Ginkgolides with anti-PAF activity from Ginkgo biloba L.

Four undescribed ginkgolides, including two rare sesquiterpene ginkgolides (compounds 1 and 2) and two diterpenoid ginkgolides (compounds 3 and 4), were isolated from Ginkgo biloba L. The structures of these four ginkgolides were identified based on extensive spectroscopic analysis, DP4+ probability analysis and X-ray diffraction. Compounds 1 and 2 exhibited excellent antiplatelet aggregation activities with IC50 values of 1.20 ± 0.25 and 4.11 ± 0.34 µM, respectively.

Pharmacokinetics of ginkgolide B-lyophilized nanoparticles after intravenous injection in rats using liquid chromatography-tandem mass spectrometry.

RATIONALE: Ginkgolide B (GB) performs diverse pharmacological activities but has poor water solubility. The currently available GB injections have a short half-life and are lethal when injected rapidly. We prepared GB-lyophilized nanoparticles (GB-NPs) using a new nonsurfactant polysaccharide polymer, ZY-010, as its carrier to regulate the release of GB in vivo. Here, the pharmacokinetics (PK) of GB-NPs after intravenous injection in rats was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: The samples were separated on an Agilent Eclipse XDB-C 18 column (2.1 × 50 mm, 1.85 µm) maintained at 30°C. The MS/MS transitions of GB and glibenclamide as the internal standard (IS) were set at m/z 423.1 → 367.1 and m/z 492.1 → 367.0, respectively. The standard curve of GB content was constructed, and the specificity, sensitivity, precision, and extraction recovery of LC-MS/MS analysis were assessed. The main PK parameters were analyzed using DAS (Drug And Statistics for Windows) software, version 2.0. RESULTS: The retention time of GB and IS at elution was 2.77 and 4.75 min, respectively. An excellent linear response across the concentration range of 0.001-100 µg/ml was achieved (r = 0.9997). The relative standard deviation value of precision was less than 10%. The total extraction recovery was above 80.76 ± 2.08%. The main PK parameters for the GB-NPs were as follows: t1/2 = 69.32 h, AUC(0 → ∞) = 188 312.97 ± 143 312.41 µg/L h, CL = 0.03 ± 0.02 L/h/kg, and V = 0.09 ± 0.05 L/kg. The t1/2 of the GB-NPs was significantly longer than that of GB solution, and AUC(0 → ∞) of GB-NPs was about 1.4 times that of GB solution. The PK data demonstrated that the blood concentration of GB in rats conformed to a three-compartment model in both GB solution and GB-NPs. CONCLUSION: A rapid and accurate LC-MS/MS method was established for the determination of GB-NPs in rats. GB-NPs exhibited a sustained-release behavior in vivo compared with GB solution.

Treatment of Ginkgo biloba with Exogenous Sodium Selenite Affects Its Physiological Growth, Changes Its Phytohormones, and Synthesizes Its Terpene Lactones.

Ginkgolide is a unique terpenoid natural compound in Ginkgo biloba, and it has an important medicinal value. Proper selenium has been reported to promote plant growth and development, and improve plant quality, stress resistance, and disease resistance. In order to study the effects of exogenous selenium (Se) on the physiological growth and the content of terpene triolactones (TTLs) in G. biloba seedlings, the seedlings in this work were treated with Na2SeO3. Then, the physiological indexes, the content of the TTLs, and the expression of the related genes were determined. The results showed that a low dose of Na2SeO3 was beneficial to plant photosynthesis as it promoted the growth of ginkgo seedlings and increased the root to shoot ratio. Foliar Se application significantly increased the content of soluble sugar and protein and promoted the content of TTLs in ginkgo leaves; indeed, it reached the maximum value of 7.95 mg/g in the ninth week, whereas the application of Se to the roots inhibited the synthesis of TTLs. Transcriptome analysis showed that foliar Se application promoted the expression levels of GbMECPs, GbMECT, GbHMGR, and GbMVD genes, whereas its application to the roots promoted the expression of GbDXS and GbDXR genes. The combined analysis results of metabolome and transcriptome showed that genes such as GbDXS, GbDXR, GbHMGR, GbMECPs, and GbCYP450 were significantly positively correlated with transcription factors (TFs) GbWRKY and GbAP2/ERF, and they were also positively correlated with the contents of terpene lactones (ginkgolide A, ginkgolide B, ginkgolide M, and bilobalide). Endogenous hormones (MeJA-ILE, ETH, and GA7) were also involved in this process. The results suggested that Na2SeO3 treatment affected the transcription factors related to the regulation of endogenous hormones in G. biloba, and further regulated the expression of genes related to the terpene synthesis structure, thus promoting the synthesis of ginkgo TTLs.

