Caffeic acid and / or cadambine phytochemicals gold nanoparticle cancer therapeutic agents

Abstract

A cancer therapeutic agent includes a caffeic acid or cadambine phytochemicals cocktail on surfaces of gold nanoparticles. The agent can include a caffeic acid and cadambine phytochemicals cocktail on the surfaces of the gold nanoparticles. The surfaces are preferably stabilized with gum Arabic. The surfaces can consist of caffeic acid stabilized with gum Arabic, cadambine stabilized with gum Arabic, or caffeic acid and cadambine stabilized with gum Arabic. A method of forming the agents uses electrons of caffeic acid and / or cadambine to reduce gold salt(s) to gold nanoparticles without the use of any external toxic chemical reducing agents.

Description

CAFFEIC ACID AN D / OR CADAMBINE PHYTOCHEM ICALS GOLDNANOPARTICLE CANCER THERAPEUTIC AGENTSPRIORITY CLAIM AN D REFERENCE TO RELATED APPLICATION

[0011] This application claims priority under 35 U.S.C. §119 and all applicable statutes and treaties from US Provisional Application 63 / 651,593, which was filed on May 24, 2024.FI ELD

[0012] Fields of the invention concerns gold nanoparticles and cancer therapeutics.BACKGROU N D

[0013] Cancer-related deaths are common despite numerous advances in cancer therapeutic technologies. The insensitivity of cancer cells toward a myriad of therapeutic agents is particularly pronounced in advance stages of the disease. Extensive pre-clinical and clinical investigations have revealed that, in most human cancers, evasion of tumor cells to treatment conferring ultimately resistance towards chemo- and radiotherapy occurs due to innate secretory factors released by the tumor cells into the microenvironment.

[0014] For example, lnterleukines-6 and 10 (IL-6 and IL-10), are the major cytokines in the tumor microenvironment, they are the most important pro-tumor cytokines which are found at high concentrations and are known to be produced in an out-of-control fashion in most cancers.Their overexpression has been reported in almost all types of tumors. The strong association between inflammation and cancer is reflected by the high IL-6 and IL-10 levels in the tumor microenvironment, where in tandem, they promote tumorigenesis by regulating all hallmarks of cancer and multiple signaling pathways, including apoptosis, survival, proliferation, angiogenesis, invasiveness, and metastasis, and, most importantly, the metabolism.

[0015] It is well-known that IL-6 protects the cancer cells from therapy-induced DNA damage, oxidative stress, and apoptosis by facilitating the repair and induction of counters signaling (antioxidant and anti-apoptotic / pro-survival) pathways.

[0016] Genetic aberrations result in severely compromised immune system, which in turn causes numerous inflammatory diseases and disorders including Fatty liver disease; Endometriosis; Type 2 diabetes mellitus; Type 1 diabetes mellitus; Inflammatory bowel disease (IBD); Asthma; Rheumatoid arthritis; Obesity; Alzheimer's and Parkinson's diseases and Cancer. The fact that the immune system is an indispensable epitome of "therapeutic" machinery further enriches the reality that immunotherapy and genomics are two faces of the same coin that control and drive numerous inflammatory functions. Genetic aberrations cause modulations of various immune suppressive or immune compromising cascade of effects that initiate and propagate various diseases through upregulating or suppressing the interconnected cytokines, macrophage phenotypes and numerous cells signaling pathways In the context of cancer and many inflammatory diseases, drugs should initiate a cross talk and modulate several genetically derived parameters.

[0017] Currently, there are no drugs that individually or in combination with other drugs, can respond to aberrations in epigenetic processes which is a primary cause of several diseases as discussed above. For example, a vast majority of cancers patients manifest significantly reduced levels of immunoglobulins— a key pathway for the reduced or fully compromised immune system. There are five major classes of Immunoglobulins: immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE). IgG is the most abundant immunoglobulin present in the blood— which is about 75% of all antibodies in the human body. Any form of IgG deficiency or IgG subclass deficiency is considered a lapse in immunity or a condition of compromised immune system which can happen in precancerous or in any stage of human cancers. IgG deficiency is frequently observed in patients going through either the chemotherapy or radiation therapy or after one or both treatments— again an indication of compromised immune system.

