**3. Occurrence of homoisoflavonoids in** *Caesalpinia* **spp.**

These structures are important for the recognition and classification of biflavonoids moieties, once they could exist as complex structures presenting aurones, isoflavonoids, neoflavonoids,

Flavonoids are products from the phenylpropanoid building block cinnamoyl‐CoA, in which chain extension is provided by three units of malonyl‐CoA [17]. Cinnamoyl‐CoA is derived from the amino acids phenylalanine and tyrosine which are converted by phenylalanine and tyrosine ammonia lyases to cinnamic acid and *para*‐hydroxycinamic acid, respectively [18]. The aromatic polyketide formed from the union of cinnamoyl‐CoA and three units of malonyl‐ CoA might form the benzo‐γ‐pyrone nucleus containing aromatic rings A, B, and a heterocy‐ clic ring C, substituted or not. This nucleus is precursor of a great number of flavonoids. In this

In addition, chalcones might undergo different cyclization with the addition of a single carbon, provided by S‐methyl moiety of methionine, which lead to the formation of the homoisofla‐ vonoid nucleus, which can be converted to the other classes of homoisoflavonoids (**Figure 2**).

**Figure 2.** Biosynthetic scheme for the formation of a flavonoid nucleus (monomeric structure of biflavonoids) and the

formation of the existing types of homoisoflavonoid nucleus.

‐C<sup>3</sup> ‐C<sup>6</sup>

skeleton.

chalcones, and other moieties as well as dimers of homoisoflavonoids.

100 Flavonoids - From Biosynthesis to Human Health

sense, flavonoids are characterized by the classic flavan nucleus presenting a C<sup>6</sup>

Homoisoflavonoids have a general structure of 16 carbons containing two phenyl rings and one heterocyclic ring. Homoisoflavonoids are biosynthesized from cinnamic acid derivatives along with malonyl‐CoA subunits. The resulting compound, an aromatic polyketide, is the precursor of chalcones. In the following step, the aromatic polyketide undergoes a Claisen and enoliza‐ tion reactions, which lead to the formation of the chalcone backbone. An additional carbon is added to the chalcone, provided by S‐methyl moiety from methionine, creating the homoisofla‐ vonoid skeleton containing 16 carbons. Thus, there is the formation of 3′‐hydroxyl‐chalcone as a precursor, which is transformed to 3‐benzylchroman‐4‐one. Subsequently, different cyclization leads to the formation of other types of homoisoflavonoids (**Figure 2**).

The existence of these compounds is associated to the genus *Caesalpinia* involving species as *C. pulcherrima* [19, 20], *C. echinata* [1, 21, 22], *C. bonduc* [3], *C. sappan* [4, 23–28], *C. japonica* [29], and *C. milletti* [30]. However, the diversity of compounds (in number and structurally) is associated to *C. sappan,* a prolific source of homoisoflavonoids with important ethnophar‐ macological applications. The crude extract of *C. sappan*, named Sappan lignum, is widely studied and used for the treatment of diverse diseases [28].

The classification of homoisoflavonoids comprises five main groups named scillascillin, bra‐ zilin, caesalpin, protosappanin, and sappanins. Homoisoflavonoids from the class scillascil‐ lins exhibit a spiro ring with four members between rings C and D. However, species from the genus *Caesalpinia* do not produce scillascillins. These compounds are encountered only in the family Asparagaceae [7].

The most common class of homoisoflavonoids in the genus *Caesalpinia* is the sappanin‐type. This class presents a 3‐benzyl chromanone unit. The diversity of these compounds is associated to a wide variation of substituents, such as hydroxyl, methoxyl, formyl, methyl groups, among others, which confer to sappanin‐type the position of the most abundant. In this chapter, the sappanin‐type homoisoflavonoids corresponded approximately to 70% of the compounds.

