Proceedings of Chemistry, Pharmacology, Pharmacokinetics and Synthesis of Biflavonoids

Biflavonoids, composed of two monoflavonoid residues, occur naturally in angiosperms, bryophytes, ferns, and gymnosperms. More than 592 biflavonoids have been structurally elucidated, and they can be classified into two groups of C-C and C-linear fragments-C, based on whether the linker between the two residues contains an atom. As the linker can be established on two arbitrary rings from different residues, the C-C type contains various subtypes, as does the C-linear fragment-C type. Biflavonoids have a wide range of pharmacological activities, including anti-inflammatory, antioxidant, antibacterial, antiviral, antidiabetic, antitumor, and cytotoxic properties, and they can be applied in Alzheimer’s disease and Parkinson’s disease. This review mainly summarizes the distribution and chemistry of biflavonoids; additionally, their bioactivities, pharmacokinetics, and synthesis are discussed.


Introduction
Flavonoids, one of the main classes of secondary metabolites in plants, and have representative scaffolds as flavones, chalcones, isoflavones, aurones, and xanthones. Biflavonoids, as members of the flavonoid family, are comprised of two monoflavonoids by a direct connection, or a linear linker. In 2017, Gontijo et al., summarized 139 biflavonoids and their medical applications [1]. In the same year, Sheng Yu et al. [2] also reviewed the phytochemistry, pharmacology, and pharmaceutics of amentoflavone in biflavonoids, including a comprehensive description and summary of the source and current situation of amentoflavone derivatives. It is known that amentoflavone can be obtained from different parts of 127 plants, 45 kinds of derivatives that belong to the same type of connection with amentoflavone. The pharmacological effects of amentoflavone are summarized, including its anti-inflammatory, antioxidation, antitumor, antiaging, antidiabetes, antiviral, central nervous, cardiovascular system, antifungal, and other pharmacological effects. The amentoflavone family is recorded in detail.
In this report, 592 biflavonoids, as well as their distribution, structural scaffolds, and chemical subtype are reviewed. In addition, the pharmacology and synthesis of biflavonoids are summarized.

C-C Type
According to the connection mode of biflavonoids, they are divided into three major groups.

Subtype
No.

Compounds Name Monomer Type
Origin (Family *) References

C-Linear Fragment-C Type
Group B (Table 9)

Complex Biflavonoids
Group C belongs to the complex biflavonoids (Tables 10 and 11). They include the simple-ring type (C-C & C-C, C-C & C-C-C, C-C & C-O-C, C-O-C & C-O-C), the bicyclic type, the atom-shared type, and spirobiflavonoids. The structure of complex biflavonoids were showed in Figure 11.

Antioxidant
Andrade et al. [272] conducted an antioxidant test on agathisflavone in 2018. Trolox was used as a control, and agathisflavone was extracted and isolated from the fresh leaves of Caesalpinia pyramidalis Tull. In the experiment of DPPH radical scavenging, it was found that agathisflavone scavenged DPPH free radicals in a concentration-dependent manner; the EC 50 of agathisflavone was 0.474 mM, and for Trolox it was 0.149 mM, within the 95% confidence interval. The ABTS scavenging assay data found that agathisflavone was EC 50 = 0.179 mM, while for Trolox, it was EC 50 = 0.311 mM. In the OH radical scavenging assay, agathisflavone also showed a concentration-dependent hydroxyl radical scavenging ability, while agathisflavone and Trolox both showed a concentration-dependent reduction in the three iron ions to ferrous iron. Through structural analysis of agathisflavone, it was found that the hydroxyl groups at positions 4 ,7,7 ,4 in its structure can provide free radical hydrogen to reduce free radicals. In addition, agathisflavone can also inhibit the production of TBARS, and has a significant ability to protect against oxidative damage, indicating that agathisflavone is likely to be a good antioxidant.
The antioxidant effect of Garcinia kola is mainly based on the biflavonoids in the extract. Through the DPPH method and the ATBS method, Lixian et al., studied the antioxidant capacity of garcinianin, kolaflavanone, GB1a, GB2, and panciflavanon. The antioxidant activity of different compounds determined by the DPPH method was garcinianin > panciflavanon > GB2 > kolaflavanone > GB1a, and the antioxidant activity of different compounds determined by the ABTS method was garcinianin > panciflavanon > GB1a > kolaflavanone > GB2. Among them, the antioxidant effect of garcinianin was more obvious [273].
In a study of the antioxidant mechanism of the neuroprotective biflavonoids, hinokiflavone, isocryptomerin, amentoflavone, ginkgetin, amentoflavone, and ginkgetin have good antioxidant capacities, can inhibit the activity of SOD, GR, Gpx, CAT, and other oxidases, reduce the content of GSH, and achieve an antioxidant effect. Ginkgetin can also act on the ERK1/2 target for antioxidants [274]. In 2013, Jia et al. [192] extracted baeckein E from Baeckea frutescens and six other known compounds, and its IC 50 value ranged from 11.8-16.1 µM in the DPPH free radical scavenging test. Baeckein A and B (IC 50 = 23.5 µM, IC 50 = 26.2 µM) showed cytotoxicity and could not be used in H 2 O 2 -induced oxidation experiments. The treatment rates of biflavonoid baeckein E, baeckein C, and baeckein D were 31.8%, 34.8%, and 36.0%, respectively, which were lower than those of nonbiflavonoids (43.0~44.7%).

