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Spinacia oleracea L., Amaranthaceae, leaves cultivated in Egypt demonstrated a potential antileukemic activity against the chronic myeloid leukemia, K562 cell line. Thus, the aim of this study is to carry out a phytochemical investigation of S. oleracea leaves as well as the isolation of its antileukemic phytoconstituents. Phytochemical investigation of S. oleracea leaves resulted in the isolation of seventeen known compounds. The biological study revealed that compounds hexaprenol, phytol, and 18-[(1-oxohexadecyl) oxy]-9-octadecenoic acid exhibited a remarkable antiproliferative activity against K562 cells in vitro. A mechanistic in silico study showed that hexaprenol, phytol, and 18-[(1-oxohexadecyl) oxy]-9-octadecenoic acid exhibited a strong binding affinity towards topoisomerase (docking score −12.50, −9.19, and −13.29 kcal/mol, respectively), and showed as well a strong binding affinity towards Abl kinase (docking score −11.91, −9.35, and −12.59 kcal/mol, respectively). Molecular dynamics study revealed that 18-[(1-oxohexadecyl) oxy]-9-octadecenoic acid produced stable complexes with both topoisomerase and Abl kinase with RMSD values of 1.81 and 1.85 Å, respectively. As a result of our findings, we recommend more in vivo and preclinical studies to confirm the potential benefit of spinach leaves for chronic myeloid leukemia patients. Graphical Abstract

Ayurvedic medicines have been tested over centuries and were developed based on the extensive knowledge on pharmacognosy, medicinal chemistry, pharmacology, and clinical case studies that are continuing to be proven useful for modern drug discovery. In recent years, drugs have been developed through the identification of targets and their validation, screening of these hit compounds, and optimizing these leads. High throughput screening (HTS) methods, molecular docking in silico methods, understanding of signal transduction pathways and their interconnection with various disorders, and advanced tools in molecular biological techniques paved a new way in drug discovery and these methodologies open huge opportunities for drug development from Ayurvedic knowledge. Interest in lead drugs from botanical products is being revitalized and, in this chapter, we suggest sequential steps that could lead to success in Ayurvedic approach-based drug discovery. Furthermore, we discuss the advantages, concerns, and challenges of integrating Ayurvedic principles into modern drug discovery. A list of new drugs developed from natural products and in particular from Ayurvedic medicine is also included. An interdisciplinary research approach involving traditional knowledge and evidence, history of use, ethnopharmacology, molecular biology, pharmacoepidemiology, reverse pharmacology, and other advanced research areas for a scientific basis are the essential steps to integrate Ayurveda into modern drug discovery.

Cannabidiol, a non-intoxicating phytocannabinoid, has potential therapeutic effects over a broad range of disorders. Recently, there has been increased interest in CBD, as several studies showed promising anticonvulsant efficacy with few side effects. In 2018, a CBD-based oral solution, Epidiolex®, was approved by the FDA to treat two severe forms of pediatric epilepsy, Dravet syndrome, and Lennox-Gastaut syndrome. Although only these two syndromes are recognized indications for CBD, it has been consumed in an unregulated fashion for a variety of indications including chronic pain, muscle stiffness, inflammation, anxiety, smoking cessation, and even cancer. While CBD legislation in the USA is confusing due to the differences in state and federal laws, CBD has proliferated in the US market in several forms such as CBD oil or capsules, hemp oil/extract, and also as an ingredient in several dietary supplements, syrups, teas, and creams. With the ever-increasing use of CBD and its widespread availability to the general public, it is important to examine and report on possible drug-drug interactions between CBD and other therapeutic agents as well as addictive substances such as alcohol and tobacco. A detailed literature search for CBD's possible interactions was conducted using online databases. As expected, CBD has been reported to interact with anti-epileptic drugs, antidepressants, opioid analgesics, and THC, but surprisingly, it interacts with several other common medications, e.g. acetaminophen, and substances including alcohol. This review provides a comprehensive list of interacting drugs. The possible mechanisms for these drug-drug interactions are presented in table format. Given the growing popularity of CBD as a medication and the dearth of available information on CBD drug-drug interactions, it is critical to be aware of current drug-drug interactions and it will be important to investigate the impact of CBD upon concomitant medication use in future randomized, controlled trials.

