Research on the Scientific Evolution of the Flavonoid Agathisflavone

Purpose: Flavonoids are a group of secondary metabolites of the polyphenols class present in several plant species. Among them, the biflavonoid agathisflavone is of interest since it bears several biological effects that include: antiviral, antitumoral, antiprotozoal and neurogenic actions. In this sense, this study aims to use the important tool of scientific prospecting to assess the level of research development concerning the flavonoid agathisflavone. Methods: The experimental design was carried out through strategic reach with keywords on the PubMed (National Center for Biotechnology Information NCBI) and Science Direct platforms. The articles were compiled and exported to Microsoft Office Excel 2007, where they were analyzed, stored and distributed in charts organized as to different countries, year of publication of scientific articles and journals RESULTS: The prospective research resulted in the identification of 81 scientific productions, published in several journals, submitted by different countries, in several areas of medical domain and in different years of publication over the last 50 years (1965 2018). It was also possible to investigate the advances in the study of agathisflavone for the development of new therapeutics. Conclusion: Although agathisflavone has been known in the literature since at least 1969, only 23 of the eligible articles found evaluated its possible therapeutic effects. The demonstrated biological activities of agathisflavone range from antiprotozoal to neurogenesis and neuroprotection, however, the molecule needs to be better studied at the in vivo and human level. __________________________________________________________________________________________


INTRODUCTION
Flavonoids account for a large group of secondary metabolites of plant origin of the polyphenol class. They are found in different plant groups and may be present in several parts of plants, such as fruits, vegetables, seeds, bark, roots, stems and flowers (1). These compounds are formed by the combination of derivatives synthesized from phenylalanine (shikimic acid metabolic pathway) and acetic acid. The basic structure of flavonoids is composed of fifteen atoms of carbon arranged in structures with two phenolic rings A and B and a heterocyclic pyran or pyrone ring C with a carbonyl group in position C-4 (2).
Several biological activities are attributed to flavonoids, such as antitumor (3), antioxidant (4), antiviral (5), and anti-inflammatory properties (6), among others, which give this group of compounds notable pharmacological significance. What makes them even more attractive is that flavonoids are integrated and abundant in diets rich in fruits and vegetables and that they have little or no adverse effects (7).
A specific class of flavonoids that has drawn the interest from scientific researchers is the class of biflavonoids, compounds composed of a combination of flavonoid dimers connected by C-C or C-O-C bonds (8). This class of flavonoids has several therapeutic effects, sometimes having higher pharmacological efficiency than their respective monomers. Thapa et al. (2011) (7) reported that the biflavonoid taiwaniaflavone was more efficient than its monomer, apigenin, in inhibiting Aβ toxicity and decreasing fibrillogenesis in in vitro models of Alzheimer's Disease (AD). In another study, morelloflavone showed a more potent antioxidant activity than its monomer, quercetin (9). Agathisflavone (Fig. 1), a flavonoid produced by oxidative coupling of two apigenins (10), has been investigated for its pharmacological potential showing promising in vitro effects, such as antibiotic (11), antiviral (12,13), antiprotozoal (14) and antitumor (15,16).
Agathisflavone has also shown positive effects in the induction of neurogenesis and neuronal differentiation (17,18), as well as neuroprotection against glutamate-induced neurotoxicity (18).
The present study presents an overview of the biflavonoid agathisflavone in the international scenario, aiming to evaluate the presence of this flavonoid in the scientific literature, as well as to identify possible uses for a therapeutic approach.

METHODS
For this research, an exploratory scientific prospection was carried taking into consideration all publications, with the objective of identifying investigations about agathisflavona in the scientific literature. We used PubMed, the database of the National Library of Medicine of the United States of America, developed by the National Center for Biotechnology Information (NCB) and maintained by the National Library of Medicine, the free version of the Medline database, available on http://www.nlm.nih.gov/citingmedicine and Science Direct, the Elsevier scientific literature platform belonging to the RELX group, available on http://www.sciencedirect.com.
PubMed comprises more than 26 million citations from Medline's biomedical literature, science journals and several online books. Science Direct includes more than 12 million citations in several fields of science, more than 3,500 scientific journals and more than 34,000 electronic books.
The research itself includes scientific articles from a number of journals. Data collection was performed in February 2018, using the keywords "agathisflavone" and "6,8-biapigenin" in the title, abstract and key words.
The articles were compiled and exported to Microsoft Office Excel 2007, where they were analyzed, stored and distributed in charts organized as to different countries, year of publication of scientific articles and journals (Fig. 1).