Anti-Inflammatory Properties
The anti-inflammatory activity of biflavonoids is mainly detected by inhibiting the expression of cyclooxygenase 2 (COX-2) and iNOS. In 2006, Park et al. [275] looked for C-C linked biflavonoids as anti-inflammatory drugs and examined the production of PGE2 and nitric oxide (NO) of synthetic biflavonoids in RAW cells treated with lipopolysaccharide (LPS). The results showed that 3 -6 , 6-6 , and 3-4 linked biflavonoids showed resistance to COX-2 -mediated significant inhibition of PGE2 production (IC 50 = 17.3 µM; IC 50 = 3.7 µM; IC 50 = 7.0 µM, respectively). Western blot and reverse transcriptionpolymerase chain reaction analyses showed that these compounds are not COX-2 downregulation mediated, but are instead COX-2 inhibition mediated. Among them, 6-6 biflavonoids have the strongest PGE2 production inhibitory activity. To ensure accuracy, PGE2 and NO tests were performed after LPS pretreatment. The IC 50 of the 6-6 is < 3.0µM, and it can be used as a synthetic leader of new anti-inflammatory agents. However, the biflavonoids 4 -6 and 3-4 can have cytotoxic effects on RAW cells.
In 2002, the anti-inflammatory mechanism of amentoflavone as a natural biflavonoid was studied. Banerjee et al. [276] found that amentoflavone can inhibit TNF-α-mediated COX-2 expression through the NF-κB pathway, thereby showing anti-inflammatory effects. In 2019, Li et al. [277] also studied the anti-inflammatory mechanism of the natural biflavonoid ginkgetin, and found that it can produce anti-inflammatory effects through the TLR4/NF-κB signaling pathway and improve ischemia/regeneration perfusion injury.
Jia et al. [255] extracted and separated root products from Baeckea frutescens in 2014 and discovered four new natural biflavonoids of baeckeins F-I. It was found that the four biflavonoids are the cyclic biflavonoids.  50 of the control indomethacin was 13.8 ± 1.29 µM, and there was no cytotoxicity. Data analysis showed that baeckein H and baeckein I had glycosylated biflavonoids that had more anti-inflammatory activity than the nonglycosylated biflavonoids. The anti-inflammatory activity of baeckein I was not much different from that of indomethacin, and it can be developed as a new antiinflammatory drug.