In this aspect, the species *C. pulcherrima*, which is a perennial large shrub, widely distributed in the tropical and subtropical areas of Americas, South India, Taiwan and South‐East Asian coun‐ tries [20, 31]. It is used in the folk medicine due to its medicinal properties for the treatment of skin diseases, tumors, and fevers, and association to antibacterial, antidiarrheal, cytotoxic, and antiulcer properties [32]. *C. pulcherrima* produces a large variety of sappanin‐type homoiso‐ flavonoids exhibiting a diverse pattern of substitution such as bonducellin **(1)**, isobonducellin **(2)**, 7‐*O*‐methylbonducellin **(3)**, 2′‐*O*‐methylbonducellin **(4)**, sappanone A **(5)**, (3*E*)‐3‐(1,3‐ benzodioxol‐5‐ylmethylene)‐2,3‐dihydro‐7‐hydroxy‐4H‐1‐benzopyran‐4‐one **(6)**, (3*E*)‐3‐ (1,3‐benzodioxol‐5‐ylmethylene)‐2,3‐dihydro‐7‐methoxy‐4H‐1‐benzopyran‐4‐one **(7)**, (*E*)‐3‐(3‐hydroxy‐4‐methoxybenzylidene)‐6,7‐dimethoxychroman‐4‐one **(8)**, (3*E*)‐2,3‐ dihydro‐7‐hydroxy‐3‐[(3‐hydroxy‐4‐methoxyphenyl)‐methylene]‐4H‐1‐benzopyran‐4‐one **(9)**, (3*E*)‐2,3‐dihydro‐3‐[(3,4‐dimethoxyphenyl)methylene]‐7‐methoxy‐4H‐1‐benzopyran‐4‐ one **(10)**, (*E*)‐7‐methoxy‐3‐(4‐methoxybenzylidene)chroman‐4‐one **(11)**, (*E*)‐7‐hydroxy‐3‐(3,4,5‐ trimethoxybenzylidene)chroman‐4‐one **(12)** [19, 20, 31]. Some of these compounds were tested against Gram‐positive microorganisms such as *Bacillus subtilis*, *Bacillus sphaericus,* and *Staphylococcus aureus* exhibiting moderate antimicrobial activity. However, they were inactive or weakly active against Gram‐negative microorganisms such as *Pseudomonas aeruginosa*, *Klebsiella aerogenes,* and *Chromobacterium violaceum*. Concerning the antifungal activity, these compounds presented moderate activity against *Aspergillus niger* and *Candida albicans* in comparison with standard compounds Clotrimazole (antifungal), Streptomycin (antibacterial), and Penicillin G (antibacterial) [19]. Compounds **5** and **6** presented moderate activity against *Staphylococcus aureus* (inhibition zone of 11–15 cm) at 100 μg/mL, while **6** and **10** presented moderate activity against *Klebsiella aerogenes* (inhibition zone of 11–15 cm) at 100 μg/mL. Streptomycin presented an inhibition zone of 21–25 cm at 100 μg/mL. Compounds **5**, **8**, and **9** exhibited moderated activity against *Aspergillus niger* (inhibition zone of 5–10 cm at 150 μg/mL). On the other hand, the compounds **4**, **8**, **7**, **9**, and **10** were moderately active against *Candida albicans* at 150 μg/mL (inhibition zone of 5–10 cm). To comparison, positive control clotrimazole was active against all strains at 100 μg/mL (inhibition zone 21–25 cm) [19].

Rao and collaborators tested the compound **2** against the inflammatory process and described that **2** inhibits the production of NO, TNF‐α, and IL‐12. In fact, **2** was the most active com‐ pound in the experiments at the concentration of 40 μM, reducing 92% of the NO production (IC50 = 20 μM) in mouse peritoneal macrophages induced by LPS + IFN‐γ. The authors sug‐ gested that the mode of action of **2** probably affects the production of NO by the induction of LPS + IFN‐γ in mouse peritoneal macrophages [20].

