**2.6.** *Smilax corbularia*

of the six major phenolic compounds active in *S*. *china*: taxifolin3*O*-glycoside (**36**), scirpusin A (**37**), piceid (**38**), oxyresveratrol (**39**), resveratrol (**40**) (**Figure 9**) and engeletin (**10**) (**Figure 3**). These compounds were extracted from the tuber of *S*. *china* with 95% aqueous ethanol and the concentrated extract was partitioned with petroleum ether, ethyl acetate and butanol. The ethyl acetate fraction was subjected to repeated silica gel chromatography. Finally, the purification of phenolic compounds was performed by HPLC [22]. Wu *et al*. also reported other study related to anticancer activity of phenolic compounds from *S*. *china* [12]. These authors obtained a 95% aqueous ethanol extract from the tuber of *S*. *china*, which was concentrated and suspended in water. The suspension was partitioned with petroleum ether, ethyl acetate and butanol. The ethyl acetate was the most bioactive fraction. This fraction was subjected to chromatographic purification. Three subfractions and six bioactive phenolic compounds bioactives: three flavonoids (kaempferol7*O*-*β*-D-glucoside (**6**), dihydrokaempferol (**9**) and dihydrokaempferol-3-*O*αLrhamnoside (**10**)) and three stilbenoids (**37**, **39** and **40**), were isolated from the ethyl acetate fraction. These compounds were found to induce apoptosis in

242 Phenolic Compounds - Natural Sources, Importance and Applications

**Figure 9.** One flavanonol (**36**) and four stilbenes (**37**–**40**) isolated from 95% ethanol extracts of *S*. *china* tubers.

*S. corbularia* is used in traditional Thai medicine for the ailments treatment caused by the menopause, as well as for ovarian and breast cancer. For this reason, Wungsintaweekul *et al*. isolated and characterized the phenolic compounds of methanol extract from *S*. *corbularia* rhizome [23]. They also evaluated the cell proliferation stimulation of the isolated compounds against human cancer cell lines MCF7 and T47D. The major compounds present in the rhizome of *S*. *corbularia* were rhamnosides dihydroflavonol derivatives, which represent 15% of methanol extract by weight. The results showed that the extract did not exhibit cytotoxicity against breast cancer cell lines MCF7 and T47D. However, the flavanonol rhamnosides (engeletin (**10**) and isoengeletin (**11**)) (**Figure 3**); as well as, astilbin (**41**), isoastilbin (**42**), neoastilbin (**43**) and neoisoastilbin (**44**) (**Figure 10**), showed a suppressive effect on estradiol at concentration of 1 μM as evidenced by human breast cancer cell proliferation.

**Figure 10.** Flavononol rhamnosides (**41**–**44**) with activity against human breast cancer isolated from methanol extract of *S*. *corbularia* rhizomes.

### **2.7.** *Smilax domingensis*

*S*. *domingensis* is used in Central America by the pharmaceutical and cosmetics industries. The most representative studies of *S*. *domingensis* are related to cytotoxicity to cancer cells [24], inhibitory activity of estrogen [25] and antioxidant activity [26]. The chemical studies of *S*. *domingensis* only cover qualitative identification of flavonoids and anthocyanins using thinlayer chromatography (TLC) [27].

### **2.8.** *Smilax excelsa*

*S*. *excelsa* is used in Turkey's traditional medicine to treat breast cancer, stomach pain and bloating [28]. Ozsoy *et al*. evaluated antioxidant activity of infusion, decoction, ethanol and ethyl acetate extracts from *S*. *excelsa* leaves using the inhibition of lipid peroxidation, metal ion chelating, reducing power, DPPH• radical scavenging, superoxide, hydroxyl radicals and hydrogen peroxide [29]. Also, total phenols, total flavonoid and anthocyanin were quantified in the extracts. The content of total phenols and total flavonoid was found in the intervals of 8.8–35.7 GAE mg/g of dry matter and 0.61–28.7 catechin equivalents mg/g of dry matter, respectively. The extract with the highest content of total phenols was the infusion. The decoction and infusion showed major DPPH• radical scavenging. These results agree with the high content of phenols and flavonoids present in the infusion and decoction. On other hand, Khaligh *et al*. isolated and elucidated three phenol compounds (*trans*-resveratrol (**40**) (**Figure 9**), 5-*O*caffeoylshikimic acid (**45**) and 6-*O*caffeoyl*β*-D-fructofuranosyl-(2→1)-*α*-Dglucopyranoside (**46**)) from ethyl acetate extract of *S*. *excelsa* (**Figure 11**) [30]. The extraction was a maceration performed at room temperature. After the solvent was removed, the extract was separated using silica gel column chromatography. In these study, also was evaluated the cytotoxicity of isolated compounds against human breast adenocarcinoma MCF-7cell lines. The 6-*O*caffeoyl*β*-D-fructofuranosyl-(2→1)αDglucopyranoside showed a promising activity against MCF-7 cell lines [30].