Antiviral Activities
To find new molecules against dengue fever virus (DV), Coulerie et al. [279] extracted four biflavonoids from the ethyl acetate extract of Dacrydium balansae, including amentoflavone, podovarpusflavone A, isoginkgetin, and hinokiflavone, and found that the biflavonoid compounds were the strongest inhibitors of the full activity of DV-NS5 RDRP and DV-NS5, with IC 50 s lower than 3.1 and 5.3 µM. The IC 50 values were as follows: hinokiflavone (IC 50 = 0.26 µM) > podovarpusflavone A (IC 50 = 0.75 µM) > amentoflavone (IC 50 = 1.40 µM) > isoginkgetin (IC 50 = 3.10 µM). Hinokiflavone was the most active biflavonoid with IC 50 = 0.26µM, but podocarpusflavone A was the strongest non-cytotoxic DV-NS5 inhibitor and could inhibit polymerase activity in the DV replicon, so podocarpusflavone A can be used as a template for the development of drugs against dengue fever virus. In addition, amentoflavone can also be developed as an antiviral drug for herpes simplex virus (HSV-1) [280], and agathisflavone can produce an anti-influenza virus effect [281].

Antibacterial and Antifungal Activities
Although the antibacterial and antifungal effects are different in mechanism, this review describes them to facilitate the summary of biflavonoids. Tang et al. [282] isolated six biflavonoids from the bark of Ochna macrocalyx. Dehydroxyhexaspermone C, and hexaspermone C are the C-C linked biflavonoids, and ochnone, cordigol, calodenin B, and 2,3-dihydrocalodenin B are all different from general biflavonoids. Calodenin B and 2,3-dihydrocalodenin B have a certain cytotoxicity, but also show strong antibacterial effects. Compared with the control drug, the antibacterial activities of calodenin B and 2,3-dihydrocalodenin B were more obvious. In addition, fukugiside can inhibit the activity of Streptococcus pyogenes [283].
The antifungal activity test mainly uses Candida albicans to test the antifungal effect of the biflavonoids. Lee et al. [284] used bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)], a traditional membrane potential dye, in a regeneration test with fungal protoplasts to study the mechanism of isocryptomerin by depolarization. In this study, amphotericin B was used as a positive control, and isocryptomerin had an MIC value of 18.11 µM, which showed antifungal activity against human pathogenic fungi (such as Candida albicans and Trypanosoma beige). The cumulative amount of the DiBAC4(3) in isocryptomerin is small and less than the value of amphotericin B, which proves that it destroys the plasma membrane of Candida albicans and causes cell death. In addition, fungal arthritis, caused by Candida albicans, and ochnaflavone can promote the expression of IL-2 and IL-10 through the T cell immune system, and inhibit the expression of inflammatory mediators such as IFN-γ and IL-2, but it does not cause hemolysis, kill redundant macrophages, or improve fungal arthritis [285].

Anti-Diabetic and Anti-Atherosclerosis
A biflavonoid composed of two molecules of kaemferol was isolated from the seeds of Semecarpus anacardium and its antihyperglycemic mechanism in diabetic mice induced by a high-fat diet plus streptozotocin, was studied showing it could reduce the content of plasma glucose and increase the level of plasma insulin [286]. At a dose of 80 mg/kg b.wt, the biflavonoid's effect is basically the same as that of metformin, and when the biflavonoid is combined with metformin, they can significantly increase liver and muscle glycogen content, maintain hemoglobin levels, and restore the glycosynthase and glycogen phosphorylase close to normal levels. The glucose metabolism is also maintained at a normal level, and it can significantly increase enzymatic antioxidants (SOD, CAT, GPx, and GST) and nonenzymatic antioxidants (vitamin C, vitamin E, and GSH) and improve the activity of enzymes, thereby curing hyperglycemia. Liu et al. [287] indicated that biflavonoids (isoginkgetin, bilobetin, ginkgetin, and sciadopitysin), which are extracted from Ginkgo biloba, have the potential to become pancreatic lipase inhibitors. Four natural biflavonoids had a strong inhibitory effect on pancreatic lipase, and their residual activities were isoginkgetin = 35.7%, bilobetin = 22.3%, ginkgetin = 41.6%, and sciadopitysin = 58.6%. Through the lipase of a concentration-dependent inhibitor of 4-MUO hydrolysis, each IC 50 value was isoginkgetin = 2.90 ± 0.98 µM, bilobetin = 3.57 ± 0.53 µM, ginkgetin = 6.90 ± 1.60 µM, and sciadopitysin = 12.78 ± 2.30 µM, showing a degree of medium to strong inhibition. Isoginkgetin can also improve the healing of foot ulcer wounds in diabetic rats [288].
There are many pathological mechanisms of atherosclerosis, but they are related to hypertension, hyperlipidemia, and other mechanisms. Therefore, the treatment of atherosclerosis is basically inseparable from the antioxidant and anti-inflammatory effects [289]. Tabares-Guevara et al. performed oxygen radical absorbance capacity (Orac) and lDl oxidation inhibition assays on three natural biflavonoids: morelloflavone, volkensiflavone, and fukugiside, and found that all of them were effective reactive oxygen scavengers, inhibited the production of reactive oxygen species and the secretion of proinflammatory factors (IL-6, IL-12p70, MCP-1, TNF-α, MIP-1α, and NLRP3, etc.) in macrophages, and they reduced the circulating levels of cholesterol and the lipid peroxidation product propylene glycol, showing the antioxidation, anti-inflammatory, hypolipidemic, and anti-atherosclerotic effects of biflavonoids in the body [290].