A new yohimbine type alkaloid N-methylarcinine (1), and the known alkaloid, holeinine (2), have been isolated from the methanolic leaf extract of Ochrosia elliptica grown in Egypt. The structures of the isolated metabolites were elucidated via 1D and 2D NMR data, as well as HRESIMS spectra and the spectral data were compared with the existing data available from literature. These two alkaloids are very different in activity from previously reported class type alkaloids, where they showed weak cytotoxic activity against K-562 leukemia cells while other compounds from this class were highly toxic in nature. Interestingly, holeinine (2) showed unique induction of FOXO expression, a cancer signaling pathway. FOXO is involved in the expression of death receptor ligands, such as TNF apoptosis ligand, where its activation plays a critical role in tumor suppression. As several anticancer leads targeting FOXO signaling gain more attention, this compound could be a candidate for drug development after the determination of its therapeutic efficacy and more mechanistic studies need to be evaluated.

The purpose of this study was to determine if a methanolic extract of the Pulsatilla patens (L.) Mill. can inhibit the progression of cancer through the modulation of cancer-related metabolic signaling pathways. We analyzed a panel of 13 inducible luciferase reporter gene vectors which expression is driven by enhancer elements that bind to specific transcription factors for the evaluation of the activity of cancer signaling pathways. The root extract of P. patens exhibited strong inhibition of several signaling pathways in HeLa cells, a cervical cancer cell line, and was found to be the most potent in inhibiting the activation of Stat3, Smad, AP-1, NF-κB, MYC, Ets, Wnt and Hdghog, at a concentration of 40 µg/mL. The methanolic extracts of P. patens enhanced apoptotic death, deregulated cellular proliferation, differentiation, and progression towards the neoplastic phenotype by altering key signaling molecules required for cell cycle progression. This is the first study to report the influence of Pulsatilla species on cancer signaling pathways. Further, our detailed phytochemical analysis of the methanolic extracts of the P. patens allowed to deduce that compounds, which strongly suppressed the growth and proliferation of HeLa cancer cells were mainly triterpenoid saponins accompanied by phenolic acids.

COVID-19 was declared an international public health emergency in January, and a pandemic in March of 2020. There are over 125 million confirmed COVID-19 cases that have caused over 2.7 million deaths worldwide as of March 2021. COVID-19 is caused by the SARS-CoV-2 virus. SARS-CoV-2 presents a surface "spike" protein that binds to the ACE2 receptor to infect host cells. In addition to the respiratory tract, SARS-Cov-2 can also infect cells of the oral mucosa, which also express the ACE2 receptor. The spike and ACE2 proteins are highly glycosylated with sialic acid modifications that direct viral-host interactions and infection. Maackia amurensis seed lectin (MASL) has a strong affinity for sialic acid modified proteins and can be used as an antiviral agent. Here, we report that MASL targets the ACE2 receptor, decreases ACE2 expression and glycosylation, suppresses binding of the SARS-CoV-2 spike protein, and decreases expression of inflammatory mediators by oral epithelial cells that cause ARDS in COVID-19 patients. In addition, we report that MASL also inhibits SARS-CoV-2 infection of kidney epithelial cells in culture. This work identifies MASL as an agent with potential to inhibit SARS-CoV-2 infection and COVID-19 related inflammatory syndromes.

