2.5.4. Drug Load Determination

FEN content was determined by solvent extraction. Approximately 10 mg of formulation was weighed into a glass vial, followed by the addition of 10 mL of methanol. Vials were sonicated for 45 min to extract FEN. Aliquots were centrifuged (44,800 rcf for 20 min) to sediment undissolved solids. Then, the supernatants were appropriately diluted and analyzed using HPLC.


**4.2 Immunophenotypic features of ovarian endometrioid carcinomas**

OECs show diffuse immunopositivity for CK7, PAX8, ER and PgR [83]. Approximately 33% of OECs display membranous and/or cytoplasmic diffuse expression for vimentin [84]. Squamous morules of endometrioid tumours demonstrate

strong CD10 cytoplasmic expression [85] and infrequently CDX2 immunopositive reaction [86]. Endometrioid ovarian carcinomas also exhibit β-catenin membranous and/ or cytoplasmic expression. Nuclear β-catenin expression is correlated with *CTNNB1* genomic alterations and favourable prognosis [77]. Differential diagnosis between high-grade endometrioid carcinomas with solid architectural patterns and high-grade serous carcinomas can be made based on the intense diffuse immunopositivity of WT1, p53 and p16 in HGSCs, whereas ECs display patchy expression for p16, wild-type or mutation-type expression for p53 and loss of WT1 immunoreactivity [87].


*5.1. Time-Integrated Polarization Measurements*

To date, the *γ*-ray polarization of a total of 31 GRBs has been published. For several GRBs, different analyses have been published, either by different groups using the same data or, in one case, using data from two different instruments. The time- and energyintegrated polarization parameters from these measurements are shown in Table 1, together with the energy range in which they were performed. It is important to note that the energy ranges mentioned here are those stated in the respective publications but that their definitions differ between experiments. The energy ranges stated by SPI for example come from an event selection based on the deposited energy, whereas, for POLAR, which cannot perform measurements of the incoming photon energy directly, the stated range is based on the energy-dependent effective area to polarization.

As can be seen from Table 1, especially for the earliest measurements, at the bottom of the table, the results indicate typically high levels of polarization, although, as explained earlier, this can in some cases be attributed to an error in the analysis. Additionally, publications of GRB polarization measurements have focused on those measurements for which a non-zero PD was found. At least several GRB measurements exist, such as some detected by GAP, for which the PD was found to be compatible with 0%; however, these were not published but only presented at conferences (https: //ttt.astro.su.se/groups/head/cost14/talks/Yonetoku.pdf talk accessed on 25 August 2014). This causes an additional bias towards higher PD values found in the list.

**Table 1.** The list of all GRBs for which a measurement has been published to date. † For GRB 160821A, several analyses were published by members of the AstroSAT collaboration. For this GRB, a time-resolved analysis found high levels of polarization with varying PA as well.

**Table 1.** *Cont.*

In recent years, data from GAP, POLAR, and Astrosat CZTI have significantly increased the number of measurements; however, the measured PD shows a large range between the different instruments. POLAR finds results that are mostly compatible with a low or unpolarized flux, whereas Astrosat CZTI reports high levels of polarization in [188], with best fitting PD for 10 out of the 11 GRBs exceeding 50%. Although in numerical form only an upper limit is provided for some of these GRBs by Astrosat CZTI (which are the numbers reported in Table 1) mthe contour plots for these GRBs in Figure 13 of [188] indicate that high levels of PD are favoured for all. In most cases, the best fitting PD is close to the upper limit. The only exception is 160623A where a best fitting PD of approximately 30% is found. It should be noted though that for GRB 160821A, two separate analyses provided different results for the main emission period. The first from [188] indicates a rather high level of polarization, whereas [231] found a time-integrated PD compatible with a lowly or unpolarized flux. The analysis methods used for both analyses were different, while additionally the selected time intervals differed (a period with low fluence was added in [231]). Although the interval selection is not discussed in detail, in [240] it is mentioned that the intervals used in [188] were optimized to maximize the significance of the PD detection, giving a possible explanation. The same analysis as applied in [188] was applied in [241] for GRB 171010A where an upper limit of 42% was reported.