A gene cluster in Ginkgo biloba encodes unique multifunctional cytochrome P450s that initiate ginkgolide biosynthesis.

The ginkgo tree (Ginkgo biloba) is considered a living fossil due to its 200 million year's history under morphological stasis. Its resilience is partly attributed to its unique set of specialized metabolites, in particular, ginkgolides and bilobalide, which are chemically complex terpene trilactones. Here, we use a gene cluster-guided mining approach in combination with co-expression analysis to reveal the primary steps in ginkgolide biosynthesis. We show that five multifunctional cytochrome P450s with atypical catalytic activities generate the tert-butyl group and one of the lactone rings, characteristic of all G. biloba trilactone terpenoids. The reactions include scarless C-C bond cleavage as well as carbon skeleton rearrangement (NIH shift) occurring on a previously unsuspected intermediate. The cytochrome P450s belong to CYP families that diversifies in pre-seed plants and gymnosperms, but are not preserved in angiosperms. Our work uncovers the early ginkgolide pathway and offers a glance into the biosynthesis of terpenoids of the Mesozoic Era.

Discovery of unreported ginkgolides of anti-PAF activity using characteristic ion and neutral loss recognition strategy in Ginkgo biloba L.

Ginkgolides are the most important bioactive components of Ginkgo biloba L, of which ginkgolide B has been successfully developed and marketed as a drug. The reported ginkgolides are very rare and exhibit a complex matrix due to the chemodiversity of Ginkgo biloba L. Herein, the global profile of characteristic ion and neutral loss recognition strategy were used for to discover eight undescribed ginkgolides, very rare cyclohexane ginkgolides R-V, ginkgolides D-F, and eight known ginkgolides. These ginkgolides were target isolated and identified using high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy, and X-ray crystallography. The undescribed and known ginkgolides exhibited antiplatelet aggregation activities. In particular, compounds U and D had IC50 values of 2.20 ± 0.15 and 6.50 ± 0.87 µM, respectively. This study has enriched the known structural diversity of ginkgolides and extended the application of mass spectrometry to the global profiling of natural products present in Ginkgo biloba L. Moreover, it could help chemists rapidly discover unreported compounds from a complex matrix.

Genome-wide transcriptome analysis reveals the regulatory network governing terpene trilactones biosynthesis in Ginkgo biloba.

Ginkgo biloba L. is currently the only remaining gymnosperm of the Ginkgoaceae Ginkgo genus, and its history can be traced back to the Carboniferous 200 million years ago. Terpene trilactones (TTLs) are one of the main active ingredients in G. biloba, including ginkgolides and bilobalide. They have a good curative effect on cardiovascular and cerebrovascular diseases because of their special antagonistic effect on platelet-activating factors. Therefore, it is necessary to deeply mine genes related to TTLs and to analyze their transcriptional regulation mechanism, which will hold vitally important scientific and practical significance for quality improvement and regulation of G. biloba. In this study, we performed RNA-Seq on the root, stem, immature leaf, mature leaf, microstrobilus, ovulate strobilus, immature fruit and mature fruit of G. biloba. The TTL regulatory network of G. biloba in different organs was revealed by different transcriptomic analysis strategies. Weighted gene co-expression network analysis (WGCNA) revealed that the five modules were closely correlated with organs. The 12 transcription factors, 5 structural genes and 24 Cytochrome P450 (CYP450) were identified as candidate regulators for TTL accumulation by WGCNA and cytoscape visualization. Finally, 6 APETALA2/ethylene response factors, 2 CYP450s and bHLH were inferred to regulate the metabolism of TTLs by correlation analysis. This study is the comprehensive in authenticating transcription factors, structural genes and CYP450 involved in TTL biosynthesis, thereby providing molecular evidence for revealing the comprehensive regulatory network involved in TTL metabolism in G. biloba.