The species *C. echinata*, commonly known as *Pau‐brasil* (brazilwood), is endemic from Brazil and played an important historical role in the country [1]. This species has been reported to contain a large range of polyphenols including the homoisolflavonoids brazilin **(13)** and brazilin **(14)**. The compound **15** is a natural dye and is also abundant in the species *C. sappan* (from 8 to 22%). The species *C. echinata*, considered the first source of brazilin, is used for diverse purposes such as healing agent, oral analgesic, and tonics. The species *C. echinata* has also demonstrated antitumor effect *in vivo* against cells strains of Ehrlich Carcinoma and Sarcoma 180. In addition, an interesting antiangiogenic effect was noticed [21]. On the other hand, compound **14**, an oxidation product of brazilin, was considered effective against the inflammatory and cytotoxic processes [22]. Compound **14** displayed cytotoxic effects against human cancer cell lines, such as HepG2 and Hep3B (liver), MDA‐MB‐231 and MCF‐7 (breast), A549 (pulmonary), and CA9‐22 (gingival) [22].

Phytochemical studies on ethanolic extracts of *C. bonduc* yielded two sappanin‐type homoi‐ soflavonoids identified as caesalpinianone **(15)** and 6‐*O*‐methylcaesalpinianone **(16)**, which exhibited different levels of GST inhibition and antifungal activities [3]. The IC50 values of compounds **15** and **16** were determined as 16.5 and 17.1 μM, respectively for GST inhibition. Ethacrynic acid, a standard substrate GST inhibitor, exhibited a IC50 = 17.6 μM, suggesting that homoisoflavonoids have significant inhibition of GST activity [3].

The species *C. sappan* is the most prolific source of homoisoflavonoids with many representa‐ tives involving brazilin‐, caesalpin‐, protosappanin‐ and sappanin‐types. Extracts of *C. sappan*, known as sappan lignum, have been used as emmenagogue, hemostatic, anti‐inflammatory and for treatment of thrombosis. There are also relates about its antimicrobial activity against *Staphylococcus*, *Diplococcus*, *Corynebacterium*, and *Shigella baydii* [24].The species *C. sappan*

afforded brazilin‐ and sappanin‐types homoisoflavonoids such as compounds **13**, **14**, caesalpin P **(17)**, 3′‐*O*‐methylbrazilin **(18),** brazilide A **(19),** 3′‐deoxy‐4‐*O*‐methylsappanol **(20)**, sappanol **(21)**, 4‐*O*‐methylsappanol (**22, Figure 3**), in which compounds **13**, **14**, and **20,** and were active to the suppression of melanin synthesis [4]. Melanin is important to the protection of the skin from UV radiation, and its excessive synthesis could lead to melasma and lentigo. Compound **13** exhibited strong suppression of melanogenesis (EC50 = 3.0 μM) and cell viability around 95%. Furthermore, compound **20** also exhibited expressive activity (EC50 = 4.6 μM) with nonsignificant toxicity (cell viability around 92%). The other compounds displayed high cytotoxicity against HMV‐II cells [4].

*Staphylococcus aureus* exhibiting moderate antimicrobial activity. However, they were inactive or weakly active against Gram‐negative microorganisms such as *Pseudomonas aeruginosa*, *Klebsiella aerogenes,* and *Chromobacterium violaceum*. Concerning the antifungal activity, these compounds presented moderate activity against *Aspergillus niger* and *Candida albicans* in comparison with standard compounds Clotrimazole (antifungal), Streptomycin (antibacterial), and Penicillin G (antibacterial) [19]. Compounds **5** and **6** presented moderate activity against *Staphylococcus aureus* (inhibition zone of 11–15 cm) at 100 μg/mL, while **6** and **10** presented moderate activity against *Klebsiella aerogenes* (inhibition zone of 11–15 cm) at 100 μg/mL. Streptomycin presented an inhibition zone of 21–25 cm at 100 μg/mL. Compounds **5**, **8**, and **9** exhibited moderated activity against *Aspergillus niger* (inhibition zone of 5–10 cm at 150 μg/mL). On the other hand, the compounds **4**, **8**, **7**, **9**, and **10** were moderately active against *Candida albicans* at 150 μg/mL (inhibition zone of 5–10 cm). To comparison, positive control clotrimazole was active against all