[0018] Genetic aberrations lead to the dysregulation of cytokine production, macrophage polarization, and cell signaling pathways, ultimately contributing to the initiation and propagation of various inflammatory diseases (autoimmune, infectious diseases, and cancer. Certain types of drugs disrupt cytokine hemostasis by suppressing the production of pro- inflammatory cytokines, such as IL-1 , TNF-a, and IFN-y, which are essential for activating and directing immune cells against tumors and suppressing the production of anti-inflammatory cytokines, such as IL-10 and TGF-0, which dampen immune responses result into a cytokine storm which leading to immunosuppression and affecting treatment efficacy.SU MMARY

[0019] A cancer therapeutic agent includes a caffeic acid or cadambine phytochemicals cocktail on surfaces of gold nanoparticles. The agent can include a caffeic acid and cadambine phytochemicals cocktail on the surfaces of the gold nanoparticles. The surfaces are preferably stabilized with gum Arabic. The surfaces can consist of caffeic acid stabilized with gum Arabic, cadambine stabilized with gum Arabic, or caffeic acid and cadambine stabilized with gum Arabic. A method of forming the agents uses electrons of caffeic acid and / or cadambine to reduce gold salt(s) to gold nanoparticles without the use of any external toxic chemical reducing agents.BRI EF DESCRI PTION OF TH E DRAWI NGS

[0020] Figs. 1A and IB summarize experimental data of the effect of present cancer therapeutic agent that includes an encapsulating phytochemicals cocktail on surfaces of gold nanoparticles on suppressed IgG levels in CPA-treated mice.

[0021] Figs. 2A and 2B summarize the prophylactic effects of present nanoparticles on suppressed IL-12 levels in CPA-treated mice.

[0022] Figs. 3A and 3B summarize the effects of present nanoparticles on suppressed IFN-Y levels in CPA-treated mice.

[0023] Figs. 4A and 4B summarize the effects of present nanoparticles on suppressed IL-10 levels in CPA-treated mice.

[0024] Figs. 5A and 5B summarize the effects of present nanoparticles on suppressed IL-6 levels in CPA-treated mice.

[0025] Figs. 6A and 6B summarize the effects of present nanoparticles on suppressed TNF-a levels in CPA-treated mice.

[0026] Figs. 7A and 7B summarize the effects of present nanoparticles on suppressed TGF-0 levels in CPA-treated mice.

[0027] Figs. 8A and 8B summarize the effects of present nanoparticles on suppressed IgG levels in CPA-treated mice.

[0028] Figs. 9A and 9B summarize the effects of present nanoparticles on suppressed IL-12 levels in CPA-treated mice.

[0029] Figs. 10A and 10B summarize the effects of present nanoparticles on suppressed IFN-y levels in CPA-treated mice.

[0030] Figs. 11A and 11B summarize the effects of present nanoparticles on suppressed IL-10 levels in CPA-treated mice.

[0031] Figs. 12A and 12B summarize the effects of present nanoparticles on suppressed IL-10 levels in CPA-treated mice.

[0032] Figs. 13A and 13B summarize the effects of present nanoparticles on suppressed IL-6 levels in CPA-treated mice.

[0033] Figs. 14A and 14B summarize the effects of present nanoparticles on suppressed TNF-a levels in CPA-treated mice.

[0034] Figs. 15A and 15B summarize the effects of present nanoparticles on suppressed iNOS levels in CPA-treated mice.

[0035] Fig. 16 shows that CA-AuNPs were synthesized using the optimum redox capabilities of caffeic acid.

[0036] Fig. 17 shows that Cad-AuNPs were synthesized by redox reaction by transferring electrons from hydroxyl groups of cadambine to the gold salt.

[0037] Fig. 18 shows that Caffeic acid and cadambine encapsulated gold nanoparticles (CCC- AuNPs) were synthesized by the combination of caffeic acid and cadambine cocktail.DETAI LED DESCRI PTION OF TH E PREFERRED EMBODIM ENTS

[0038] A preferred cancer therapeutic agent includes a caffeic acid or cadambine phytochemicals cocktail on surfaces of gold nanoparticles. The surfaces preferably include both a caffeic acid and cadambine phytochemicals cocktail. The surfaces are preferably stabilized with gum Arabic. In a preferred embodiment, the cocktail consists of caffeic acid stabilized with gum Arabic. In a preferred embodiment, the cocktail consists of cadambine stabilized with gumArabic. In a preferred embodiment, the cocktail consists of caffeic acid and cadambine stabilized with gum Arabic. The cocktail encapsulates surfaces of the nanoparticles.

[0039] The combination of caffeic acid and cadambine on gold nanoparticles, collectively or singularly, imparts immunomodulatory features to these therapeutic gold nanoparticles. Such immunomodulatory gold nanoparticles can be used in treating various inflammatory diseases including cancer, diabetes, arthritis, and various inflammatory diseases and disorders.