Neuroregulatory role of ginkgolides.

The application of ginkgolides as a herbal remedy reaches ancient China. Over time many studies confirmed the neuroprotective effect of standard Ginkgo biloba tree extract-the only available ginkgolide source. Ginkgolides present a wide variety of neuroregulatory properties, commonly used in the therapy process of common diseases, such as Alzheimer's, Parkinson's, and many other CNS-related diseases and disorders. The neuroregulative properties of ginkgolides include the conditioning of neurotransmitters action, e.g., glutamate or dopamine. Besides, natural compounds induce the inhibition of platelet-activating factors (PAF). Furthermore, ginkgolides influence the inflammatory process. This review focuses on the role of ginkgolides as neurotransmitters or neuromodulators and overviews their impact on the organism at the molecular, cellular, and physiological levels. The clinical application of ginkgolides is discussed as well.

Advances in Supercritical Carbon Dioxide Extraction of Bioactive Substances from Different Parts of Ginkgo biloba L.

Ginkgo biloba L. has always been a popular area of research due to its various active ingredients and pharmacological effects. Ginkgo biloba is rich in ginkgo flavonoids, ginkgolides, and ginkgolic acid, with anti-inflammation, antioxidation, neuroprotection, anti-platelet agglutination, hypolipidemic effect, anti-cancer, and anti-radiation properties. There are many methods to extract and separate the active components of ginkgo. Among them, supercritical carbon dioxide fluid extraction (SFE-CO2) is known for its green, clean, and environment-friendly properties. In this paper, the pharmacological activities, the active components, and structures of different parts of ginkgo, the extraction methods of its effective ingredients, and the application of the SFE-CO2 method for the extraction and separation of active ingredients in Ginkgo biloba from leaves, seeds, pollen, and roots were reviewed, in order to make best use of ginkgo resources, and provide support and references for the development of SFE-CO2 of active components from Ginkgo biloba.

Diterpene Ginkgolides Meglumine Injection inhibits apoptosis induced by optic nerve crush injury via modulating MAPKs signaling pathways in retinal ganglion cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Diterpene Ginkgolides Meglumine Injection (DGMI) is made of extracts from Ginkgo biloba L, including Ginkgolides A, B, and K and some other contents, and has been widely used as the treatment of cerebral ischemic stroke in clinic. It can be learned from the "Compendium of Materia Medica" that Ginkgo possesses the effect of "dispersing toxin". The ancient Chinese phrase "dispersing toxin" is now explained as elimination of inflammation and oxidative state in human body. And it led to the original ideas for today's anti-oxidation studies of Ginkgo in apoptosis induced by optic nerve crush injury. AIM OF THE STUDY: To investigate the underlying molecular mechanism of the DGMI in retinal ganglion cells (RGCs) apoptosis. MATERIALS AND METHODS: TUNEL staining was used to observe the anti-apoptotic effects of DGMI on the adult rat optic nerve injury (ONC) model, and flow cytometry and hoechst 33,342 staining were used to observe the anti-apoptotic effects of DGMI on the oxygen glucose deprivation (OGD) induced RGC-5 cells injury model. The regulation of apoptosis and MAPKs pathways were investigated with Immunohistochemistry and Western blotting. RESULTS: This study demonstrated that DGMI is able to decrease the conduction time of F-VEP and ameliorate histological features induced by optic nerve crush injury in rats. Immunohistochemistry and TUNEL staining results indicated that DGMI can also inhibit cell apoptosis via modulating MAPKs signaling pathways. In addition, treatment with DGMI markedly improved the morphological structures and decreased the apoptotic index in RGC-5 cells. Mechanistically, DGMI could significantly inhibit cell apoptosis by inhibiting p38, JNK and Erk1/2 activation. CONCLUSION: The study shows that DGMI and ginkgolides inhibit RGCs apoptosis by impeding the activation of MAPKs signaling pathways in vivo and in vitro. Therefore, the present study provided scientific evidence for the underlying mechanism of DGMI and ginkgolides on optic nerve crush injury.

