). Based on this preliminary screening, the white and orange flowers of L. leonurus var. albiflora and L. leonurus were selected for downstream optimization and phytochemical analyses. Table 1 : Preliminary positively identified cannabimimetic compounds of L. leonurus and L. leonurus var. albiflora

Optimized HPTLC phytochemical profiles of L. leonurus and L. leonurus var. albiflora flower extracts

Based on the preliminary HPTLC screening for pCBs (Figure 1 ), the flower extract samples of both L. leonurus and leonurus var. albiflora were selected for HPTLC optimization and downstream phytochemical analyses. The developed HPTLC plates (optimized extract purification and increased sample loading volume) plates were first visualized under UV254 light (Figure 2), followed by pCB-specific derivatization with FBBS (Figure 3). The UV254 HPTLC profiles presented 5 potential aromatic compounds within both the white ( leonurus var. albiflora) and orange (L. leonurus) flower extracts (Figure 2, Table 2; a: Rf 0.10, b: Rf 0.35, c: Rf 0.59, d: Rf 0.70, e: Rf 0.98, f: Rf 0.10, g: Rf 0.41 , h: Rf 0.62, i: Rf 0.71 , j: Rf 0.98). The same HPTLC plates produced a variety of potential positive tracks for pCBs for both the L. leonurus var. albiflora white flower (Figure 3, Table 3; a: Rf 0.05; purple hue, b: Rf 0.21 ; yellow hue: c: Rf 0.33; pink/brown hue, d: Rf 0.40; purple hue, e: Rf 0.58; orange/red hue, f: Rf 0.63; purple hue, g: Rf 0.69; brown hue, h: Rf 0.81 ; purple hue, i: Rf 0.90; pink hue) and the L. leonurus orange flower acetonitrile extracts (Figure 3, Table 3; j: Rf 0.08; purple/brown hue, k: Rf 0.13; brown hue: I: Rf 0.22; brown hue, m: Rf 0.41 ; orange hue, n: Rf 0.46; purple hue, 0: Rf 0.56; orange/brown hue, p: Rf 0.61 ; orange hue, q: Rf 0.70; brown hue, r: Rf 0.81 ; purple/brown hue, s: Rf 0.90; pink hue). Table 2: U V254 derivatized HPTLC compound tracks of L. leonurus and L. leonurus var. albiflora

Table 3: Tentatively identified cannabimimetic compounds of L. leonurus and L. leonurus var. albiflora flower extracts

Tandem mass spectrometry LC-MS/MS analysis

LC-MS/MS analyses were performed by the Central Analytical Facility (CAF) at Stellenbosch University, as previously described with a Waters Synapt G2 quadrupole time-of- flight mass spectrometer (Waters Corporation, Milford, MA, USA; Loedolff et aL, 2017) equipped with a Waters Acquity UPLC. Samples were separated on a Waters UPLC BEH C18 column at a controlled flow rate and temperature (2.1 x 100 mm, 1.7 pm; 0.25 ml/min, 55 C). Solvent A consisted of 0.1% (v/v) formic acid in water and solvent B was 0.1% (v/v) formic acid in acetonitrile. The mobile phase gradient was initiated at 100% solvent A and linearly reduced to 28% solvent A (1 min at 100%; 22 min linear reduction). Subsequently, the mobile phase was changed to 40% solvent B followed by a wash step in 100% solvent B before the column was reequilibrated to the initial conditions (8 min at 40%; 1 min wash; 4 min equilibrate). Electrospray ionization was applied and samples were analysed in a negative mode run and a positive mode run. Data was acquired in MSE mode, which consist of a high collision energy scan range of m/z 125-1500 and a low collision energy scan from m/z 40-1500. The photo diode array detector was set to scan from 220-600 nm. The capillary voltage was set at 3.5 kV and the collision energy either at 6 V (low collision energy scan from) or 30-60 V (high collision energy scan). The cone voltage, source temperature, and desolvation temperature were set (15 V, 120 °C, 275 °C). The desolvation and cone gas (nitrogen) flows were similarly set and monitored (650 L/h and 50 L/h, respectively). Sodium formate was used for calibration and leucine encephalin was infused in the background as lock mass for accurate mass determinations. Metabolites were monitored using their deprotonated quasi-molecular ions.

Compounds were tentatively identified using the Metabolomics workbench (Sud et aL, 2016). The database was searched, using the m/z mass obtained from total ion chromatograms, with parameters set to the negative [M-H]- and positive ion [M+H] ⁺ mode, respectively, and a mass tolerance of +/- 0.2 m/z. Tentative identification was based on the accurate mass and fragment ions of the specific peaks compared to literature and was carried out in conjunction with independent metabolite repositories, Metabolomics Workbench, PubChem, and METLIN Metabolite and Chemical Entity Database.