[0040] The preferred cancer therapeutic agents are capable of targeting the tumor microenvironment to: A) Elevate levels of antitumor IL-12 cytokine; B) Block IL-6 and IL-10 cytokines or inhibit their associated pro-tumor cell signaling; C) Promote regeneration of IgG to catalyze over expression of a number of antitumor cytokines and cause skewed macrophage polarization toward anti-tumor Ml-like phenotype from the pro-tumor M2 macrophages; and D) Modulate, IFN-y, TNF-a, TGF-fJ and levels of Nitric oxide (NO).

[0041] The preferred cancer therapeutic agents can reactivate a severely compromised immune system, which happens due to either inflammatory diseases (including cancer) or as a result of using myriad of drugs to treat diseases. Reactivation of severely compromised immune system is the only pathway to make therapies work on the human body and save lives. The preferred cancer therapeutic agents are nanoayurveda drugs capable of reactivating suppressed or fully compromised immune system which occurs in several inflammatory diseases or through treatment with various pharmaceuticals.

[0042] Testing of preferred cancer therapeutic agents has demonstrated the modulation of all the immunomodulatory factors including IL-12, IL-10, IL-6, IFN-y, TNF-a, TGF-0 and levels of nitric oxide (NO) in severely compromised immune systems. Testing has shown efficacy against breast, pancreatic, prostate and a host of cancers. Testing also showed effective prophylactic activities in preventing cancer and other diseases.

[0043] A preferred embodiment provides a method of forming uses electrons of caffeic acid or cadambine to reduce gold salt(s) to gold nanoparticles without the use of any external toxic chemical reducing agents. The creation of combinatorial caffeic acid and cadambine ingredients on gold nanoparticles is facilitated because electrons from these chemicals are used to reduce gold salt(s) to gold nanoparticles. Preferred methods select plant ingredients that occlude caffeic acid and cadambine cocktail— naturally.

[0044] A preferred embodiment method obtains a solution of caffeic acid and / or cadambine phytochemicals from leaves, bark, or flowers rich in caffeic acid and cadambine and uses thesolution o reduce gold salts. Nanofilters can be used to isolate gold nanoparticles encapsulated with caffeic acid and / or cadambine phytochemicals.

[0045] Preferred embodiments of the invention will now be discussed with respect to experiments. Broader aspects of the invention will be understood by artisans in view of the general knowledge in the art and the description of the experiments that follows.

[0046] A preferred embodiment consists of an encapsulating caffeic acid or cadambine cocktail on gold nanoparticles (CA-AuNP or Cad-AuNP)), and caffeic acid and cadambine cocktail on gold nanoparticles (CCC-AuNP). Both enhance the levels of immunoglobulin G (IgG) in both serum and spleen. Caffeic acid or cadambine on gold nanoparticles (CA-AuNP and Cad-AuNP)) as well as caffeic acid and cadambine cocktail on gold nanoparticles (CCC-AuNP)— individually and collectively— immunomodulate by enhancing immunoglobulin G (IgG) in both serum and spleen, immunoglobulin G (IgG) in both serum and spleen.

[0047] The caffeic acid and cadambine cocktail on gold nanoparticles (CCC-AuNP) resulted in significant enhancement of the cytokine tumor necrosis factor a (TNF-a). This is a significant feature because high concentrations of TNF-a have the propensity to damage tumor vasculature and subsequently inducing cancer cell killing. Increased levels of TNF-a, as seen upon treatments with CCC-AuNP, promoted blood vessel permeability and thus aided cancer therapy.

[0048] The caffeic acid and cadambine cocktail on gold nanoparticles enhance interferon-y (IFN-y) levels in vivo and thus aid cancer immune therapy through cytostatic, pro-apoptotic and antiproliferative functions. The nanoparticles induce regulatory T-cell apoptosis, and thus stimulates the activity of anti-tumor Ml macrophage phenotype to overcome tumor progression.

[0049] The caffeic acid and / or cadambine cocktail on gold nanoparticles target the tumor microenvironment and thus potentiates the release of antitumor cytokine IL-12. The nanoparticles engage in anti-tumor immunity by activating the effector Thl response— a prerequisite for the activation of cytotoxic T and NK cells in immunomodulatory cancer therapy.

[0050] The caffeic acid and / or cadambine cocktail on gold nanoparticles target the tumor microenvironment and thus control the over expression of tumor promotion IL-10, and control the over expression of immunosuppressive iNOS.