The overall impression given by the Astrosat CZTI results is that GRBs are rather highly polarized. From the POLAR results this is not the case as no significant PD was detected, and all results are compatible with an unpolarized flux within the 99% confidence interval. The POLAR results favors low polarization degrees, with PD values exceeding 50% excluded by five of the brightest GRBs with a 99% confidence level. The results from GAP show both GRBs with a high level of polarization, as well as those with a low level, while COSI, the last of the four detectors, which was well calibrated on ground, additionally excludes high values of PD.

Despite the significant increase in available measurements, no clear conclusion on the PD of GRBs has emerged. It therefore appears that simply continuing to push for more measurements with the current generation of instruments might not be the best way forward. Rather, detailed studies scrutinizing the different results found by different instruments are an easier and more promising way forward. One way to achieve this, which is discussed later on, is the use of more standardized analyses methods as well as by making the polarization data public for an independent analysis by different groups.


**1. Introduction**

Angiogenesis is the process of forming new blood vessels. Physiologically, angiogenesis is pivotal for tissue growth and regeneration [1] which is beneficial for many processes including embryogenesis and wound healing. Regulation of angiogenesis is complex and is maintained by the balance between endogenous stimulators (e.g., vascular endothelial growth factor (VEGF), platelet derived growth factors (PDGFs) and hypoxia-inducible factors (HIFs)), and inhibitors (e.g., angiostatin and endostatin). Other body conditions also contribute to the regulation of angiogenesis under physiological conditions. For example, certain metabolic demands such as the need for more oxygen can induce VEGF secretion and angiogenesis in heart and brain tissues [2,3]. Since angiogenesis affects many organs and tissues in the body, an imbalance in its regulation has been associated with different pathologies [4]. For instance, cancer, rheumatoid arthritis and diabetic retinopathy feature an upregulation of proangiogenic factors [5]. Conversely, if antiangiogenic factors were upregulated, several cardiovascular diseases are more likely to happen [6]. The use of drugs like Bevacizumab

(Avastin®, Genentech) and Aflibercept (Eylea®, Regeneron) for the treatment of cancer and ocular diseases, emphasizes the imperative medicinal applications of antiangiogenic agents [7,8].

Flavonoids are widely distributed in fruits, vegetables and nuts. They are one of the most important chemical classes of natural compounds showing various pharmacological profiles that include anticancer [9–11], anti-inflammatory [12], cardioprotective [13] and neuroprotective activities [14].

The antiangiogenic activity of flavonoids has been extensively studied over the last two decades. Several studies document the ability of flavonoids to inhibit the proliferation and migration of endothelial cells by interfering with key angiogenesis signaling cascades such as the mitogen activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways. Nevertheless, they can inhibit the expression of major proangiogenic factors such as VEGF and matrix metalloproteinases (MMPs) [2,7,15].

Researchers rely on different in vitro and in/ex vivo assays to quantitatively assess the effects of chemical compounds on angiogenesis [16,17]. Each of these assays can probe one or more of the different steps involved in the angiogenesis process such as cell proliferation, migration and tubulogenesis.

Despite considerable research concerning the antiangiogenic activities of flavonoids, to date they have neither progressed to the market nor clinical trials for that purpose. Therefore, the aim of this review is to systematically assess the antiangiogenic activities of flavonoids to provide greater insight into their potential as therapeutic agents. This study is comprised of two parts: Section 1 provides a systematic overview of the classes of flavonoids that have been investigated for their antiangiogenic activities, along with a summary of the different in vitro and/or in/ex vivo angiogenesis assays that have been used; Section 2 is a meta-analysis study of a quantitatively comparative subset of data, based on the in vivo chick chorioallantoic membrane (CAM) assay, to statistically evaluate the antiangiogenic effects of flavonoids.