Phytochemical composition of L. leonurus and L. leonurus var. albiflora flower extracts The L. leonurus var. albiflora white flower acetonitrile extract exhibited a range of 30 tentatively identified compounds within the groups of terpenes and terpene derivatives, iridoid glucosides, flavonoids, flavonoid glycosides, fatty acids, and endocannabinoids (Figure 4, Table 4). Individual compounds of the groups coumarins (Scopoletin), furans (Methanofuran), ethanolamines (1 -Eicosyl-glycero-3-phosphoethanolamine), and diacylglycerophosphates (1 - Dodecanoyl-2-(4Z,7Z, 10Z, 13Z, 16Z, 19Z-docosahexaenoyl)-glycero-3-phosphate) were also predicted to occur. Within the L. leonurus orange flower acetonitrile extract, 32 compounds were tentatively predicted to occur (Figure 5, Table 5). These compounds included various terpenes and derivatives, fatty acids, flavonoids, flavonoid glycosides, spermidine hydroxycinnamic acids, eicosanoids, phytocannabinoids, and endocannabinoids. Also inclusive were the individual compounds within the groups of proline derivatives (4-Hydroxystachydrine) and ethanolamines (1 -Eicosyl-glycero-3-phosphoethanolamine). All predicted compounds were presented by their ion adduct m/z and retention time, as observed by the LC-MS/MS spectral analyses (Figures 4 and 5). With the use of the Metabolomics Workbench repository compounds were further arranged by their experimental ion m/z, mass error, neutral m/z, molecular formula, isotope fragments, and compound group (Tables 4 and 5). From the observed HPTLC plate of the white flower extract of L. leonurus var. albiflora, one compound track for further characterization, based on its distinct orange/red hue (track e, Figure 3) was isolated. LC-MS/MS assessment of this isolated compound exhibited a single peak at maximum intensity matching a corresponding peak from the whole white flower extract LC-MS/MS spectra (Figure 6). This isolated compound was tentatively identified as adrenoyl-EA.

Table 5: Tentatively identified compounds within the orange flowers of L. leonurus

EXAMPLE 2

Analysis of the antioxidant capacity and chemoprotective ability of Leonitis leonorus flower extracts

Total antioxidant capacity (TAC) of both the white (var. albiflora) and orange flower extracts of L. leonurus

Samples of the L. leonurus white (var. albiflora) and orange whole flower extracts were prepared from the extract stocks as used in Example 1 and diluted in ddH ₂ O (0.5 mg/mL). Samples were subsequently analyzed using the Total Antioxidant Capacity Assay Kit (Cat# MAK187, Sigma-Aldrich, South Africa). The protocol was carried out as per manufacturer’s instructions, including the use of Trolox (a vitamin E derivative) as a standard control.

Acetonitrile extracts of both the white (var. albiflora) and orange flowers of L. leonurus were further analyzed to determine their total antioxidant capacity (TAC), as expressed in Trolox equivalent units, a vitamin E antioxidant standard (Figure 7). The orange flower acetonitrile extract of L. leonurus exhibited a significantly greater TAC, displaying a 2.91 -fold increase when compared to that of the white flower acetonitrile extract of L. leonurus var. albiflora (0.5 mg/mL acetonitrile whole flower extract: 7.75 ± 0.07 nmole/pL and 2.66 ± 0.04 nmole/pL for the orange and white flower extracts, respectively).

Human genomic DNA protection assay

Human genomic DNA (gDNA) damage/protection assays were performed as previously described (Jiang et aL, 2012; Xonti et aL, 2020) The flower extract samples were first prepared in a dilution range using ddH ₂ O (0.5 mg/mL; 0.25 mg/mL; 0.125 mg/mL; 0.0625 mg/mL; 0.03125 mg/mL whole extract). Human gDNA (0.5 pg/pL; Cat#11691112001 , Sigma-Aldrich, South Africa) was diluted in phosphate buffer (50 mM, pH 7.4) and aliquoted into microfuge tubes on ice. The human gDNA was subsequently damaged using Fenton’s reagent (1 mM FeSO4, 0.1 mM H ₂ O ₂ ) both in the presence and absence of the flower extract samples, as well as a Trolox (vitamin E derivative) gDNA protecting control sample (1 mM Trolox). The samples were incubated and subsequently placed on ice to prevent further DNA damage (45 min, 37 °C). Human gDNA protection was assessed by agarose gel electrophoresis (1% agarose, 10 mg/mL ethidium bromide; 100 V, 10 min) and subsequently visualized using a gel documentation system (G:BOX, Syngene, United Kingdom).