Synthetic, Mechanistic, and Biological Interrogation of Ginkgo biloba Chemical Space En Route to (-)-Bilobalide.

Here we interrogate the structurally dense (1.64 mcbits/Å3) GABAA receptor antagonist bilobalide, intermediates en route to its synthesis, and related mechanistic questions. 13C isotope labeling identifies an unexpected bromine migration en route to an α-selective, catalytic asymmetric Reformatsky reaction, ruling out an asymmetric allylation pathway. Experiment and computation converge on the driving forces behind two surprising observations. First, an oxetane acetal persists in concentrated mineral acid (1.5 M DCl in THF-d8/D2O); its longevity is correlated to destabilizing steric clash between substituents upon ring-opening. Second, a regioselective oxidation of des-hydroxybilobalide is found to rely on lactone acidification through lone-pair delocalization, which leads to extremely rapid intermolecular enolate equilibration. We also establish equivalent effects of (-)-bilobalide and the nonconvulsive sesquiterpene (-)-jiadifenolide on action potential-independent inhibitory currents at GABAergic synapses, using (+)-bilobalide as a negative control. The high information density of bilobalide distinguishes it from other scaffolds and may characterize natural product (NP) space more generally. Therefore, we also include a Python script to quickly (ca. 132 000 molecules/min) calculate information content (Böttcher scores), which may prove helpful to identify important features of NP space.

A new bilobalide isomer and two cis-coumaroylated flavonol glycosides from Ginkgo biloba leaves.

A new bilobalide isomer (1), together with two flavonol glycosides (2, 3), have been isolated and elucidated from the extract of Ginkgo biloba leaves. Significantly, 1 was a new sesquiterpene lactone with two lactone ring groups, both 2 and 3 were two flavonol glycosides with a same cis-coumaroylated fragment. Their chemical structures were elucidated by NMR and MS spectroscopic date and the absolute configuration of 1 was specific established by Cu-Kα X-ray crystallographic analyses. However, 1-3 showed no obvious anti-platelet aggregation activity.

Bilobalide and PC12 cells: A structure activity relationship study.

Ginkgo biloba extracts have been postulated to beneficial for improving cognitive function and as such they have been used as a potential treatment of Alzheimer's disease. The main active ingredients of the extract are terpene trilactones (TTLs), such as bilobalide (BB) and ginkgolides. Several structure-activity relationship (SAR) studies using ginkgolide scaffolds produced more biologically potent species by modification of the lactone moieties. However, modifications of BB scaffold have been limited, and no SAR studies on BB have been accomplished to date. Thus, the aim of this study was to elucidate how the modification of the lactone moieties of BB would affect their biological activities in a number of assays, including proliferating cell activity, neuroprotective effects against Aß (1-40) peptides, and neurite outgrowth effects in PC12 neuronal cells. It appeared that the derivatives containing lactone groups showed similar biological activity to native BB, while those that possessed no lactone moieties exhibited lower neurite outgrowth effects. Thus, the results suggested that the lactone moieties of BB played an important role in exerting neurite outgrowth effects in PC12 cells.

Theoretical design, preparation, and evaluation of Ginkgolide B molecularly imprinted polymers.