Alzheimer's Disease and Parkinson's Disease
Alzheimer's disease in terms of anti-inflammatory, antioxidative stress, and neurodegenerative damage overlaps to a large extent with the treatment pathway of biflavonoids [291] so biflavonoids have great potential in the treatment of Alzheimer's disease [292]. Moreover, due to the aromatic interaction of biflavonoids, their therapeutic effect is better than that of a flavonoid [293], indicating that biflavonoids can be used as lead compounds for the development of treatments for Alzheimer's disease. In particular, the amentoflavone type includes amentoflavone (1) and its monomethoxy derivatives. They can inhibit the formation and accumulation of amyloid β, thereby preventing Alzheimer's disease [294].
Choi et al. used the peptide of Aβ1-42 to inhibit the aggregation of Aβ1-42 in vitro by thioflavin T fluorescence analysis of biflavonoids (amentoflavone, bilobetin, sequoiaflavone, sotetsuflavone, podocarpuflavone, ginkgetin, isoginkgetin, and sciadopitysin), and found that amentoflavone has the strongest comprehensive strength in inhibiting the formation of Aβ1-42 fibers and reducing the formation of Aβ1-42 fibers among the eight biflavonoids, and it has great potential as a lead compound for treating Alzheimer's disease [295]. CGY-1 [82], GB1, and other gambogic biflavonoids [296] also have the potential to treat Alzheimer's disease.
Biflavonoids extracted from Impatiens balsamina can prevent the production of NO, have neuroprotective activity, and improve neurodegenerative diseases [61]. Amentoflavone can improve Parkinson's disease through the PI3K/Akt and ERK signaling pathways [297], while ginkgetin can improve Parkinson's disease nerve damage through neuroprotection [298].