Rao and collaborators tested the compound **2** against the inflammatory process and described that **2** inhibits the production of NO, TNF‐α, and IL‐12. In fact, **2** was the most active com‐ pound in the experiments at the concentration of 40 μM, reducing 92% of the NO production (IC50 = 20 μM) in mouse peritoneal macrophages induced by LPS + IFN‐γ. The authors sug‐ gested that the mode of action of **2** probably affects the production of NO by the induction of

The species *C. echinata*, commonly known as *Pau‐brasil* (brazilwood), is endemic from Brazil and played an important historical role in the country [1]. This species has been reported to contain a large range of polyphenols including the homoisolflavonoids brazilin **(13)** and brazilin **(14)**. The compound **15** is a natural dye and is also abundant in the species *C. sappan* (from 8 to 22%). The species *C. echinata*, considered the first source of brazilin, is used for diverse purposes such as healing agent, oral analgesic, and tonics. The species *C. echinata* has also demonstrated antitumor effect *in vivo* against cells strains of Ehrlich Carcinoma and Sarcoma 180. In addition, an interesting antiangiogenic effect was noticed [21]. On the other hand, compound **14**, an oxidation product of brazilin, was considered effective against the inflammatory and cytotoxic processes [22]. Compound **14** displayed cytotoxic effects against human cancer cell lines, such as HepG2 and Hep3B (liver), MDA‐MB‐231 and MCF‐7 (breast),

Phytochemical studies on ethanolic extracts of *C. bonduc* yielded two sappanin‐type homoi‐ soflavonoids identified as caesalpinianone **(15)** and 6‐*O*‐methylcaesalpinianone **(16)**, which exhibited different levels of GST inhibition and antifungal activities [3]. The IC50 values of compounds **15** and **16** were determined as 16.5 and 17.1 μM, respectively for GST inhibition. Ethacrynic acid, a standard substrate GST inhibitor, exhibited a IC50 = 17.6 μM, suggesting

The species *C. sappan* is the most prolific source of homoisoflavonoids with many representa‐ tives involving brazilin‐, caesalpin‐, protosappanin‐ and sappanin‐types. Extracts of *C. sappan*, known as sappan lignum, have been used as emmenagogue, hemostatic, anti‐inflammatory and for treatment of thrombosis. There are also relates about its antimicrobial activity against *Staphylococcus*, *Diplococcus*, *Corynebacterium*, and *Shigella baydii* [24].The species *C. sappan*

that homoisoflavonoids have significant inhibition of GST activity [3].

strains at 100 μg/mL (inhibition zone 21–25 cm) [19].

102 Flavonoids - From Biosynthesis to Human Health

LPS + IFN‐γ in mouse peritoneal macrophages [20].

A549 (pulmonary), and CA9‐22 (gingival) [22].

Species *C. sappan* constitute a source of sappanin‐type homoisoflavonoids. Related compounds such as 4‐(7‐hydroxy‐2,2‐dimethyl‐9βH‐1,3,5‐trioxa‐cyclopenta[α]naphthalene‐3‐lymethyl)‐ben‐ zene‐1,2‐diol **(23)**, 7,3′,4′‐trihydroxy‐3‐benzyl‐2H‐chromene **(24)** exhibited moderate activity as inhibitors of viral neuraminidases. Viral neuraminidases are considered essential to viral repli‐ cation cycle and a valid therapeutic target for antiviral drugs. Compound **5** presented the best activity against H1N1 (IC50 = 0.7 μM); H3N2 (IC50 = 1.1 μM); and H9N2 (IC50 = 1.0 μM) [23].