Ginkgolide B is in great demand worldwide on account of its extensive and excellent pharmacological effects, however, it is difficult to separate and purify ginkgolide B. In this study, ginkgolide B molecularly imprinted polymers were prepared by combining software simulation and molecular imprinting technique, and its characterization and adsorption performed evaluation were performed to understand the adsorption behavior of the polymers. The adsorption equilibrium concentration of molecularly imprinted polymers was 0.70 mg/mL, and the adsorption equilibrium time was 4 h. Meanwhile, the adsorption isotherm of the polymers for ginkgolide B fitted well with the Langmuir model, and the adsorption kinetics was in line with the pseudo-second-order kinetics. In contrast, the adsorption capacity of molecularly imprinted polymers on ginkgolide B was higher than that of non-molecular imprinted polymers, with better selectivity and better adsorption after repeated use for six times. The application experiments showed that molecular imprinted polymers have a good adsorption effect in low purity samples. Therefore, the polymers reported herein can be expected to apply in the adsorption and separation of ginkgolide B samples.

Pharmacokinetics and pharmacodynamics analysis of XQ-1H and its combination therapy with clopidogrel on cerebral ischemic reperfusion injury in rats.

Ischemic stroke is the main cause of disability and mortality worldwide. 10-O-(N N-dimethylaminoethyl)-ginkgolide B methane-sulfonate (XQ-1 H) is a novel drug based on the remedial approach for ischemic stroke. Clopidogrel, a widely used anti-platelet drug, is often co-prescribed in the clinic. In this study, we established an UPLC-MS/MS spectrometry method for the determination of XQ-1H and investigated the pharmacokinetic effect of clopidogrel on XQ-1H in rats subjected to middle cerebral artery occlusion (MCAO). Meanwhile, the anti-apoptotic and neuroprotective effects of XQ-1H and its combination with clopidogrel were also studied. The results revealed that XQ-1H and its combination with clopidogrel abridged brain infarct volume, cerebral edema and alleviated neurological dysfunction caused by cerebral ischemic reperfusion injury. Further study demonstrated that XQ-1H combined with clopidogrel lessened TUNEL positive cells, up-regulated bcl-2 expression notably and down-regulated bax expression as compared to both XQ-1H and clopidogrel individually. In addition, a rapid, sensitive UPLC-MS/MS method was developed to quantify the concentration of XQ-1H in MCAO/R rats. Our pharmacokinetic results showed that clopidogrel significantly increased the exposure of XQ-1H, increased the peak plasma concentration (Cmax), area under the curve (AUC) and slowed elimination of XQ-1H in the co-administered group. Besides, for further exploring which CYP450 isoforms are involved in the XQ-1H metabolism, XQ-1H was incubated in human liver microsomes (HLMs) system with or without P450 isoform-selective inhibitors. Our results revealed that clopidogrel altered pharmacokinetics of XQ-1H potentially and subsequently enhanced the pharmacological effect of XQ-1H. Moreover, XQ-1H could be applied as an efficacious neuroprotective agent for ischemic stroke because of its considerable effect on averting neuronal apoptosis.

Ginkgolide B and bilobalide promote the growth and increase ß-catenin expression in hair follicle dermal papilla cells of American minks.

The regeneration of proliferation potential of dermal papilla (DP) cells contributes to the treatment of hair loss disorders. Ginkgolide B (GKB) and bilobalide (BB) are two functional components isolated from Ginkgo biloba that can promote hair growth. In the current study, the effect of GKB or BB on DP cell viability and the related signaling was assessed. Hair follicles were isolated from minks, and the growth of hair follicles was measured under the administration of GKB or BB. DP cells isolated from minks were also subjected to GKB or BB. The administration of GKB or BB induced the growth of hair follicles. The viability of DP cells was also increased by GKB or BB as illustrated by methyl thiazolyl tetrazolium and flow cytometry detection. Moreover, the secretion of VEGF was enhanced by GKB or BB. At molecular level, the activities of Akt, ERK1/2, and ß-catenin were induced by GKB, whereas BB only increased the activities of Akt and ß-catenin. In conclusion, although the two components influenced the ß-catenin signaling activity in distinct mechanisms, they both increased the viability of DP cells and promoted the cycle of hair follicles.