[0051] The caffeic acid and / or cadambine cocktail on gold nanoparticles reactivate fully compromised immune system— showing significantly elevated levels of every immunomodulatory cytokine IL-12, IL-6, IL-10, IFN-y, TNF-a, TGF- , and Nitric oxide (NO).

[0052] The caffeic acid and / or cadambine cocktail on gold nanoparticles reactivate the fully compromised immune system to prevent / reduce immunosuppressive effects caused by cancer and various other inflammatory disease.

[0053] The caffeic acid and / or cadambine cocktail on gold nanoparticles exert immunomodulatory curative effects through significant elevation of anti-tumor IL-12 cytokine on a variety of tumor types including prostate, breast, pancreatic cancers, and a host of cancers.

[0054] The caffeic acid and / or cadambine cocktail on gold nanoparticles exert immunomodulatory curative effects through significant elevation of anti-tumor IL-12 cytokine in animals with breast cancers and thus result in effective treatment and arrestation of tumor growth and exert immunomodulatory curative effects through significant elevation of antitumor IL-12 cytokine in animals with breast, and a host of cancers.

[0055] The caffeic acid and / or cadambine cocktail on gold nanoparticles exert immunomodulatory curative effects through significant elevation of anti-tumor interferon-y (IFN-y) cytokine. This cytokine is vitally important for its role in the activation of cellular immunity and to stimulate antitumor immune response through cytostatic, pro-apoptotic and antiproliferative functions.

[0056] The caffeic acid and / or cadambine cocktail on gold nanoparticles exert immunomodulatory curative effects through significant elevation of anti-tumor interferon-y (IFN-y) cytokine as evidenced by effective treatment of breast tumors in animals and from results of MTT assays showing significant antitumor response on a variety of tumor types including prostate, breast, pancreatic cancers, and a host of cancers.

[0057] The caffeic acid and / or cadambine cocktail on gold nanoparticles demonstrate unprecedented cancer therapeutic efficacies attributed to the ability of IFN-y to inhibit angiogenesis in tumor tissue, and thereby induce regulatory T-cell apoptosis as well as induce and stimulate the activity of anti-tumor Ml proinflammatory macrophages to overcome tumor progression.

[0058] The caffeic acid and / or cadambine cocktail on gold nanoparticles target the tumor microenvironment and initiate synergistic immunomodulatory roles in modulating both TGF- and TNF-a to control and treat cancers.

[0059] Experimental Formation Method

[0060] Natural plant ingredients were prepared as followed. 5 g plant powder in 100 mL doubly deionized (DI) water, was stirred at 700 rpm and heated at 90 °C for 15 min. The solutionwas allowed to cool and centrifuged at 10, 000 rpm for 15 min. The supernatant was characterized by LC-MS / MS to determine phytonutrient composition and used for synthesis of the gold nanoparticles.

[0061] Experiments produced A) Caffeic acid encapsulated gold nanoparticles (CA-AuNPs) and corresponding CA vehicle (caffeic acid); B) Cadambine encapsulated gold nanoparticles (Cad- AuNPs) and corresponding Cad vehicle (cadambine);C) Caffeic acid and cadambine cocktail encapsulated gold nanoparticles (CCC-AuNPs) and corresponding CCC vehicle (caffeic acid and cadambine cocktail.

[0062] The caffeic acid gold nanoparticles (CA-AuNPs) were produced by mixing 1.5 mg caffeic acid (CA) from various plant ingredients that occlude caffeic acid naturally and 12 mg gum arabic in 6 mL of doubly deionized (DI) water. The solution was stirred at 80°C for 10 min to dissolve the CA into water to get a clear solution. A preferred temperature range is 80°C-100°C. The gold salt (100 pL of 0.1 M) was added to the reaction mixture to produce the gold nanoparticles (AuNPs). A preferred concentration range is reflected by the use of 1.5-2 mg of Caffeic acid, 12- 15 mg Gum Arabic; and 80-100 micro liters of Gold salt with 6 mL of doubly deionized (DI) water. The color was changed to ruby-red within a second and developed the homogenous AuNPs. The AuNPs were centrifuged twice in 2 mL Eppendorf tube at 12,000 rpm at 12°C for 15 min to remove the unreacted CA and gold salt and was stored at 4°C for further use. The CA-AuNPs were characterized by various techniques.