**Figure 11.** Two phenylpropanoids (**45** and **46**) derivates of caffeic acid with activity against human breast cancer.

### **2.9.** *Smilax fluminensis*

*S. fluminensis* has a wide geographical distribution in Brazil, and propagation studies have shown it to grow relatively easily. Hence, it is a promising species for growing demand from the pharmaceutical industry [31, 32] have published the only chemical study of *S*. *fluminensis* thus far. They obtained extracts from leaves and isolated phenolic compounds. Two flavonol glycosides were isolated and characterized, rutin (**25**) (**Figure 6**) and quercetin-3-*O*-*β*-Dgalactopyranoside (**47**) (**Figure 12**).

**Figure 12.** Flavonol glycoside (**47**) isolated from branches of *S*. *fluminensis*.

### **2.10.** *Smilax glabra*

ion chelating, reducing power, DPPH• radical scavenging, superoxide, hydroxyl radicals and hydrogen peroxide [29]. Also, total phenols, total flavonoid and anthocyanin were quantified in the extracts. The content of total phenols and total flavonoid was found in the intervals of 8.8–35.7 GAE mg/g of dry matter and 0.61–28.7 catechin equivalents mg/g of dry matter, respectively. The extract with the highest content of total phenols was the infusion. The decoction and infusion showed major DPPH• radical scavenging. These results agree with the high content of phenols and flavonoids present in the infusion and decoction. On other hand, Khaligh *et al*. isolated and elucidated three phenol compounds (*trans*-resveratrol (**40**) (**Figure 9**), 5-*O*caffeoylshikimic acid (**45**) and 6-*O*caffeoyl*β*-D-fructofuranosyl-(2→1)-*α*-Dglucopyranoside (**46**)) from ethyl acetate extract of *S*. *excelsa* (**Figure 11**) [30]. The extraction was a maceration performed at room temperature. After the solvent was removed, the extract was separated using silica gel column chromatography. In these study, also was evaluated the cytotoxicity of isolated compounds against human breast adenocarcinoma MCF-7cell lines. The 6-*O*caffeoyl*β*-D-fructofuranosyl-(2→1)αDglucopyranoside showed a promising

*S. fluminensis* has a wide geographical distribution in Brazil, and propagation studies have shown it to grow relatively easily. Hence, it is a promising species for growing demand from the pharmaceutical industry [31, 32] have published the only chemical study of *S*. *fluminensis* thus far. They obtained extracts from leaves and isolated phenolic compounds. Two flavonol glycosides were isolated and characterized, rutin (**25**) (**Figure 6**) and quercetin-3-*O*-*β*-D-

**Figure 11.** Two phenylpropanoids (**45** and **46**) derivates of caffeic acid with activity against human breast cancer.

activity against MCF-7 cell lines [30].

244 Phenolic Compounds - Natural Sources, Importance and Applications

**2.9.** *Smilax fluminensis*

galactopyranoside (**47**) (**Figure 12**).

The second most-studied species, after *S*. *china* is *S*. *glabra*. This species has been used in Chinese folk medicine for the treatment of acute bacterial dysentery, syphilis, acute and chronic nephritis [33], hyperinsulinemia [34] and cancer [35]. She *et al*. evaluated the effect of aqueous extract of *S*. *glabra* on cancer cell adhesion, migration and invasion of HepG2, MDA-MB-231 and T24 cells *in vitro* and the metastasis suppression of MDA-MB-231 cells *in vivo* [36]. Gao *et al*. showed 95% aqueous ethanol extracts of *S*. *glabra* rhizomes to be effective against cancer via mitochondrial apoptosis in human breast cancer MCF7, colon carcinoma HT-29 and gastric cancer cell line BGC-823 [3]. The results obtained by these authors point out that the aqueous extract of *S*. *glabra* possibly promotes cell adhesion by increasing the size and strength of focal adhesions and inhibits the invasion of HepG2, MDA-MB-231 and T24 cells.