Ginkgolides-loaded soybean phospholipid-stabilized nanosuspension with improved storage stability and in vivo bioavailability.

The purpose of this study was to investigate the effects of soybean phospholipid, as a steric stabilizer, on improving dissolution rate, storage stability and bioavailability of ginkgolides. The ginkgolides coarse powder, hydroxypropyl methylcellulose (HPMC), soybean phospholipid and sodium dodecyl sulfate (SDS) were mixed and wet-milled to prepare nanosuspension S1. Nanosuspension S2 was obtained by the same technique except adding the soybean phospholipid. Results of particle size showed that particle size (D50) of S1 significantly decreased from 44.25 µm to 0.373 µm. Results of differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and transmission electron microscope (TEM) showed that ginkgolides in nanosuspension still maintained its crystallinity, and the nanoparticles were all nearly circular and uniformly dispersed. Then, pellets F1 and F2 were prepared by layering S1 and S2 onto the microcrystalline cellulose (MCC) spheres, respectively. The dissolution rate of ginkgolide A (GA) and ginkgolide B (GB) in F1 was 98.3% and 97.7% in 30 min, respectively. It was much higher than F2 (89.0% and 86.5%) and coarse powder of ginkgolides (22.3% and 24.6%). According to the results of stability test, the storage stability of F1 was improved compared with F2. In addition, compared with coarse powder of ginkgolides, the relative bioavailability of GA and GB in F1 were up to (221.84 ±â€¯106.67) % and (437.45 ±â€¯336.43) %, respectively. The above results demonstrated that soybean phospholipid added to the nanosuspension played an important role in improving drug dissolution rate, storage stability and in vivo bioavailability: (1) The amphiphilic soybean phospholipid interacted with the drug, with the hydrophobic part adsorbed on the surface of the poorly soluble drug and the hydrophilic part exposed to the aqueous medium. This increases the wettability of the nanoparticles, which ensure a good redispersibility of the drug particles. (2) It could self-assemble to form an interfacial phospholipid film by surrounding the individual nanoparticles, which can produce enough steric hindrance to prevent nanoparticles from aggregation and ensure a rapid dissolution rate. (3) Soybean phospholipid and its hydrolysate formed strong micellar solubilizing vehicles with bile salts in vivo, stimulated the absorption process of ginkgolides. Thus, soybean phospholipid was a promising steric stabilizer in nanosuspension drug delivery system.

Design, Synthesis and Anti-Platelet Aggregation Activity Study of Ginkgolide-1,2,3-triazole Derivatives.

Ginkgolides are the major active component of Ginkgo biloba for inhibition of platelet activating factor receptor. An azide-alkyne Huisgen cycloaddition reaction was used to introduce a triazole nucleus into the target ginkgolide molecules. A series of ginkgolide-1,2,3-triazole conjugates with varied functional groups including benzyl, phenyl and heterocycle moieties was thus synthesized. Many of the designed derivatives showed potent antiplatelet aggregation activities with IC50 values of 5~21 nM.

RETRACTED: Ginkgolides B alleviates hypoxia-induced PC-12 cell injury by up-regulation of PLK1.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief as panels within Figures 2D and 5D appear similar to each other. Given the comments of Dr Elisabeth Bik regarding this article "This paper belongs to a set of over 400 papers (as per February 2020) that share very similar Western blots with tadpole-like shaped bands, the same background pattern, and striking similarities in title structures, paper layout, bar graph design, and - in a subset - flow cytometry panels", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request.

Studies of the Anti-amnesic Effects and Mechanisms of Single and Combined Use of Donepezil and Ginkgo Ketoester Tablet on Scopolamine-Induced Memory Impairment in Mice.