PURPOSE: Oral cancer causes over 120,000 deaths annually and affects the quality of life for survivors. Over 90% of oral cancers are derived from oral squamous cell carcinoma cells (OSCCs) which are generally resistant to standard cytotoxic chemotherapy agents. OSCC cells often exhibit increased TGFβ and PDPN receptor activity compared to nontransformed oral epithelial cells. Maackia amurensis seed lectin (MASL) can target the PDPN receptor and has been identified as a novel agent that can be used to treat oral cancer. However, mechanisms by which MASL inhibits OSCC progression are not yet clearly defined. METHODS: Here, we performed cell migration and cytotoxicity assays to assess the effects of MASL on OSCC motility and viability at physiologically relevant concentrations. We then performed comprehensive transcriptome analysis combined with transcription factor reporter assays to investigate the how MASL affects OSCC gene expression at these concentration. Key data were then confirmed by western blotting to evaluate the effects of MASL on gene expression and kinase signaling activity at the protein level. RESULTS: MASL significantly affected the expression of about 27% of approximately 15 thousand genes found to be expressed by HSC-2 cells used to model OSCC cells in this study. These genes affected by MASL include members of the TGFβ-SMAD, JAK-STAT, and Wnt-βCTN signaling pathways. In particular, MASL decreased expression of PDPN, SOX2, and SMAD5 at the RNA and protein levels. MASL also inhibited SMAD and MAPK activity and exhibited potential for combination therapy with doxorubicin and 5-fluorouracil. CONCLUSIONS: Taken together, results from this study indicate that MASL decreases activity of JAK-STAT, TGFβ-SMAD, and Wnt-βCTN signaling pathways to inhibit OSCC growth and motility. These data suggest that further studies should be undertaken to determine how MASL may also be used alone and in combination with other agents to treat oral cancer.

Enthusiasm for the use of dietary bioactive compounds as chemopreventive agents and adjuvants for current therapies has increased laboratory research conducted on several types of cancers including Head and Neck Squamous Cell Carcinoma (HNSCC). The green chemoprevention movement is a modern approach to highlight healthy lifestyle changes that aim to decrease the incidence of HNSCC. A healthy diet can be an effective way to prevent the development of oral cancers. Discovery of the naturally occurring plant based compounds called phytochemicals has facilitated the development of new treatment strategies for patients that are at risk for, or have developed HNSCC. Many of these compounds have been shown to elicit very potent anti-carcinogenic properties. While there are many compounds that have been studied, the compounds from two specific categories of phytochemicals, phenolics (resveratrol, EGCG, curcumin, quercetin, and honokiol) and glucosinolates (sulforaphane, PEITC and BITC), are emerging as potent and effective inhibitors of oral carcinogenesis. These compounds have been shown to inhibit HNSCC growth through a variety of mechanisms. Research has demonstrated that these compounds can regulate cancer cell proliferation through the regulation of multiple cell signaling pathways. They can impede cell cycle progression, induce differentiation and apoptosis, prevent angiogenesis, and inhibit cancer cell invasive and metastatic properties. They can protect normal cells during treatment and reduce the damage caused by chemotherapy and radiotherapy. This review aims to provide an overview of some of the most effective phytochemicals that have the potential to successfully prevent and treat head and neck squamous cell carcinoma.

COVID-19 was declared an international public health emergency in January, and a pandemic in March of 2020. There are over 125 million confirmed COVID-19 cases that have caused over 2.7 million deaths worldwide as of March 2021. COVID-19 is caused by the SARS-CoV-2 virus. SARS-CoV-2 presents a surface "spike" protein that binds to the ACE2 receptor to infect host cells. In addition to the respiratory tract, SARS-Cov-2 can also infect cells of the oral mucosa, which also express the ACE2 receptor. The spike and ACE2 proteins are highly glycosylated with sialic acid modifications that direct viral-host interactions and infection. Maackia amurensis seed lectin (MASL) has a strong affinity for sialic acid modified proteins and can be used as an antiviral agent. Here, we report that MASL targets the ACE2 receptor, decreases ACE2 expression and glycosylation, suppresses binding of the SARS-CoV-2 spike protein, and decreases expression of inflammatory mediators by oral epithelial cells that cause ARDS in COVID-19 patients. In addition, we report that MASL also inhibits SARS-CoV-2 infection of kidney epithelial cells in culture. This work identifies MASL as an agent with potential to inhibit SARS-CoV-2 infection and COVID-19 related inflammatory syndromes.