Xia *et al*. performed an evaluation of the protective effect of 60% aqueous ethanol extract of *S*. *glabra* rhizome against leadinduced oxidative stress in rats and quantified total phenols and total flavonoids [37]. The results of this study proved that the extract of *S*. *glabra* could minimize damages caused by the lead. The protective effect can be attributed to high concentrations of total phenols and total flavonoids in the extract. Total phenols reported were 262 ± 12.7 mg gallic acid equivalents (GAE)/g dry weight of the extract and total flavonoids were 203.4 ± 9.1 mg rutin equivalents/g dry weight of the extract [37].

Trinh *et al*. evaluated the antioxidant activity of 95% aqueous ethanol extract of *S*. *glabra* roots. They obtained two fractions from a partitioning with hexane and ethyl acetate, and astilbin (**41**) (**Figure 10**) isolated from the ethyl acetate fraction [38]. The fractions were obtained from 95% aqueous ethanol previously dried. The extract was suspended in 50% aqueous ethanol and partitioned with hexane and ethyl acetate. The extract, hexane and ethyl acetate fractions, and **41** were subjected to evaluation of DPPH• radical scavenging activity, thiobarbituric acid-reactive species (TBARS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay for hepatoprotective effect via H<sup>2</sup> O2 injured mouse hepatocytes. The astilbin was isolated from the ethyl acetate fraction. The results of this study showed the ethyl acetate fraction has the principal antioxidant activity and MTT, attributed to the presence of astilbin, the main phenolic compound present in *S*. *glabra* rhizome [38]. Astilbin (**41**), a rhamnosyl flavanonol, besides exhibiting antioxidant activity, has coenzyme A reductase-inhibiting [39], aldose reductase-inhibiting [40], hepatoprotective [41], anti-oedemaogenic [42] and anti-arthritis [43] activities.

Zhang *et al*. obtained two aqueous and methanolic extracts, from *S*. *glabra* rhizomes and evaluated their antioxidant activity using DPPH• radical scavenging, ABTS radical cation scavenging, reducing power, superoxide anion radical scavenging activity and antioxidant activity in a linoleic acid emulsion system. They carried out quantification of total phenols [44]. The results showed that the methanol extract had the highest content of total phenols (152.28 ± 10.57 mg GAE/g of extract) and astilbin (245.65 ± 8.21 mg/g of extract). In general, the methanol extract has more antioxidant activity than aqueous extract and this behavior is attributed to the presence of **41** in the methanol extract [44]. Lu *et al*. evaluated antioxidant and antiinflammatory activities of 70% aqueous ethanol extract from *S*. *glabra* rhizomes [45]. The methods used to evaluate antioxidant activity were DPPH• radical scavenging, ABTS radical cation scavenging and reducing power. Antiinflammatory activity was evaluated with MTT cell viability, measuring of nitric oxide/nitrite and enzymelinked immunosorbent assay for IL6 and TNFα cytokines detection. The study also involved quantification of total phenols and total flavonoids. This was done by separation and identification of major phenols using ultrahigh pressure liquid chromatography coupled to electrospray mass spectrometry (U-HPLC-ESI-MS). The results obtained showed the 70% aqueous ethanol extract of *S*. *glabra* rhizome has a radical scavenging on DPPH• statistically equal to ascorbic acid (P > 0.05). The results regarding antiinflammatory activity showed that accumulation of NO, IL6, and TNFα in lipopolysaccharides (LPS)stimulated groups was higher than the group used as the positive control. Dexamethasone was employed as a positive control. Finally, 17 phenolic compounds were isolated and identified from the 70% aqueous ethanol extract of *S*. *glabra* rhizome, including engeletin (**10**) and isoengeletin (**11**) (**Figure 3**); astilbin (**41**), isoastilbin (**42**), neoastilbin (**43**), neoisoastilbin (**44**) (**Figure 10**); and 5-*O*caffeoylshikimic acid (**45)** (**Figure 11**).