In the absence of Fenton’s reagent, the undamaged human gDNA control exhibits a distinct and intact band on the agarose gel following electrophoresis (Lane U; Figure 8). In comparison, exposure to Fenton’s reagent results in the damaging of human gDNA, as observed by the smeared DNA and lack of an intact band on the visualized agarose gel (Lane D; Figure 8). However, in the presence of the Trolox antioxidant control, the human gDNA band remained intact after the administration of Fenton’s reagent (Lane P; Figure 8). Varying levels of human gDNA protection were observed in proportionately increased concentrations of both flower extracts, with the highest extract concentration clearly exhibiting DNA protection capacities as discerned by the intact DNA band in both the white and orange flower extract samples (0.125 mg/mL, 0.25 mg/mL, and 0.5 mg/mL; Lanes 3, 2, and 1 , respectively; Figure 8). It is surprising that despite the orange flower extract displaying a significantly greater total antioxidant capacity than the white flower extract, there were comparable levels of DNA protection in the two extracts. This may indicate that it is not only the presence of antioxidants in the extracts that determine the DNA protective capacities of the white and orange flower extracts.

EXAMPLE 3

Cytotoxicity and viability of the extracts

MCF12-A cell line culture and treatment preparation

Cells were cultured in 96-well plates Dulbecco’s Modified Eagle Medium containing Ham’s F12 (DMEM: F12, 1 :1 ) (Cat# 11320033; Highveld Biologicals, Lyndhurst, United Kingdom). For regular cell growth (non-supplemented DMEM), fetal bovine serum (FBS, 10% v/v) and penicillin/streptomycin (Pen/Strep, 5%) were added to the medium. For enhanced cell growth (supplemented DMEM), the medium was supplemented with fetal bovine serum (FBS), penicillin/streptomycin blend (Pen/Strep), epidermal growth factor (EGF), choleratoxin, hydrocortisone, and insulin (5% v/v, 1 % v/v, 20 ng/mL, 0.1 pg/mL, 0.5 pg/mL, and 10 pg/mL, respectively). Cells were maintained in an air-humidified incubator (5% CO2, 37 °C). Medicalgrade cisplatin (NAPPI # 720382001 ; Cisacor 50, Accord Healthcare, RSA) was used as the control cytotoxic agent throughout the study. Working concentrations of cisplatin and whole flower extract treatments were prepared fresh in non-supplemented DMEM and filter sterilized before use.

Cytotoxicity and viability assay

Cytotoxicity and cellular viability were assessed using the 3-(4,5 dime-thylthiazol-2-yl)-2,5- diphenyltrazolium bromide (MTT) assay, according to the manufacturer’s instructions (Cat#M2181 G; Sigma-Aldrich, South Africa). Cellular viability after each treatment was determined relative to an untreated vehicle control (non-supplemented DMEM). Cisplatin was used as a damaging control and assessed at a range of concentrations (5 pg/mL; 10 pg/mL; 20 pg/mL; 30 pg/mL) over 48 h. Both the white (var. albiflora) and orange L. leonurus flower extracts were also initially assessed at a range of concentrations (1 mg/mL; 0.5 mg/mL; 0.25 mg/mL; 0.125 mg/mL). The final assessments of cytotoxicity/viability using the MTT assay was carried out at individual selected concentrations for the cisplatin control and the flower extracts (16.43 pg/mL and 0.5 mg/mL, for cisplatin and both flower extracts, respectively). For the combined treatment assessment (plant extract treatment and subsequent treatment with cisplatin) the MCF-12A cells were exposed to the respective flower extract for 24 h prior to cisplatin treatment, after which the MTT assay was assessed 48 h following treatment with cisplatin, as previously conducted. Absorbance was measured using a scanning microplate reader at 570 nm. MCF-12A cell morphology assessment was performed after treatment, immediately prior to measuring absorbance, using the EVOS™ XL Core Configured Cell Imager (Cat# AMEX1 100; ThermoFisher, USA) at the Prince Group Cell Biology labs (UCT).