Prospective analysis
We found a total of 81 scientific publications between 1969 and 2018, with 16 articles referred on the Pubmed platform, 55 on the Science Direct platform and 10 appearing on both platforms, as shown in figure 2. In the period from 2012 to 2018, there was an increase in the number of scientific productions mentioning agathisflavone (Fig. 3).
The journal with the highest number of publications mentioning agathisflavone is Phytochemistry, with 19 publications, followed by Journal of Ethnopharmacology and Tetrahedron Letters, with 9 and 6 publications, respectively (Fig.  4). As for the countries responsible for the publications, Brazil was the country with the largest number, with 16 publications, followed by China and Germany, with 11 publications each (Fig. 5).
An important aspect to note in this prospection is that publications frequently found on the Pubmed database were not available on Science Direct and vice versa. Sixteen of the papers found on Pubmed appear only on this database, and so do 55 of the papers found on Science Direct. Only 10 of the 81 publications were found on both databases. It demonstrates the importance of collecting data for scientific research using more than one source, because publications of a particular subject do not always appear universally on all databases.

Background of extraction and identification of agathisflavone
The literature reports that the flavonoid agathisflavone is present in plants of different genus, in different parts of plants, and can be extracted by several methods, being found in extracts of various solvents. The first article found in this study only mentions agathisflavone as one of the flavonoids that can be identified in the plant Searsia succedanea (Rhus succedanea) (19). Chexal et al. (1970) (20) reported that thin-layer chromatography on silica gel, using benzenepyridine formic acid as a solvent, allowed the easy identification and separation of 28 distinct biflavonoids, among them agathisflavone. Yuh-Meei and Fa-Ching (1974) (21) used thin-layer chromatography on polyamide and methanol solvent, followed by nuclear magnetic resonance (NMR) spectroscopy, to isolate and identify agathisflavone from Searsia succedanea (Rhus succedanea). The authors reported that this method allowed the extraction of larger amounts of flavonoid than did the methods used in articles published in the literature until then. Ilyas 1969 1970 -1974 1975 -1979 1980 -1984 1985 -1989 1990 -1994 1995 -1999     Agathisflavone also showed moderate cytotoxic effect in HL60 cells (promyelocytic acute leukemia),   (13), agathisflavone was shown to be a non-competitive inhibitor of dengue virus serotype 2 NS2B-NS3 protease, with Ki values of 11 μM. This protease is essential for the replication process of the virus and has been studied as a promising molecular target for anti-dengue virus medications.

Antibiotic activity
The first test to investigate the antibiotic potential of agathisflavone was by Lin et al. (2001) (39). Several biflavonoids were evaluated for their inhibitory activity against Mycobacterium tuberculosis (tuberculosis agent) in vitro, but agathisflavone had no effect on the concentration used, 12. Anacardium occidentale showed moderate inhibitory activity with a minimum inhibitory concentration of 1.0 mg / mL against most of the pathogens tested (B. subtilis, C. sporogenes, E. coli, K. pneumonia, P. fluorescences, P. aeruginosa and S. aureus). These authors were also the only ones in the period evaluating the antioxidant capacity of agathisflavone using the DPPH free radical scavenging assay, the total antioxidant capacity determination test and the antioxidant determination power reduction test. Agathisflavone did not demonstrate significant antioxidant capacity in any of the tests performed.