PURPOSE: Daphne mucronata Royle grown in Iran has shown anticancer activities against different cancer cell lines. Therefore, within this study, we investigate the phytochemical pattern of this plant. METHOD: Phytochemical investigation was done using standard column chromatography system: The structures were recognized by the interpretation of one and two-dimensional nuclear magnetic resonance (NMR) spectra and the help of High-Resolution Electrospray Ionization Mass spectroscopy (HR-ESIMS) and Infrared spectroscopy (IR) data. Stereochemistry was determined using 2D and 3D NOESY, and comparison of coupling constant values with literature. The absolute configuration was determined and confirmed using specific rotation and electronic circular dichroism experiments. Cytotoxicity was done against HeLa cells by standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Luciferase assay was used to check if the compounds can inhibit the activation of cancer-related signaling pathways. Molecular docking simulation was done for biological activity evaluation and to examine the interaction of the ligand with each of the proteins. RESULTS: A new sesquiterpenoid, 4,11(12)-guiadiene-1-ol-3-one (4), together with eight specialized metabolites, betulinic acid (1), coniferyl aldehyde (2), oleanolic acid (3), daphnetoxin (5), apigenin (7), syringin (8), and genkwanol A (9) were isolated and reported for the first time from the shoots of the plant. Compound 4 as an undescribed compound was submitted for cytotoxicity assay and showed moderate activity with the IC(50) value of 51.3 ± 4.2 μM against HeLa cancer cells. It showed selective inhibition of Interleukin-6 mediated signal transducer and activator of transcription 3 pathway (STAT-3/ IL-6), and Smad protein / transforming growth factor beta (TGF-β) transcription factors when screened through an array of cancer signaling pathways. Molecular docking confirmed biological tests and showed the interaction with STAT3 and Smad proteins. CONCLUSION: An undescribed sesquiterpenoid: 4,11(12)-guiadiene-1-ol-3-one in addition to eight known compounds were isolated. The new sesquiterpene was evaluated for the luciferase assay on 14 main cancer-related signaling pathways and showed selective inhibition of STAT3/IL6, and Smad/ TGF-β transcription factors. Molecular docking simulation showed more interactions with STAT3 than Smad, which confirms better interaction of compound 4 with STAT3 than Smad proteins. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s40199-020-00336-x) contains supplementary material, which is available to authorized users.

The anticancer activities of Rubia cordifolia and its constituents have been reported earlier, but their influence on the crosstalk of complex cancer-related signaling metabolic pathways (i.e., transcription factors, TF) has not yet been fully investigated. In this study, R. cordifolia root extract was subjected to the cancer signaling assay based bioactivity-guided fractionation, which yielded the following compounds viz., three anthraquinones, namely alizarin (1), purpurin (2), and emodin (3), two lignans, namely eudesmin (4) and compound 5, and two cyclic hexapeptides, namely deoxybouvardin RA-V (6), and a mixture of 6+9 (RA-XXI). The structures of the isolated compounds were determined by NMR spectroscopy and HRESIMS. The isolated compounds 1, 2, 3, 6, and a mixture of 6+9 were tested against a panel of luciferase reporter genes that assesses the activity of a wide-range of cancer-related signaling pathways. In addition, reference anthraquinones viz., chrysophanol (11), danthron (12), quinizarin (13), aloe-emodin (14), and &alpha, lapachone (15) were also tested. Among the tested compounds, the cyclic hexapeptide 6 was found to be very active against several signaling pathways, notably Wnt, Myc, and Notch with IC50 values of 50, 75, and 93 ng/mL, respectively. Whereas, the anthraquinones exhibited very mild or no inhibition against these signaling pathways. Compound 6 being the most active, we tested it for stability in simulated intestinal (SIF) and gastric fluids (SGF), since the stability in biological fluid is a key short-coming of cyclic hexapeptides. The anticancer activity of 6 was found to remain unchanged before and after the treatment of simulated gastric/intestinal fluids, indicating that RA-V was stable. As a result, it could be bioavailable when orally used in therapeutics and possibly a drug candidate for cancer treatment. The mechanism for the preferential inhibition of these pathways and the possible crosstalk effect with other previously reported signaling pathways has been discussed.