Cytotoxic effect of the chemotoxin cisplatin on healthy human breast epithelial cells

The cellular damaging effect of cisplatin (chemotoxin control) on human breast epithelial cells (MCF-12A cell line) was observed in an MTT cell viability assay (Figure 9). Following treatment with varying concentrations of cisplatin over 48 h, the percentage viability of the respective MCF-12A cells was measured relative to an untreated control cell line (5 pg/mL cisplatin: 83.62% ± 1.31% viability, 10 pg/mL cisplatin: 69.56% ± 1.94% viability, 20 pg/mL cisplatin: 37.80% ± 1.56% viability, 30 pg/mL cisplatin: 9.74% ± 0.50% viability; Figure 9). The IC ₅ o value (concentration of cisplatin exhibiting 50% relative cell viability) of cisplatin against the MCF-12A cell line was determined for use as a damaging control treatment in downstream analyses (16.43 pg/mL cisplatin, Figure 9). Parallel to the MTT assay, changes in physical cell morphology were also observed at each respective cisplatin treatment concentration (Figure 9). When compared to the untreated control (exhibiting highly adhered, oblong and transparent cells) increased cellular death was observed proportionate to the concentration of cisplatin as exhibited by a decrease in adherence, cellular rounding and darkened dead cells (UTC: untreated control, A-D: increasing cisplatin concentration; Figure 9).

Therapeutic and chemoprotective effect of L. leonurus var. albiflora white flower and L. leonurus orange flower extract on human breast epithelial cells

Preliminary determination of the protecting or damaging effect of both the white (L. leonurus var. albiflora) and orange (L. leonurus) flower extracts was conducted using an MTT cell viability assay (Figure 10). When compared to untreated MCF-12A cells, cell death resulting from cisplatin treatment was observed by the significant reduction in percentage viability (16.43 pg/mL cisplatin: 62.94% ± 1 .84% viability; Figure 10, WF). Comparatively, MCF-12A cell treatment with varying concentrations of L. leonurus (L.) R. Br. var. albiflora white flower extracts exhibited a proportionate increase in percentage cell viability, relative to the untreated control cell line (1 mg/mL extract: 133.22% ± 1.37% viability; 0.5 mg/mL extract: 119.98% ± 1.15% viability; 0.25 mg/mL extract: 1 15.34% ± 1 .09% viability; 0.125 mg/mL extract: 109.12% ± 1 .33% viability; Figure 10 WF). Similarly, treatment with varying concentrations of leonurus (L.) R. Br. orange flower extract showed a proportionate increase in MCF-12A cell viability, when compared to the untreated control cell line (16.43 pg/mL cisplatin: 61.32% ± 0.96%; 1 mg/mL extract: 152.15% ± 2.08% viability; 0.5 mg/mL extract: 129.41% ± 1.47% viability; 0.25 mg/mL extract: 118.46% ± 1 .57% viability; 0.125 mg/mL extract: 109.97% ± 1.1 1% viability; Figure 10 OF). The potential capability of the white and orange flower extracts to protect healthy MCF- 12A cells against exposure to cisplatin was further examined in an MTT assay (Figure 1 1 ). Independent controls included MCF-12A cell treatments with cisplatin only, white flower extract only, and orange flower extract only, with both the white flower and orange flower extract controls showing significantly increased cellular viability, when compared to the untreated control cell line (0.5 mg/mL white flower extract: 131.55% ± 1.94% viability; 0.5 mg/mL orange flower extract: 141.95% ± 2.18% viability; 16.43 pg/mL cisplatin: 64.86% ± 1.12% viability, Figure 11 ). The protecting capacities of both flower extracts was determined by initially exposing MCF-12A cells to the respective flower extract (0.5 mg/mL acetonitrile extract) for 24 h, followed by treatment with cisplatin (16.43 pg/mL cisplatin). Both the white and orange flower extracts showed significant levels of cellular protection against cisplatin, when compared to the cisplatin only treatment control (cisplatin control: 64.86% ± 1 .22%; white flower extract with cisplatin: 89.00% ± 0.81 % viability, orange flower extract with cisplatin: 85.31% ± 1.35% viability; Figure 1 1 ). The percentage protection of each flower extract relative to the cisplatin only control was also discerned, indicating the level of cellular proliferation (white flower extract with cisplatin: +24.14% viability, orange flower extract with cisplatin: +20.45%, relative to the cisplatin control; Figure 1 1 ). The chemoprotective effect of both flower extracts was further observed by the significant level of MCF-12A cell viability even in the presence of cisplatin, as discerned by adhered and transparent viable cells in the respective combined flower extract and cisplatin treatments, when compared to the rounded and darkened cells present in the cisplatin only control treatment (Cis, Figure 11 ). Despite the difference in metabolite composition between orange and white flower extracts, it is surprising that both extracts display the ability to protect and increase healthy cell viability, even in the presence of a chemotherapeutic drug.