Cytotoxic Activity and Antitumor Activities
The cytotoxicity of flavonoids with different structures is also different. A review had summarized that the flavonoids with flavone(B) units (galangin, kaempferol, quercetin, myricetin, apigenin, and chrysin) had the ability to antihepatoma; the flavonoids with chalcone(G) units (flavokavain C) could cause hepatic failure; the flavonoid with isoflavone(E) units (genistein) had an antiestrogen, increasing the risk of breast cancer and the flavonoids with flavan(A) units (catechin) had no effect on tumor cells, but had the hemolytic anemia thrombocytopenia [299]. Biflavonoids are composed of two flavone monomers, so the toxicity study of flavonoids is also helpful to the toxicity activity of biflavonoids. The structure of these flavonoids are shown in Figure 12.
For the toxicity of biflavonoids, a study found that amentoflavone, sciadopitysin, ginkgetin, isoginketin, and bilobetin extracted from ginkgo can reduce the cell viability of human renal tubular epithelial cells (HK-2 cells) in a dose-dependent manner. Ginkgetin, isoginkgetin, and bilobetin showed the cell viability of HK-2 cells were less than 50% at 10 and 100 µg/mL. At the dose of 100 µg/mL, amentoflavone, ginkgetin, isoginkgetin, and bilobetin injured the human normal hepatocytes (L-02 cells), moreover, the cell viability of isoginkgetin and bilobetin were less than 50%. After HE staining of mouse liver sections, it was found that bilobetin and ginkgetin were more toxic to hepatocytes. In renal tissue, these five biflavonoids caused acute renal injury, and renal interstitial hemorrhage was a common pathological phenomenon [300]. Therefore, Ginkgo biloba extract preparation should pay attention to its hepatorenal toxicity. A study found that hinokiflavone, as the cytotoxic principle, its ED 50 value was 2.0 µg/mL in KB cells. It was proven that the ether bond between the two flavonoid monomers had a significant cytotoxicity. However, other biflavonoids with C-C linkages, being hexamethyl ethers of ring C/A-linked dimers between two flavonoid units, also showing the cytotoxic activity (the ED 50 value was 3.0~4.0 µg/mL) [301]. A non-clinical toxicological study in 2019 revealed that there were no reported fatalities after agethisflavone acted on the female mice, and it has an LD 50 larger than 2000 mg/kg [302].
Adem et al. [10] used the caspase-Glo assay to test the cytotoxicity of three biflavonoids (chamaejasmin, 7,7 -di-O-methylchamaejasmin, and campylospermone A) and other compounds. The cell cycle, apoptosis, mitochondrial membrane sites, and reactive oxygen species were analyzed by flow cytometry. The model cells were CCRF-CEM leukemia cells and CEM/ADR5000 cells and seven other cancer cells including U87MG. = EGFR glioblastoma, HepG2 liver cancer cells, U87MG. = EGFR cells, MDA-MB-231/BCRP breast cancer cells, MDA-MB-231 cells, and HepG2 cells. The IC 50 values of chamaejasmin in CCRF-CEM cells and CEM/SDR5000 cells were both greater than 61 µM, and the IC 50 value of campylospermone A to CEM/ADR5000 cells was also greater than 61 µM. Therefore, chamaejasmin and campylospermone A were considered to be less cytotoxic. However, 7, 7 -di-O-methylchamaejasmin had an IC 50 = 3.58 ± 0.09 µM for CCRF-CEM cells, and an IC 50 = 5.69 ± 0.51 µM for CEM/ADR5000 cells, and the IC 50 values of the other cancer cells were less than 8 µM, indicating that it had greater cytotoxicity, and could inhibit the growth of cancer cells.
Due to the cytotoxicity of biflavonoids, they have great potential in the treatment of cancer. For example, delicaflavone can inhibit the PI3K/Akt/mTOR and Ras/MEK/Erk signaling pathways in rectal cancer cells through the mitochondrial ROS pathway [303], inhibit the MSPK signaling pathway in HeLa cervical cancer cells, and induce cell apoptosis in G2/M phase [304]; hinokiflavone inhibits the induction of apoptosis of the NF-κB signaling pathway in liver cancer cells by activating the mitochondrial ROS/JNK/caspase pathway [305]. However, the spirobiflavonoids of abiesinolA-F extracted from Abies sachalinensis can effectively inhibiting the activation of NOR1, thereby inhibiting the activity of skin cancer [306]. In the literature on the toxicology of biflavonoids, only the toxicological experiments of biflavonoids with Aunits, B units, and spirobiflavones were included. For instance, 6-8 linkage biflavonoid (agathisflavone) had no cytotoxic activity [302], but 3 -8 linkage biflavonoids (amentoflavone, ginkgetin, isoginkgetin, and bilobetin) impaired the liver and renal cells [300] and 3-3 linkage biflavonoids (chamaejasmin, 7,7 -di-Omethylchamaejasmin, and campylospermone A) had the capacity to inhibit the growth of cancer cells [10]. The biflavonoids with the ether bond between two flavonoid monomers (delicaflavone [301,304], hinokiflavone [305], and spirobiflavone [306]) had the ability of anticancer. Additionally, the biflavonoids with the hexamethyl ether substituents could reduce cell activity [301].