Ayurveda is a major traditional system of Indian medicine that is still being successfully used in many countries. Recapitulation and adaptation of the older science to modern drug discovery processes can bring renewed interest to the pharmaceutical world and offer unique therapeutic solutions for a wide range of human disorders. Eventhough time-tested evidences vouch immense therapeutic benefits for ayurvedic herbs and formulations, several important issues are required to be resolved for successful implementation of ayurvedic principles to present drug discovery methodologies. Additionally, clinical examination in the extent of efficacy, safety and drug interactions of newly developed ayurvedic drugs and formulations are required to be carefully evaluated. Ayurvedic experts suggest a reverse-pharmacology approach focusing on the potential targets for which ayurvedic herbs and herbal products could bring tremendous leads to ayurvedic drug discovery. Although several novel leads and drug molecules have already been discovered from ayurvedic medicinal herbs, further scientific explorations in this arena along with customization of present technologies to ayurvedic drug manufacturing principles would greatly facilitate a standardized ayurvedic drug discovery.

Structure activity relationships of aristolochic acid analogues: Toxicity in cultured renal epithelial cells. Background Aristolochia species are nephrotoxic and carcinogenic. Recent studies showed that aristolochic acid (AA) could induce acute renal failure and tubular lesions in several species and available evidences demonstrate the unequivocal role of AA in so called Chinese herbs nephropathy. Methods A series of AA derivatives isolated from Aristolochia spp. were analyzed for their nephrotoxic potential using the neutral red dye exclusion assay in cultures of LLC-PK 1 cells. The structural relationships between AA I and its analogues were compared with their cytotoxic effects to predict structural determinants for AA toxicity. Further, caspase-3 assay was performed on toxic compounds to determine if caspases, the enzymes that play a critical role in apoptosis are involved in AA-induced cytotoxicity. Results AA I was found to be most toxic followed by AA II, AA VIIIa, and AA Ia in decreasing levels of toxicity. The other compounds, nitrophenanthrene carboxylic acid analogues of AA I, aristolactams, and other derivatives did not exhibit considerable toxicity. The results showed significant relationships between cytotoxicity of AA compounds and the localization of functional groups in their structure. Analogues containing hydroxyl groups diminished cytotoxicity. The demethylated analogues of AA I are markedly less active. The negative impact on cytotoxicity was found on nitroreduction of AA I. AA induced caspase activation was also observed. Conclusion These cytotoxic data suggest that the nitro and methoxy groups are critical determinants of nephrotoxicologic potency of AA.

An integrated approach is needed to manage cancer using the growing body of knowledge gained through scientific developments. Thousands of herbal and traditional compounds are being screened worldwide to validate their use as anti-cancerous drugs. The science of Ayurveda is supposed to add a step on to the curative aspects of cancers that have resemblance with clinical entities of arbuda and granthi mentioned in Sushrutha samhita. Hence, an attempt is made in this review to discuss about the pathology and therapeutic management of various cancers described in Ayurveda. Review of literature on anticancer drugs of plant origin revealed identification of newer ayurvedic drugs that are not mentioned in the ancient texts. These new findings add up to ayurvedic science that has been developed through ages. In addition, details of experimental and clinical studies conducted on single and compound ayurvedic preparations for their anticancer efficacy strongly emphasize ayurvedic therapy as a scientifically driven one and not simply unconventional.