#### **2. Results**

#### *2.1. Section 1: Systematic Overview*

#### 2.1.1. Search Results

For Section 1 of the study, 3708 records were initially identified in three electronic databases (1555 from ScienceDirect, 1984 from PubMed and 169 from Web of Science). Search results were then limited to research articles, review articles, short communications and systematic reviews and the remaining 3380 articles were subjected to title and abstract screening. 2556 records were found to be irrelevant of the subject in focus or did not fulfill the inclusion criteria. After the removal of duplicates (422), 402 articles were finally included in the qualitative analysis for Section 1 of this study (Figure 1).

#### 2.1.2. Study Characteristics

The pool of studies included was classified with respect to: (a) flavonoid class (Figure 2), (b) flavonoid name, (c) disease, (d) in vitro test and (e) in/ex vivo tests. Characteristics of the included studies are summarized in Table 1 (flavonols are used as a representative example in Table 1 and Table S1 contains similar data for all classes of flavonoids). A total of 402 research and review articles were considered. All of the included articles reported angiogenesis related in vitro and/or in/ex vivo assays for different classes of flavonoids.

**Figure 1.** PRISMA flow diagram of study search and selection process of Section 1.

**Figure 2.** Chemical structures of classes of flavonoids.

**Table 1.** Characteristics of the studies included in Section 1 for flavonols subclass (see Table S1 for all 9 subclasses).

 Data Analysis

The majority of articles (332, 82%) focused on the implications of angiogenesis on cancer growth and metastasis. 7% of the articles studied antiangiogenic effects of flavonoids on other diseases such as diabetes, bone and eye diseases, whilst 11% focused on the antiangiogenic activity of flavonoids without application to a specific pathology (Figure 3a). A profiling of the studies retrieved with respect to chemical class of flavonoids is shown in Figure 3b.

**Figure 3.** Profiling of papers retrieved in Section 1 with respect to: (**a**) pathology type; (**b**) chemical class of flavonoid.

Figure 4 summarizes the types of in vitro and in vivo assays that were utilized in the studies. From a pool of 342 research articles included in this study, 152 articles (44%) reported a combination of in vitro and in/ex vivo assays in their studies. The percentage of research articles that depended only on in/ex vivo tests to evaluate antiangiogenic activity of flavonoids were comparatively low compared to those conducting only in vitro assays (3% vs. 53%, respectively).

**Figure 4.** Types of assays used for in vitro and in vivo antiangiogenic evaluation of flavonoids.

#### *2.2. Section 2: Meta-Analysis*

#### 2.2.1. Search Results

The second subset search, which is the basis of the meta-analysis forming Section 2 of this study, followed the same general methodology as detailed in the initial overview. 960 records were identified from four electronic databases (381 from ScienceDirect, 496 from PubMed, 65 from Web of Science and 18 from Google Scholar). 25 research articles were finally included in the quantitative analysis after the sequential steps of screening and sifting, as shown in Figure 5.

**Figure 5.** PRISMA flow diagram of study search and selection process of Section 2.

#### 2.2.2. Study Characteristics

The main study characteristics of the research articles included in Section 2 for the meta-analysis are summarized in Table 2 by study name.

 Meta-Analysis (Antiangiogenic Effect of Flavonoids on CAMs)

25 studies reporting the CAM assay for the in vivo evaluation of flavonoids were eligible for the meta-analysis. The number of blood vessels relative to the control was used as the outcome measure, the lower the ratio the higher the antiangiogenic activity. The studies were grouped into 3 sub-sets based on the controls used. In the first set (12 studies), the normal vasculature of the CAM was used as a control without any interventions that would induce angiogenesis. The second and third sets, however, tested the antiangiogenic activity of flavonoids on CAMs with abnormal angiogenesis using either proangiogenic factors for set 2 (4 studies) or cancer cell lines for set 3 (9 studies). 10 studies [18,95,100,132,137,139–141,145,149] out of the 25 eligible studies were not included in any of the conducted meta-analyses as they failed to report the required data outcomes or did not fit under any particular subgroup.