[0063] The cadambine gold nanoparticles (Cad-AuNPs) were produced by mixing 1.5 mg cadambine (Cad) from various plant ingredients that occlude cadambine naturally and 12 mg gum arabic in 6 mL of doubly deionized (DI) water. The solution was stirred at 80°C for 10 min to dissolve Cad into water to get a clear solution. . A preferred temperature range is 80°C- 100°C. The gold salt (100 pL of 0.1 M) was added to the reaction mixture to produce the gold nanoparticles (AuNPs). A preferred concentration range is reflected by the use of 1.5-2 mg of Cadambine, 12-15 mg Gum Arabic; 80-100 micro liters of Gold salt with 6 mL of doubly deionized (DI) water. The color was changed to ruby-red within a second and developed the homogenous AuNPs. The AuNPs were centrifuged twice in 2 mL eppendorf tube at 12,000 rpm at 12°C for 15 min to remove the unreacted Cad and gold salt and was stored at 4°C for further use. The Cad-AuNPs were characterized by various instrumentation techniques.

[0064] The caffeic acid and cadambine cocktail gold nanoparticles (CCC-AuNPs) were produced by using 12 mg of gum arabic added to a solution containing 1 mL of plant ingredients thatocclude caffeic acid and cadambine cocktail— naturally and 5 mL of doubly deionized (DI) water. The solution was stirred at 25°C for 10 min to dissolve the CA and Cad into water to get a clear solution. This temperature of 25°C refers to only to dissolve Caffeic acid and Cadambaine into water. The temperature used to produce gold nanoparticles is in the range of 80-100°C. The gold salt (100 pL of 0.1 M) was added to the reaction mixture to produce gold nanoparticles (AuNPs). The color was changed to ruby-red within a second and developed the homogenous AuNPs. The AuNPs were centrifuged twice in 2 mL eppendorf tube at 12,000 rpm at 12°C for 15 min to remove the unreacted CA, Cad, and gold salt and was stored at 4°C for further use. The CCC-AuNPs were characterized by various instrumentation techniques.

[0065] Experimental results.

[0066] Prophylactic effects of present nanoparticles in immune-suppressed mice.

[0067] Using standard ELISA methods, the ability of the present nanoparticles to prevent / reduce immunosuppressive effect of CPA was evaluated by measuring levels of proinflammatory cytokines and levels of IgG in serum and spleen. CPA is a powerful immunosuppressant and is known to downregulate both cellular and humoral immune components, represented by biomarkers.

[0068] Figs. 1A and IB summarize the prophylactic effects of present nanoparticles in immune- suppressed mice. Data shown are mean + / - SD of values derived from 4-6 animals. Data were analyzed using the GraphPad Prism version 8 statistical program (GraphPad Software, Inc.). Statistical differences between groups were evaluated using one-way analysis of variance followed by the Dunnett's test or t-test. P<0.05 was considered to indicate a statistical significance (where **** refers to p<0.0001, *** refers to p<0.001,** refers to p<0.01 and * refers to<0.05. In serum (Fig. 1A) CPA treatment caused a significant reduction in circulating IgG levels (~55% reduction). The CCC-AuNP helped to maintain IgG level in serum (~90% of normal). CCC vehicle treatment did not improve suppressed IgG level in CPA-treated mice. In spleen (Fig. IB) CPA-suppressed level of IgG (~37% of normal) was significantly corrected by CCC-AuNP (~77% of normal) treatment.

[0069] Figs. 2A and 2B summarize the effects of present nanoparticles on suppressed IL-12 levels in CPA-treated mice. In serum (Fig. 2A) of normal animals, IL-12 levels were almost undetectable. This remained same in CPA-treated animals as well. CCC vehicle treatments did not elevate IL-12 levels in CPA-treated animals. The CCC-AuNP treatment caused dramatic elevation of IL-12 levels (78-fold higher than normal untreated). In spleen (Fig. 2B) IL-12expression was observed in normal untreated animals (~53 ng / mL). CPA-treatment caused significant reduction in basal IL-12 levels (~31% reduction). CCC vehicle treatment improved suppressed IL-12 levels in CPA-treated animals. The CCC-AuNP (~152% of normal) treatment caused significant elevation in IL-12 levels than in untreated control animals.

[0070] Figs. 3A and 3B summarize the effects of present nanoparticles on suppressed IFN-Y levels in CPA-treated mice. In serum (Fig. 3A), CPA treatment caused ~40% reduction in IFN-y expression. Vehicle treatments had only negligible effect in correcting lowered expression. The CCC-AuNP ("'145% of control) not only corrected suppressed IFN-y but elevated it higher than normal circulating level. This is most likely due to the effect of increased IL-12 level in nano particle treated animals. In spleen (Fig. 3B) CPA-treatment caused only a modest suppression in IFN-y level in spleen of CPA-treated animals (~24% reduction over control). While CCC vehicle treatment had only a modest effect in improving IFN-y level (118% of control), CCC-AuNP elevated IFN-y levels far greater than untreated control animals (>170% of control). The expression levels of IFN-y upon treatment with nanoparticles appeared similar to observations made for IL-12.