**Figure 3.** Sappanin‐type and brazilin‐type homoisoflavonoids from *Caesalpinia* spp.

Other sappanin‐type compounds such as (3*R*,4*S*)‐3‐(4′‐hydroxybenzyl)‐3,4‐dihydro‐2″,3″‐ dimethyl‐3H‐[1,3]dioxolo[4,5‐c]chromen‐7‐ol **(25)**, and (3a*R*,9b*S*)‐3a‐(4‐hydroxy‐3‐methoxy‐ benzyl)‐2,2‐dimethyl‐4,9b‐dihydro‐3aH‐[1,3]dioxolo[4,5‐c]chromen‐7‐ol **(26)** are associated to the inhibition of NO production [27]; **21** and **22** associated to the inhibition of melanin synthesis [4]. Sappanol derivatives were also identified from *C. sappan* as in the case of 3′‐*O*‐ methylsappanol **(27)**, 3′‐*O*‐methylepisappanol **(28)**, and a unique lactone‐based homoisofla‐ vonoid named caesalpiniaphenol B **(29)** [6]. In addition, the compound caesalpin J **(30)**, one of the only seven caesalpin‐type homoisoflavonoids reported in the literature, was isolated from *C. sappan*. Caesalpin J exhibited weak to moderate antimicrobial effects [25].

The species *C. japonica* is considered another source of biologically active homoisoflavonoids in which diverse homoisoflavonoids including **5**, **13**, **20**, **21**, **22**, protosappanin A‐C **(31–33)**, 4‐*O*‐methylepisappanol **(34)**, episappanol **(35)**, and sappanone B (**36, Figures 4**–**6**) have been isolated and characterized [29].

**Figure 4.** Sappanin‐, casealpin‐, and protosappanin‐type structures of homoisoflavonoids from *Caesalpinia* spp.

Homoisoflavonoids from *Caesalpinia* spp.: A Closer Look at Chemical and Biological Aspects http://dx.doi.org/10.5772/67723 105

**Figure 5.** Bihomoisoflavonoids from species of the genus *Caesalpinia*.

Other sappanin‐type compounds such as (3*R*,4*S*)‐3‐(4′‐hydroxybenzyl)‐3,4‐dihydro‐2″,3″‐ dimethyl‐3H‐[1,3]dioxolo[4,5‐c]chromen‐7‐ol **(25)**, and (3a*R*,9b*S*)‐3a‐(4‐hydroxy‐3‐methoxy‐ benzyl)‐2,2‐dimethyl‐4,9b‐dihydro‐3aH‐[1,3]dioxolo[4,5‐c]chromen‐7‐ol **(26)** are associated to the inhibition of NO production [27]; **21** and **22** associated to the inhibition of melanin synthesis [4]. Sappanol derivatives were also identified from *C. sappan* as in the case of 3′‐*O*‐ methylsappanol **(27)**, 3′‐*O*‐methylepisappanol **(28)**, and a unique lactone‐based homoisofla‐ vonoid named caesalpiniaphenol B **(29)** [6]. In addition, the compound caesalpin J **(30)**, one of the only seven caesalpin‐type homoisoflavonoids reported in the literature, was isolated

The species *C. japonica* is considered another source of biologically active homoisoflavonoids in which diverse homoisoflavonoids including **5**, **13**, **20**, **21**, **22**, protosappanin A‐C **(31–33)**, 4‐*O*‐methylepisappanol **(34)**, episappanol **(35)**, and sappanone B (**36, Figures 4**–**6**) have been

from *C. sappan*. Caesalpin J exhibited weak to moderate antimicrobial effects [25].

**Figure 4.** Sappanin‐, casealpin‐, and protosappanin‐type structures of homoisoflavonoids from *Caesalpinia* spp.

isolated and characterized [29].

104 Flavonoids - From Biosynthesis to Human Health

**Figure 6.** Scheme of synthesis of sappanin‐type homoisoflavonoids obtained from *C. pulcherrima*. Reagent and conditions: (i) CH<sup>3</sup> I, K2 CO<sup>3</sup> , acetone, 2h, reflux, (91%); (ii) substituted benzaldehyde, piperidine, 2h (58–69%). Adapted from Ref. [19].

Homoisoflavonoids classified as protosappanins are commonly associated to the species *C. sappan* and *C. japonica*. These compounds are resulting from the connection of C‐4 and C‐4a atoms forming an eight‐membered ring. There are only eleven protosappanins reported so far [26]. Compounds **31**–**33** did not show significant cytotoxicity against MCF7, A549, LN229 cell lines. In addition, compounds **32** and **33** were also tested against the inflammatory process exhibiting weak to moderate activity [27].