Set 1: Antiangiogenic effect of flavonoids under normal conditions

To ensure consistency in our comparison, for the meta-analysis of set 1, the concentrations were grouped into three ranges i.e., low (10–20 μM), medium (40–50 μM) and high (100 μM). Flavonoids were sub grouped based on their chemical class as shown in Figure 6. Pooled results indicate that all subclasses, except for anthocyanidines, demonstrate concentration dependent antiangiogenic activity expressed as a reduction in the number of blood vessels in a CAM. For the flavonols subgroup, for instance, the overall means ratios (summary estimates of antiangiogenic activity of a subgroup of flavonoids relative to control) were 0.74 (95%CI: 0.69, 0.79; *p*-value < 0.00001), 0.50 (95%CI: 0.46, 0.56; *p*-value < 0.00001) and 0.26 (95%CI: 0.19, 0.35; *p*-value < 0.00001) for the low, mid and high concentrations, respectively. On the other hand, the anthocyanidines subgroup exhibited only a minor overall reduction of 18% at the highest concentration and a slightly proangiogenic effect (overall means ratio: 1.07; 95%CI: 0.86, 1.33; *p*-value: 0.53) at 20 μM.

In addition to the forest plot analysis that gives a general idea about the overall in vivo antiangiogenic activity of flavonoids and identifies trends of activity among the different subclasses, some structure activity relationship (SAR) conclusions were drawn from the pooled results (Figure 7).

First, there was no correlation between the number of hydroxyl groups and antiangiogenic activity. However, the position of the hydroxyl groups appeared to be of importance as most of the highly active flavonoids had hydroxyl groups at positions 3, 5 and 7 and/or 4- (e.g., as demonstrated for 3-OH flavone, acacetin, biochanin A, apigenin, silibinin and kaempferol). The 7-OH group can be considered to be of greatest importance for activity since 7-OH flavone showed higher activity in the low and medium concentrations compared to the 5-OH analogue. Absence of the 3-OH group caused up to a 14% decrease in activity at the 50 and 100 μM concentrations, as demonstrated, for example, for 3-OH flavone vs. flavone, kaempferol vs. apigenin and 3,7-diOH flavone vs. 7-OH flavone. This was also true for quercetin vs. luteolin but with only a trivial drop of activity of 1 to 2%. However, this was not the case for 3,6-diOH flavone vs. 6-OH flavone where removal of the 3-OH group slightly increased the activity by 1 to 5% at the mid and high concentrations.

Secondly, unsaturation of the C2 and C3 bond is a common feature of most of the highly active flavonoids and is important for activity. 7-OH flavone and 7-OH flavanone are two good examples that exemplify this, as demonstrated by a reduction of the number of vessels: 27%, 32% and 52% for 7-OH flavone and 10%, 22% and 39% for 7-OH flavanone at 10 μM, 50 μM, and 100 μM, respectively.

Third, there are examples of where the presence of a methoxy group at position 4 increases activity (e.g., biochanin A, diosmin and formononetin). However, the presence of a methoxy group at C7 caused a decrease in the activity when compared to the unsubstituted analogue (ie for the 3-OH flavone vs. 3-OH-7-OCH3 flavone, reduction of number of vessels: 35% and 20% at 10 μM, 64% and 42% at 50 μM, 79% and 69% at 100 μM, respectively). On the other hand, conversion of the 7-OH group in 3,7-diOH flavone to a 7-OCH3 group in 3-OH-7-OCH3 flavone slightly improved the activity (reduction of number of vessels) from 18% to 20% at 10 μM and from 63% to 69% at 100 μM, respectively. Finally, glycosylation at positions 3 or 7 showed neither a pronounced nor a consistent effect on the antiangiogenic activity of flavonoids. While a decrease in activity was observed with

quercetin vs. rutin, hesperitin vs. hesperidin and cyanidin vs. cyanidin-3-*O*-glucoside, an increase was observed in the cases of naringin vs. naringenin and delphinidin vs. delphinidin-3-*O*-glucoside.

#### (**a**) Low conc. (10–20 μM)

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Figure 6. Cont.
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(**b**) Mid conc. (40–50 μM)

**Figure 6.** *Cont.*