Activity on the nervous system
In an article published in 2006, Svenningsen et al. (28) reported for the first time the affinity of agathisflavone for the benzodiazepine/GABAA receptor, an important molecular target for antiepileptic medications. Agathisflavone extracted from the plants Searsia dentata (Rhus dentata), Searsia penteri (Rhus penteri) and Searsia pyroides (Rhus pyroides) underwent a [3H] -Flumazenil binding assay to evaluate the affinity to GABAA receptor. Agathisflavone showed significant affinity for the receptor in this assay, with a Ki value of 28 nM, whereas apigenin, also tested, showed much lower activity with Ki of only 7.6 μM. In this case, the biflavonoid was shown to be a more active compound than its monomer. Shrestha (18) tested the effects of agathisflavone from this same plant on primary glia and neuron co-cultures from the cerebral cortex of neonatal rats. Compared to the control, treatment with agathisflavone at 10 μM increased the number of mature neurons and neural progenitors, without affecting the number of astrocytes and microglia. These effects were suppressed by estrogen receptor antagonists MPP dihydrochloride and PHTPP (antagonists for the receptors ERα and ERβ, respectively), suggesting that this is, at least partially, the mechanism of action of the neurogenic effects of the flavonoid. In these same cultures, when subjected to glutamate-induced excitotoxicity (an important cause of neurodegeneration in several pathologies such as Alzheimer's and Parkinson's diseases), treatment with agathisflavone at 10 μM significantly reduced glutamate-induced cell death, in addition to reducing the expression of pro-cytokine inflammatory agents (including TNF, IL1β and IL6) associated with neurotoxicity. It was also able to increase the expression of IL-10 cytokine and arginase-1 enzyme, which are associated with anti-inflammatory and neuroprotective microglia. The authors also observed that the flavonoid induced increased expression of neuroprotective trophic factors such as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-4 (NT4) and glial cell line-derived neurotrophic factor (GDNF). The neuroprotective effects of agathisflavone against glutamate-induced excitotoxicity have been associated with the increased expression of glutamate regulatory proteins in astrocytes, glutamine synthetase (GS) and excitatory amino acid transporter 1 (EAAT1).

Other pharmacological effects
The first and only in vivo test using agathisflavone reported in the literature was made by Anand et al. (1992) (25) and published under the title "Structure and hepatoprotective activity of a biflavonoid from Canarium manii" in the journal Planta Medica. In this study, the authors report that in Charles-Foster rats and Swiss albino mice undergoing carbon tetrachloride-induced hepatic injury, agathisflavone given orally (50 mg and 100 mg) one hour before the injury provided dose-dependent hepatoprotective effects, decreasing serum levels of aspartate aminotransferase and alanine aminotransferase in animals compared to the untreated group, suggesting that the flavonoid contributed to preserve hepatocyte integrity.
De Souza et al. (2015b) (14) published the only report found in the literature on the antiprotozoal effect of the flavonoid agathisflavone. L-cathepsin rCPB2.8 is present in Leishmania mexicana (one of the parasites that cause leishmaniasis) and is an important protease for the suppression of the immune response of infected individuals and is, therefore, a promising molecular target in the therapy against this protozoan. Agathisflavone was able to partially and non-competitively inhibit the enzyme rCPB2.8 with an EC50 of 0.43 ± 0.04 μM and Ki values of 0.14 ± 0.04 μM.
Agathisflavone extracted from the plant Ouratea polyantha Engl. (Ocnhaceae) demonstrated in vitro at a concentration of 2.5 ppm the ability to inhibit the glucose-6-phosphatase enzyme present in murine hepatocytes, which is a potential target for the treatment of diabetes (29). Campana et al. (2015) (44) tested the ability of several flavonoids extracted from Ouratea semiserrata to decrease the release of TNF-α by monocytic THP-1 cells activated by LPS. Agathisflavone did not demonstrate antiinflammatory effects in this study at any of the concentrations adopted but showed cytotoxicity at concentrations greater than 62.5 μM.

CONCLUSIONS
Based on this prospective study, 81 publications were identified on the PubMed and Science Direct databases from the key descriptor agathisflavone and, although the flavonoid has been known in the literature since at least 1969, only 23 of the articles found evaluated its possible therapeutic effects. The research also revealed an increase in the number of publications mentioning agathisflavone over the last decade, indicating a possible increased interest in the flavonoid. The demonstrated biological activities of agathisflavone range from antiprotozoal to neurogenesis and neuroprotection. However, with one exception, the molecule has only been tested in vitro and needs to be better studied at the in vivo and human level.