Anti-Angiogenesis
Li et al. [307] correlated zebrafish angiogenesis measurement with ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF/MS) as the base chemometric analysis to identify the potential antiangiogenic active compounds of Garcinia xanthochymus. Preliminary biological activity results showed that amentoflavone can significantly inhibit the growth of subintestinal vessels at 10 and 20 µM, and downregulate the expression of the Angpt2 and Tie2 genes in zebrafish embryos. In addition, the zebrafish model was used to evaluate the structure-activity relationship of seven biflavonoids (volkensiflavone, fukugetin, fukugeside, GB 1a, GB 1a with glycosides, GB 2a and GB 2a with glycosides) isolated from Garcinia. Fukugetin, which has anticancer effects, and can effectively inhibit the growth of subintestinal vessels. Both amentoflavone and fukugetin showed antiangiogenic effects on zebrafish for the first time [308].

Pharmacokinetics
LC-MS/MS is a sensitive method used in pharmacokinetics, and it is also used in the pharmacokinetics of biflavonoids. It is used in the study of amentoflavone pharmacokinetics by different drug intake modes, including oral gavage (p.o.), intravenous (i.v.), or intraperitoneal (i.p.) injection in rat models. As a result, 90.7% ± 8.3% of the total amount of amentoflavone (300 mg/kg) by p.o., 73.2% ± 6.29% of amentoflavone (10 mg/kg) by i.v., and 70.2% ± 5.18% of the total amentoflavone (10 mg/kg) by i.p. could be detected. The total amentoflavone was found to circulate as conjugated metabolites in the plasma of rats after different modes of administration [320].
Amentoflavone was used as the standard of the study of pharmacokinetics of biflavonoids in LC-MS/MS. For instance, the pharmacokinetics of total hinokiflavone in rat plasma was studied by LC-MS/MS. It was discovered that T 1/2 was 6.10 ± 1.86 h [321].
However, there are other ways to calculate the main index of pharmacokinetics. The main components of Platycladus orientalis leaf extract include amentoflavone and hinokiflavone. Therefore, their pharmacokinetics in the plasma of a rat model were evaluated by UFLC-MS/MS. Their T 1/2 and Tmax were 2.60 ± 1.34 h and 1.5 ± 0.00 h (amentoflavone), and 2.11 ± 0.29 h and 1.92 ± 0.20 h (hinokiflavone), respectively [322]. All the pharmacokinetics data of biflavonoids were showed in Table 12.

The Biosynthesis of Biflavonoids
There were few references about the biosynthesis of biflavonoids, but it involves the oxidative coupling of two flavonoid units; therefore, the biosynthesis of flavonoids was a significant step to shape biflavonoids in plants. Alzand et al. [323] had reviewed the major pathways of flavonoid biosynthesis. Starting from phenylpropanoid metabolism and then giving the chalcone (trihydroxychalcone and tetrahydroxychalcone). The tetrahydroxychalcone is isomerised to naringenin, a key intermediate, which can transform to several end-flavonoids ( Figure 13).
Furthermore, promoting the biosynthesis of biflavonoids can improve the yield of biflavonoids in plants by changing different catalytic enzymes or elicitors. Kicia Karinne Pereira Gromes-Copeland et al. [324] had converted the elicitors of 30 g/L of sucrose and 5 mg/L of 2,4-dichlorophenoxyacetic acid in Poincianella pyramidalic. Providing a higher accumulation of amentoflavone (16.44 mg/L) and agathisflavone (0.58 mg/L). Subsequently, they found that the amentoflavone biosynthesis is superior to agatisflavone. It seems to be related to the linkage type between two flavonoid units.