[0071] Figs. 4A and 4B summarize the effects of present nanoparticles on suppressed IL-10 levels in CPA-treated mice. In serum (Fig. 4A), CPA treated mice showed ~65% reduction in IL-10 levels. CCC vehicle treatment did not improve the suppressed expression. The CCC-AuNP (62% of normal) increased IL-10 expression in CPA-treated mice. In spleen (Fig. 4B), reduced expression of IL-10 was observed in CPA-treated animals (27% reduction compared to untreated). CCC vehicle treatment did not improve suppressed expression. CCC-AuNP also showed no change in IL-10 expression in CPA-treated mice.

[0072] Figs. 5A and 5B summarize the effects of present nanoparticles on suppressed IL-6 levels in CPA-treated mice. Basal expression of IL-6 in serum was low in untreated animals ("'26 ng / mL) (Fig. 5A). CPA treated mice showed almost a complete inhibition of expression ("'89% reduction). CCC vehicle treatment did not improve the suppressed expression. The CCC-AuNP (77% of normal) increased IL-6 expression in CPA-treated mice. In spleen (Fig. SB), reduced expression of IL-6 in CPA-treated animals was elevated by both CCC-AuNP ("'110% of normal). CCC vehicle treatment also caused a betterment of IL-6 levels, but levels achieved with nanoparticles was better than vehicle treatment.

[0073] Figs. 6A and 6B summarize the effects of present nanoparticles on suppressed TNF-a levels in CPA-treated mice. In serum (Fig. 6A) basal TNF-a level was low in untreated controlgroup. CPA treatment caused almost 88% reduction in basal TNF-a expression. The reduced level was not corrected by CCC vehicle treatment. The CCC-AuNP was effective in improving suppressed TNF-a levels to 58 % of normal. Similar characteristics were seen in spleen TNF-a levels (Fig. 6B).

[0074] Figs. 7A and 7B summarize the effects of present nanoparticles on suppressed TGF- levels in CPA (Cyclophosphamide) -treated mice. In serum ( Fig.7A), CPA treatment caused ~45% reduction in TGF-fJ expression. The reduced level was corrected to normal level by both vehicle (CCC) and nanoparticle treatments (CCC-AuNP). In spleen (Fig. 7B), a similar pattern was observed, with both vehicle (CCC) and nanoparticle (CCC-AuNP) treatments restoring cytokine level to normal.

[0075] Figs. 8A and 8B summarize the effects of present nanoparticles on suppressed IgG levels in CPA-treated mice. In serum (Fig. 8A) CPA treatment caused a significant reduction in circulating IgG levels (>75% reduction). Levamisole (positive control) treatment improved IgG levels (~75% of normal). The CCC-AuNP treatment improved IgG level in serum (~40% of normal). CCC vehicle treatment did not improve suppressed IgG level in CPA-treated mice. In spleen (Fig. 8B) ssuppressed level of IgG was partially corrected by CCC-AuNP treatment. Levels were almost similar to that achieved with Levamisole treatment.

[0076] Figs. 9A and 9B summarize the effects of present nanoparticles on suppressed IL-12 levels in CPA-treated mice. In serum (Fig.9A) CPA-treatment caused significant reduction in basal IL-12 levels (~60% inhibition). Levamisole treatment improved IL-12 levels to ~84% of normal. CCC vehicle treatments did not improve suppressed IL-12 levels in CPA-treated animals. The CCC-AuNP treatment not only corrected, but elevated IL-12 levels (129% of normal) greater than normal level seen in untreated control animals. In spleen (Fig. 9B) CPA-treatment caused significant reduction in basal IL-12 levels (~40% inhibition). Levamisole treatment improved & elevated IL-12 levels to "'135% of normal. CCC vehicle treatments significantly improved suppressed IL-12 levels in CPA-treated animals (~87% of normal). The CCC-AuNP treatment not only corrected, but elevated IL-12 levels ("'165% of normal) greater than normal levels seen in untreated control animals.