The amiloride-sensitive epithelial sodium channel (ENaC), a multimeric plasma membrane protein composed of alpha-, beta-, and gamma-ENaC subunits, mediates Na(+) reabsorption in epithelial tissues, including the distal nephron, colon, lung, and secretory glands, and plays a critical role in pathophysiology of essential hypertension and cystic fibrosis (CF). The function of ENaC is tightly regulated by signals elicited by aldosterone, vasopressin, agents that increase intracellular cAMP levels, ions, ion channels, G-protein-coupled mechanisms, and cytoskeletal proteins. In this paper, the effects of Ca(2+) on the expression of the human ENaC subunits expressed in human embryonic kidney cells (HEK-293 cells) were examined. Incubation of cells with increased extracellular Ca(2+) and treatment of cells with A23187 and thapsigargin stimulated the expression of the monomeric ENaC subunits. Treatment of cells with Ca(2+)-chelating agents, EGTA and BAPTA-AM, reduced the levels of ENaC subunit expression. The pulse-chase experiments suggested that a rise in the intracellular Ca(2+) increases the ENaC subunit expression. Immunoblot analysis using the anti-ubiquitin antibody indicated that ENaC undergoes ubiquitination. A correlation between the processes that regulate ENaC function with the intracellular Ca(2+) was discussed.

The amiloride-sensitive epithelial sodium channels (ENaC) mediate Na(+) reabsorption in epithelial tissues including distal nephron, colon, lung, and secretory glands and plays a critical role in pathophysiology of hypertension and cystic fibrosis. The ENaC is a multimeric protein composed of alpha-ENaC, beta-ENaC, and gamma-ENaC subunits. To study the biochemical properties of the channel, the subunit cDNAs of rat colon ENaC (rENaC) were subcloned into baculoviruses, and the corresponding proteins were expressed in Sf9 insect cells. The functional characteristics of the expressed rENaC were studied in planar lipid bilayers. The results show that expression of alpha-rENaC and alphabetagamma-rENaC in Sf9 insect cells results in the generation of cation-selective large conductance channels. Although the large conductance channels observed in the alpha-rENaC-containing membranes were unaffected by amiloride, the large conductance channels found in alphabetagamma-rENaC complex-containing membranes exhibited voltage-dependent flickering in the presence of micromolar amiloride. Possible implications of these observations are discussed.

Bioassay-guided fractionation of the leaves of Eugenia rigida yielded three stilbenes, (Z)-3,4,3',5'-tetramethoxystilbene (1), (E)-3,4,3',5'-tetramethoxystilbene (2), and (E)-3,5,4'-trimethoxystilbene (3). Their structures were determined using 1D- and 2D-NMR spectroscopy and HRESIMS. The sterically hindered Z-stereoisomer 1, a new natural product, was prepared by time-dependent photoisomerization of the E-isomer (2) under UV irradiation at λ254 nm, while 2,3,5,7-tetramethoxyphenanthrene (5) was identified at λ365 nm by UHPLC/APCI-MS and NMR spectroscopy. Compounds 1-3 were tested against a panel of luciferase reporter gene assays that assess the activity of many cancer-related signaling pathways, and the Z-isomer (1) was found to be more potent than the E-isomer (2) in inhibiting the activation of Stat3, Smad3/4, myc, Ets, Notch, and Wnt signaling, with IC50 values between 40 and 80 μM. However, both compounds showed similar inhibition against Ap-1 and NF-κB signaling. In addition, 1 demonstrated cytotoxic activity toward human leukemia cells, solid tumor cells of epidermal, breast, and cervical carcinomas, and skin melanoma, with IC50 values between 3.6 and 4.3 μM, while 2 was weakly active against leukemia, cervical carcinoma, and skin melanoma cells. Interestingly, 2 showed antioxidant activity by inhibition of ROS generation to 50% at 33.3 μM in PMA-induced HL-60 cells, while 1 was inactive at 100 μM (vs Trolox 1.4 μM).