The Synthesis of Biflavonoids
Biflavonoids have great medicinal value and great development prospects. Therefore, the quantity needed in treatment and research will increase. However, it is impossible to obtain a large number of single and high-quality biflavonoids by simply extracting and separating the biflavonoids. In the process of synthesizing biflavonoids, Xue Ying et al. [325] reviewed the previous synthesis methods of biflavonoids in 2010, compared the differences between the various methods, and concluded that the synthesis method of biflavonoids is mainly to synthesize a flavonoid monomer. Then, two molecules of flavonoids coupled with boron-containing flavonoids are chosen by Suzuki or iodide-biflavonoids to obtain the final product, or the two molecules are coupled with the catalyst. The related C-C biosynthesis and reverse synthesis analysis, and the Ullmann ether condensation reaction of C-O-C, are also introduced. In the case of the literature that has been previously summarized, this review will conduct a general analysis of the new biflavonoid synthesis method, and compare the old method with the new one, so that readers can be more intuitive.
Until 2017, the syntheses of biflavonoids were the construction of a biflavonoid skeleton, and different types of dimers were synthesized under different synthesis conditions. First, the biflavonoid skeleton, bichalcones (S3) is obtained by Claisen-Schmidt aldol condensation from the different dialdehyde molecules (S1) with the corresponding acetophenone (S2). Second, the bichalcone skeleton can obtain biflavones (S4), through iodine-mediated or produce biaurones (S6) by mercury acetate oxidation [27]. Biflavans can be obtained by oxalic acid with EtOH [326], but biflavans will change to biflavones in MeOH with HCl [327]. (Scheme 1). These methods can synthesize different types of biflavonoids as long as different dialdehydes can be provided. For example, the di-aldehydes S1 are 4,4 -biphenyldicarboxaldehyde, 4,4 -diaryletherdicarboxaldehyde, or 4,4 -bitoluenedicarboxaldehyde, and the biflanonoids are C-C, C-O-C, or C-C-C. It can be said that this Claisen-Schmidt aldol condensation of dialdehyde and acetophenone can be the synthesis route of most symmetric biflavonoids. According to the difference in the final product, bichalcones, biflavones, biflavans, and biaurones can also be obtained by autonomously controlling the conditions. Scheme 1. Total synthesis of C-O-C, C-C-C, and C-C biflavonoids.
Due to the large number and types of biflavonoids connected to C-C, there are many related synthetic studies. Among them, Chen et al., achieved the synthesis of C-C biflavonoids through the construction of two flavonoid analogs in 2006: one flavonoid analog substituted by a halogen atom (bromide), and the other substituted by a group coupled by a transition metal-catalyzed cross-coupling method, namely two typical methods: the Suzuki coupling reaction and the Stille coupling reaction. The two flavonoid monomers are connected through the biaryl group. In addition, they synthesized a series of C-C 4'-4'linkage biflavonoids a-f and compared the inhibition of sPLA2-IIA among them. Amentoflavone and ochnaflavone were used as controls. Subsequently, they found that the inhibitory potency of the synthesis biflavonoid a(IC50 = 3.0 + 0.9 M) was slightly better than ochnaflavone(IC50 = 3.5 + 0.6 M), the biflavonoids b(IC50 = 15.5 + 3.7 M), d(IC50 = 19.9 + 4.6 M), and f(IC50 = 23.2 + 3.1 M) possessed the comparative inhibitory potency with amentoflavone(IC50 = 23.8 + 3.4 M) [328]. The C-C 4'-4' linkage biflavonoids a-f are shown in Figure 14. However, due to the low yield of the above method, it is impossible to obtain highyield biflavonoids on a large scale; as a result, researchers have found other ways to synthesize C-C type biflavonoids. Brominated, iominated, or chlorine substituted flavones (S7) and commercially available bis(pinacolato) diboron are reacted to obtain the corresponding pinacolato boronates (S8), and then S8 (120 mol%) and S7 under standard conditions (Pd(PPh 3 ) 4 (5 mol%), NaOH (400 mol%), and DMF-water (9:1), 100 • C) are reacted to obtain C-C biflavones. R1, R2, R3, R4, R 1, R 2, R 3, and R 4 are the positions attacked by brominated, iominated, chlorine, or bis(pinacolato) diboron. Moon et al., adjusted the reaction conditions to catalytic PdCl 2 (dppf) and K 2 CO 3 in DMF at 90 • C, to reduce the loss of products [329] (Scheme 2). Scheme 2. The synthesis of C-C biflavonoids.
According to the above method, Lim et al., processed chrysin into the precursor product required for the reaction and then performed the relevant synthesis under standard conditions to obtain a C-C (6-6 ) anti-inflammatory biflavonoid G168 [330], which had the potency of inhibiting COX-2 mediated PGE2 production. For G168, the IC 50 value of inhibiting PGE2 production and againsting iNOS-mediated NO production were 0.1 µM and 50 µM. Furthermore, 5 mg/kg G168 was able to inhibit the paw edema in mice (30% inhibition) and 1~5 mg/kg G168 had the capacity to inhibit writhing in mice (57.3~82.9%). It has been proven that this method can synthesize amentoflavone-type biflavonoids [331] and Wikstrol A and B [332].
In addition to the synthesis shown in Scheme 1, C-C-C-type biflavonoids can be obtained by the Ullmann condensation reaction of the corresponding flavonoid monomers; assuming the relevant conditions are controlled, and the yield is generally high. For instance, the flavonoid monomer chrysin was used to obtain 7-hydroxy-8-hydroxymethyl-4 -methoxyisoflavonoid [333]. Therefore, the synthesis of C-C-C can be summarized as follows: flavone monomer S9 reacts with formaldehyde to form an intermediate, and then it reacts with another flavone monomer S'9 to form C-C-C type biflavone S10 (Scheme 3). However, the yield of the synthesis in the above conditions is low. Thus, Xue Ying et al., modified the method in 2010; they used daidzein as the raw material, the catalyst was concentrated sulfuric acid, and the feed ratio of the raw material was daidzein. When the amount of catalyst was 10% of the molar ratio of daidzein, and the reaction temperature was 80 • C for 24 h, the highest yield of daidzein biflavonoid derivative was obtained [334]. Later, in 2011, Xue Ying et al., further improved the method: using 9% Lewis acid as a catalyst, isoflavones and formaldehyde as raw materials, and controlling the reaction temperature to 90 • C for 20 h; the yield can reach 82~85% [335]. Scheme 3. The synthesis of C-C-C biflavonoids.
In 2015, Baron and Mead first synthesized 3-benzylidene-dihydrofurochromen2-ones (S14), a flavan-chalcone type biflavonoid [336]. This was the first time de novo synthesis was attempted. The raw material of this synthetic route was flavonoids and chromene (S11). In the presence of catalytic Rh2(S-TBSP)4, the researchers treated S11 with a diazo derivative to obtain the donor-acceptor cyclopropane, and then used Sn(OTf) 2 to rearrange the donoracceptor cyclopropane to obtain the α-carbomethoxy lactone (S12). Then, removal from the hydroxyl protection and treatment with enolate lithium will result in a mixture of stereoisomers with a high hydroxyl alcohol ratio. The alcohol base was protected by TESCl and oxidized by DDQ to selectively oxidize the protected allyl alcohol to obtain aldehyde S13. After adding the aryl lithium reagent, 71% of the final product S14 was obtained, which was an inseparable isomer mixture, but it had all of the functions of target biflavonoids (Scheme 4). Compared with the synthetic methods of Scheme 1, the required conditions are more difficult to control, but it may become one kind of synthetic method that can control the separation of intermediate stereoisomers to better obtain a pure single product and heterobiflavonoids with different types of flavonoid monomers.