[0077] Figs. 10A and 10B summarize the effects of present nanoparticles on suppressed IFN-y levels in CPA-treated mice. In serum (Fig. 10A), CPA treatment caused ~30% reduction in IFN-y expression. Levamisole effectively improved the expression levels to near normal (85% of control). CCC vehicle treatment had no effect in correcting lowered expression. The CCC-AuNPnot only corrected suppressed IFN-y levels in CPA-treated animals but elevated it higher than normal circulating levels. This is most likely due to the effect of increased IL-12 levels in nanoparticle treated animals. In spleen (Fig. 10B) CPA-treatment caused only a modest suppression in IFN-y levels in spleen of CPA-treated animals, but treatment of animals with nanoparticles (CCC-AuNP) resulted in higher-than-normal circulating levels in untreated animals.

[0078] Figs. 11A and 11B summarize the effects of present nanoparticles on suppressed IL-10 levels in CPA-treated mice. CPA treated mice showed ~60 % reduction (Fig. 11A) of IL-10 levels in serum. Levamisole treatment improved IL-10 levels (~78 % of normal). CCC vehicle treatment did not improve the suppressed expression. The CCC-AuNP increased IL-10 expression in CPA- treated mice (64 % of normal). In spleen (Fig. 11B), reduced expression of IL-10 in CPA-treated animals was significantly elevated by CCC-AuNP. The stimulatory effect seen was even better than Levamisole treatment.

[0079] Figs. 12A and 12B summarize the effects of present nanoparticles on suppressed IL-10 levels in CPA-treated mice. CPA treated mice showed "'2.5-fold reduction in IL-10 levels in serum. Levamisole treatment improved expression levels to 64 % of normal. CCC vehicle treatment did not correct the low expression. The CCC-AuNP increased IL-10 expression in CPA- treated mice (51 % of normal)(Fig. 12A). In spleen (Fig. 12B), reduced expression of IL-10 in CPA treated animals was corrected to near-normal levels by nanoparticle treatment.

[0080] Figs. 13A and 13B summarize the effects of present nanoparticles on suppressed IL-6 levels in CPA-treated mice. In serum (Fig. 13A) CPA-treatment caused ~50 % decline in IL-6 content in the serum. Levamisole treatment improved expression levels to near normal (~78 % of normal). CCC vehicle had minimal effect on correcting. The CCC-AuNP significantly reversed the suppressive effect of CPA (78 % of normal level). In spleen (Fig. 13B) the CCC-AuNP also improved levels of IL-6 to reverse the effect of CPA.

[0081] Figs. 14A and 14B summarize the effects of present nanoparticles on suppressed TNF-a levels in CPA-treated mice. In serum (Fig. 14A) basal TNF-a level was very low in general in all experimental groups. CPA treatment caused a 50 % reduction in TNF-a expression. The reduced level was not corrected by CCC vehicle treatment. Levamisole improved TNF-a level to nearnormal ("'90 % of normal). The CCC-AuNP was effective in improving TNF-a levels to ~75 % of normal. In spleen (Fig. 14B) results reflect that of observations made for serum.

[0082] Figs. 15A and 15B summarize the effects of present nanoparticles on suppressed iNOS levels in CPA-treated mice. CPA treated mice showed ~40% reduction in iNOS levels in serum(Fig. 15A). CCC treatment (vehicle) modestly improved expression. The CCC-AuNP increased iNOS expression in CPA-treated mice to near-normal levels. In spleen also the CCC-AuNP reversed suppressed expression of iNOS in CPA-treated animals. Levels reached were almost like that achieved with Levamisole treatment.

[0083] Fig. 16 shows that CA-AuNPs were synthesized using the optimum redox capabilities of caffeic acid. Caffeic acid is a powerful electron injector, the reduction of Au3+ions from sodium tetrachloroaurate (III) dihydrate (NaAuCI4-2H2O) by caffeic acid is facilitated by the phenolic hydroxyl groups in the molecule which contributes a strong bidentate chelating bond with zero- valent gold (Au°).

[0084] The core size of CA-AuNPs, obtained by TEM, indicated that the nanoparticles are spherical, mono-disperse and homogenous with the core size of 37±4 nm. The results obtained by dynamic light scattering instrument revealed that CA-AuNPs showed hydrodynamic size of 219±4 nm and the zeta potential ( ) -30±2 mV. XRD analysis of CA-AuNPs revealed four prominent peaks at 20 values of 38.1°, 44.6°, 64.5°, and 77.7° align with the standard Bragg reflections (111), (200), (220), and (311) characteristic of the face-centered cubic (fee) lattice structure for gold.