We have found that the bacterial components lipopolysaccharides (LPS) and Braun-type lipoproteins are responsible for the majority of the in vitro macrophage activation exhibited by extracts of Echinacea and other immune enhancing botanicals. Furthermore, we show that endophytic bacteria are the principal source of these components. We also show that total bacterial load within Echinacea samples are strongly correlated with the innate immune stimulatory activity (NF-κB activation in THP-1 cells) and content of bacterial LPS within extracts of this plant material. Differences in the levels of these two bacterial components are responsible for the substantial variation observed (˜200-fold) in innate immune cell activation from E. purpurea material obtained from six major growers/commercial suppliers in North America. This substantial variability in bacterial component content/activity could be a factor contributing to the inconsistent outcomes exhibited in Echinacea clinical trials. We also demonstrate that the oral consumption of extracts rich in these bacterial components is therapeutically relevant with respect to enhancing immune parameters and for impacting disease outcomes in both animal models and in humans. Acknowledgements: This research was partially funded by grants from the National Institutes of Health R21AT01207, R01AT002360 and R01AT007042 (NCAAM) to DSP and by the USDA, Agricultural Research Service Specific Cooperative Agreement No. 58 – 6408 – 02 – 1-612.

Immulina®, a commercial extract of Arthrospira (Spirulina) platensis is a potent activator of THP-1 monocytes and CD4+ T cells IN VITRO and enhances several immunological functions in mice. We further characterized Immulina® by determining that Braun-type lipoproteins are responsible for a major portion of the IN VITRO monocyte activation exhibited by this material. In order to understand the effect of Immulina® on NK cell activity, a pilot study was conducted on ten healthy North American individuals who supplemented their diet with Immulina® (400 mg/day) for seven days. We observed a 40% average increase in the killing of K562 tumor cells by NK cells (p 0.01) after Immulina® supplementation. In a separate placebo-controlled, crossover study involving 11 healthy Danish subjects, we observed increased mRNA expression of the NK cell marker NKG2D by 37% (p = 0.02) and by 55% (p = 0.0003) after administration of Immulina® (200 mg and 400 mg per day, respectively) for seven days. The mRNA expression of the NK- and T-cell marker perforin increased by 75% (p = 0.008) after administration of 400 mg Immulina® per day. Both markers displayed significant dose-dependent effects (p = 0.0003 and p = 0.02, respectively). The ratio between CD56 (bright) and CD56 (dim) NK cells was not affected by Immulina® administration. In summary, two independent studies showed enhancement of NK cell activity following administration of Immulina® for seven days.

Immulina is a commercial extract of Spirulina (Arthrospira) platensis that is standardized by biological activity. We previously reported that this extract is a potent activator of THP-1 monocytes in vitro and that oral consumption enhanced several immunological functions in mice [1]. In this study we further characterized Immulina by determining that Braun type lipoproteins are responsible for a major portion of the in vitro monocyte activation exhibited by this material. In order to understand the effect of Immulina on the human immune system, a pilot study was conducted on ten healthy individuals who supplemented their diet with Immulina (400 mg/day) for seven days. Blood was drawn from the participating individuals at two time points: before and after seven days of Immulina supplementation. Changes in mononuclear and polymorphonuclear phagocytosis were determined in heparinized whole blood as well as the cytotoxicity exhibited by natural killer (NK) and lymphokine activated killer cells. We observed statistically significant increases both in tumor cell killing by NK cells (p = 0.0019) and in phagocytosis by blood mononuclear cells (p = 0.0124) after Immulina supplementation. Acknowledgements: This research was partly funded by a USDA, Agricultural Research Service Specific Cooperative Agreement No. 58-6408-012. Immulina capsules were supplied by Scandinavian Clinical Nutrition Denmark A/S, Greve, Denmark. References: [1] Balachandran P, et al. (2006), Inteternational Immunopharmacology, 6: 1808–1814.