Conclusions
In recent years, the method of extracting active ingredients from herbs and using them in research experiments has been a key research direction, and also a huge challenge. As the components in plants are complex, there are many metabolites, and current extraction and separation technologies are still insufficient. A suitable method to efficiently extract, purify, and apply the required active ingredients is the goal we need to achieve. There are many kinds of biflavonoids, and there is an increasing number of synthetic biflavonoids; they are used as anti-inflammatory and antioxidant therapeutics, as treatments for Alzheimer's disease and Parkinson's disease, and for other therapeutic applications. Their use is more significant in anticancer and antiviral treatment. Moreover, Qiu-xia et al. [337] developed and applied amentoflavone based on antisolvent freeze-drying technology, and studied its stability during storage and the stable type of drug efficacy to solve the problem of poor water solubility of amentoflavone micropowder, and improve the oral availability of the drug. In particular, Ginkgo biloba has been proven to be useful in clinical treatment [338]. In summary, there is still much room for developing the pharmacology and synthesizing of biflavonoids, but there is a large gap in the research on dosage forms that needs to be supplemented by additional research. This review mainly provides a more detailed report on the classification, pharmacology, pharmacokinetics, synthesis, and other aspects of biflavonoids, to assist researchers in exploring biflavonoids.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
All the authors declare no conflicts of interest.