[0085] Fig. 17 shows that Cad-AuNPs were synthesized by redox reaction by transferring electrons from hydroxyl groups of cadambine to the gold salt. The formation of Cad-AuNPs was confirmed by the observation of red color solutions. The chemical structure of Cad plays a significant role in the production of stable gold nanoparticles. Cad molecule comprises of monoterpenoid, indole, and glucoside moieties. The reaction between cadambine and Au3+ions from sodium tetrachloroaurate (III) dihydrate (NaAuCI4-2H2O) is a complex process of collaborative chemistry. The glucoside provides solubility of Cad, while monoterpenoid moiety provides a platform for Au3+ions to nucleate and the nitrogen atoms in the indole ring drives the reactivity of Cad, absorbs to Au3+ions and facilitates the reduction to produce the corresponding gold nanoparticles. TEM indicated that the nanoparticles are spherical, mono-disperse and homogenous with the core size of 9±2 nm). The results obtained from DLS measurements revealed that Cad-AuNPs showed hydrodynamic size of 51+4 nm and a zeta potential ( ) -28+2 mV. XRD analysis of Cad-AuNPs showed the main characteristic peaks corresponding to the (111), (200), and (220) planes, confirming the presence of cubic (FCC) gold.

[0086] Fig. 18 shows that Caffeic acid and cadambine encapsulated gold nanoparticles (CCC- AuNPs) were synthesized by the combination of caffeic acid and cadambine cocktail. Caffeicacid, a potent antioxidant, modulates immune responses by scavenging free radicals, reducing oxidative stress, and inhibiting inflammatory mediators. Cadambine, on the other hand, directly interacts with immune cells, regulating their activation, proliferation, and differentiation. In this respect, the combination of caffeic acid and cadambine cocktail through embedding them on gold nanoparticles (CCC-AuNPs). Various plant ingredients that occlude caffeic acid and cadambine cocktail— naturally used for the synthesis of caffeic acid and cadambine cocktail encapsulated gold nanoparticles (CCC-AuNPs) were evaluated. The results revealed that these plant ingredients composed of caffeic acid, cadambine, and cadambine derivatives (30- Dihydrocadambine and 3a-lsodihydrocadambine) - thus collectively referred to as 'cadambine' due to the cadambine backbone, produced (CCC-AuNPs). The CCC-AuNPs were characterized by various instrumentation techniques. CCC-AuNP FTIR absorption data corresponded to the CCC phytoextract cocktail. Interestingly the absorption bands from the CC were quite similar and had quite a bit of overlap. There is the O-H stretching seen around 3,400 cm'1and C-H stretching seen around 2,900 cm1. There is also the "*1,600 cm1peak and "*1,400 cm1corresponding to the COOH stretching and O-H bending respectively. The peak around "*1100 cm'1peak corresponds to the C-0 stretching. These are all indicative of the functional groups in gum arabic and the phytoextracts. After reduction for the nanoparticle synthesis and conjugation, there are minor changes indicated by the CCC-AuNP FTIR.

[0087] While specific embodiments of the present invention have been shown and described, it should be understood that other modifications, substitutions and alternatives are apparent to one of ordinary skill in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the invention, which should be determined from the appended claims.

[0088] Various features of the invention are set forth in the appended claims.

Claims

Claims1. A cancer therapeutic agent comprising a caffeic acid or cadambine phytochemicals cocktail on surfaces of gold nanoparticles.

2. The agent of claim 1, comprising a caffeic acid and cadambine phytochemicals cocktail on the surfaces of the gold nanoparticles.

3. The agent of claim 1 or 2, wherein the surfaces are stabilized with gum Arabic.

4. The agent of claim 1, wherein the cocktail consists of caffeic acid stabilized with gum Arabic.

5. The agent of claim 1, wherein the cocktail consists of caffeic acid and cadambine stabilized with gum Arabic.

6. The agent of claim 1, wherein the cocktail consists of cadambine stabilized with gum Arabic.

7. The agent of any previous claim, wherein the cocktail encapsulates the surfaces of the gold nanoparticles.

8. A method of forming the agents of any of claims 1-2, comprising using electrons of caffeic acid and / or cadambine to reduce gold salt(s) to gold nanoparticles without the use of any external toxic chemical reducing agents.

9. The method of claim 3, comprising using electrons of caffeic acid and cadambine to reduce the gold salt(s).

10. A method of treatment of cancer, comprising administration of the agents of any of claims 1-2.

11. The method of claim 8, comprising obtaining a solution of caffeic acid and / or cadambine phytochemicals from leaves, bark, or flowers and using the solution to reduce gold salts.

12. The method of claim 11, comprising filtering the solution with nanofilters to isolate gold nanoparticles encapsulated with caffeic acid and / or cadambine phytochemicals.

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