We previously reported that the majority of in vitro monocyte/macrophage activation exhibited by extracts of Echinacea and other botanicals depends on bacterial lipopolysaccharides and Braun type bacterial lipoproteins. We determined the contribution made by these bacterial components to the overall immune enhancing activity detected in E. purpurea and E. angustifolia from bulk root and aerial material obtained from six major growers/suppliers in North America. Substantial variation in activity (up to 200-fold) was observed in extracts of these materials when tested in two monocyte/macrophage cell lines. The majority of activity was negated by treatment with agents that target bacterial lipoproteins (lipoprotein lipase) and lipopolysaccharides (polymyxin B). Experiments comparing the activity of freeze dried, freshly harvested Echinacea plants with those harvested and dried using various commercially relevant conditions, suggest that post-harvesting procedures do not substantially contribute to the variation observed in the commercial material.

We have identified potent monocyte/macrophage activating bacterial lipoproteins within commonly used immune enhancing botanicals such as Echinacea, American ginseng and alfalfa sprouts. These bacterial lipoproteins, along with lipopolysaccharides, were substantially more potent than other bacterially derived components when tested in in vitro monocyte/macrophage activation systems. In experiments using RAW 264.7 and mouse peritoneal macrophages the majority (85–98%) of the activity within extracts from eight immune enhancing botanicals was eradicated by treatment with agents (lipoprotein lipase and polymyxin B) known to target these two bacterial components. Alfalfa sprouts exhibited the highest activity of those botanicals tested but the appearance of this activity during the germination of surface sterilized seeds was abolished by the presence of antibiotics. These studies indicate that the majority of the in vitro macrophage activating properties in extracts from these botanicals can be attributed to the presence of lipoproteins and lipopolysaccharides derived from bacteria and that bacterial endophytes may be a significant source of these components.

We reported previously that a high molecular weight polysaccharide fraction (Immulina) from Spirulina was a potent activator of NF-kappa B and induced both IL-1 beta and TNF-alpha mRNAs in THP-1 human monocytes. In the present study, we show that NF-kappa B activation by Immulina is suppressed by antibodies to CD14 and TLR2 but not by antibodies to TLR4. Similarly, NF-kappa B directed luciferase expression was enhanced by Immulina treatment when cells were co-transfected with vectors expressing proteins supporting TLR2- (CD14 and TLR2) but not TLR4-(CD14, TLR4, and MD-2) dependent activation. Mice that consumed a chemically defined chow mixed with an extract containing Immulina exhibited changes in several immune parameters. The ex vivo production of IgA and IL-6 from Peyer's patch cells was enhanced 2-fold and interferon-gamma production from spleen cells was increased 4-fold in Immulina-treated mice. The enhanced production of these factors was most notable with mice that had consumed this extract for 4 or 5 days. These studies shed light on how Immulina activates cells of the innate immune system and suggests that oral consumption of this polysaccharide can enhance components within both the mucosal and systemic immune systems.

The agents responsible for the therapeutic effects of many botanical supplements have not been established in spite of their popularity. Here we show that melanin is a previously unrecognized immunostimulatory compound that is a major component of botanicals traditionally used to enhance immune function. While melanin is present in commonly consumed vegetables, its specific activity is several orders of magnitude less than melanin extracted from these botanicals. The major reason that this agent has eluded detection is its solvent-specific requirement for extraction/solubility. Melanin activates NF-kappa B in monocytes in vitro through a toll-like receptor 2-dependent process. Ingestion of melanin by mice for four days increases production ex vivo of interferon-gamma by spleen cells and IgA and interleukin-6 by Peyer's patch cells. The identification of this new class of mucosal immune stimulants will allow further characterization of botanical products and advances our understanding of the basis for their traditional use.