Ginkgo ketoester tablets (GT) and donepezil were a clinically used combination for the treatment of Alzheimer's disease (AD). The aim of the study was undertaken to investigate the antiamnesic effects of the two drugs alone and in combination through in vivo models of the Morris water maze along with in vitro antioxidants, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). The potential mechanisms were speculated by the activities of acetylcholine (ACh), AChE, superoxide dismutase (SOD), and malondialdehyde (MDA) and the protein expression of brain-derived neurotrophic factor (BDNF) and tyrosine protein kinase B (TrkB). The combination group showed a concentration-dependent inhibition of cholinesterase and antioxidation. As far as its mechanism was concerned, the combination of two drugs exerted excellent effects on oxidative stress, cholinergic pathway damage, and inactivation of the BDNF-TrkB signaling pathway. Additionally, to elucidate the binding mechanism of GT active ingredients into the structure of AChE, the results of molecular docking studies indicated that hydrogen and/or hydrophobic bonds might play an important role in their binding process. Thus, the combination of drugs could treat AD perfectly and further verify the scientific rationality of clinical medication.

Carnosic Acid, Tangeretin, and Ginkgolide-B Anti-neoplastic Cytotoxicity in Dual Combination with Dexamethasone-[anti-EGFR] in Pulmonary Adenocarcinoma (A549).

BACKGROUND: Traditional chemotherapeutics of low-molecular weight diffuse passively across intact membrane structures of normal healthy cells found in tissues and organ systems in a non-specific unrestricted manner which largely accounts for the induction of most sequelae which restrict dosage, administration frequency, and duration of therapeutic intervention. Molecular strategies that offer enhanced levels of potency, greater efficacy and broader margins-of-safety include the discovery of alternative candidate therapeutics and development of methodologies capable of mediating properties of selective "targeted" delivery. MATERIALS AND METHODS: The covalent immunopharmaceutical, dexamethasone-(C21-phosphoramidate)-[anti- EGFR] was synthesized utilizing organic chemistry reactions that comprised a multi-stage synthesis regimen. Multiple forms of analysis were implemented to vadliate the successful synthesis (UV spectrophotometric absorbance), purity and molar-incorporation-index (UV spectrophotometric absorbance, chemical-based protein determination), absence of fragmentation/polymerization (SDS-PAGE/chemiluminescent autoradiography), retained selective binding-avidity of IgG-immunoglobulin (cell-ELISA); and selectively "targeted" antineoplastic cytotoxicity (biochemistry-based cell vitality/viability assay). RESULTS: The botanicals carnosic acid, ginkgolide-B and tangeretin, each individually exerted maximum antineoplastic cytotoxicity levels of 58.1%, 5.3%, and 41.1% respectively against pulmonary adenocarcinoma (A549) populations. Dexamethasone-(C21-phosphoramidate)-[anti-EGFR] formulated at corticosteroid/ glucocorticoid equivalent concentrations produced anti-neoplastic cytotoxicity at levels of 7.7% (10-9 M), 26.9% (10-8 M), 64.9% (10-7 M), 69.9% (10-6 M) and 73.0% (10-5 M). Ccarnosic acid, ginkgolide-B and tangeretin in simultaneous dual-combination with dexamethasone-(C21-phosphoramidate)-[anti-EGFR] exerted maximum anti-neoplastic cytotoxicity levels of 70.5%, 58.6%, and 69.7% respectively. DISCUSSION: Carnosic acid, ginkgolide-B and tangeretin botanicals exerted anti-neoplastic cytotoxicity against pulmonary adenocarcinoma (A549) which additively contributed to the anti-neoplastic cytotoxic potency of the covalent immunopharmaceutical, dexamethasone-(C21-phosphoramidate)-[anti-EGFR]. Carnosic acid and tangeretin were most potent in this regard both individually and in dual-combination with dexamethasone-(C21- phosphoramidate)-[anti-EGFR]. Advantages and attributes of carnosic acid and tangeretin as potential monotherapeutics are a wider margin-of-safety of conventional chemotherapeutics which would readily complement the selective "targeted" delivery properties of dexamethasone-(C21-phosphoramidate)-[anti-EGFR] and possibly other covalent immunopharmaceuticals in addition to providing opportunities for the discovery of combination therapies that provide heightened levels of anti-neoplastic efficacy.