Moreover, there have been several chemical studies to isolate and characterize phenolic compounds from different parts of *S*. *glabra*. Chen *et al*. obtained a methanol extract of *S*. *glabra* rhizome. After solvent removal, the dry extract was suspended in water and partitioned with ether petroleum and ethyl acetate. The ethyl acetate extract was purified by chromatographic column to separate the phenolic compounds. The compounds isolated were five flavonoids (engeletin (**10**), astilbin (**41**), smitilbin (**48**), taxifolin or dihydroquercetin (**49**) and eucryphin (**50**), **Figure 13**), and two phenylpropanoids, resveratrol (**40**) and 5-*O*caffeoylshikimic acid (**45**) [33]. Cheng *et al*. isolated new five phenylpropanoid glycosides, containing a sucrose core, smiglasides A–E (**35**, **51**–**54**, (**Figures 8** and **13**) from *S*. *glabra* rhizomes, [46]. The extraction and isolation procedures were followed exactly as was described by Chen *et al*. [33].

Xu *et al*. conducted a comprehensive chemical study of *S*. *glabra* rhizomes [47]. The air-dried and powdered rhizomes of *S*. *glabra* were extracted with 95% aqueous ethanol and 50% aqueous ethanol under reflux, consecutively. The extracts were combined and evaporated until to dryness and the residue was suspended in water and partitioned with petroleum ether, ethyl acetate and butanol. The ethyl acetate and butanol fractions were subjected to chromatographic separation. The purification allowed to the isolation 13 flavanones (dihydrokaempferol (**9**), engeletin (**10**), astilbin (**41**), isoastilbin (**42**), neoastilbin (**43**), neoisoastilbin

**Figure 13.** Flavonoids and smiglasides isolated from *S*. *glabra* rhizome.

A reductase-inhibiting [39], aldose reductase-inhibiting [40], hepatoprotective [41], anti-oede-

Zhang *et al*. obtained two aqueous and methanolic extracts, from *S*. *glabra* rhizomes and evaluated their antioxidant activity using DPPH• radical scavenging, ABTS radical cation scavenging, reducing power, superoxide anion radical scavenging activity and antioxidant activity in a linoleic acid emulsion system. They carried out quantification of total phenols [44]. The results showed that the methanol extract had the highest content of total phenols (152.28 ± 10.57 mg GAE/g of extract) and astilbin (245.65 ± 8.21 mg/g of extract). In general, the methanol extract has more antioxidant activity than aqueous extract and this behavior is attributed to the presence of **41** in the methanol extract [44]. Lu *et al*. evaluated antioxidant and antiinflammatory activities of 70% aqueous ethanol extract from *S*. *glabra* rhizomes [45]. The methods used to evaluate antioxidant activity were DPPH• radical scavenging, ABTS radical cation scavenging and reducing power. Antiinflammatory activity was evaluated with MTT cell viability, measuring of nitric oxide/nitrite and enzymelinked immunosorbent assay for IL6 and TNFα cytokines detection. The study also involved quantification of total phenols and total flavonoids. This was done by separation and identification of major phenols using ultrahigh pressure liquid chromatography coupled to electrospray mass spectrometry (U-HPLC-ESI-MS). The results obtained showed the 70% aqueous ethanol extract of *S*. *glabra* rhizome has a radical scavenging on DPPH• statistically equal to ascorbic acid (P > 0.05). The results regarding antiinflammatory activity showed that accumulation of NO, IL6, and TNFα in lipopolysaccharides (LPS)stimulated groups was higher than the group used as the positive control. Dexamethasone was employed as a positive control. Finally, 17 phenolic compounds were isolated and identified from the 70% aqueous ethanol extract of *S*. *glabra* rhizome, including engeletin (**10**) and isoengeletin (**11**) (**Figure 3**); astilbin (**41**), isoastilbin (**42**), neoastilbin (**43**), neoisoastilbin (**44**) (**Figure 10**); and 5-*O*caffeoylshikimic acid (**45)** (**Figure 11**).

Moreover, there have been several chemical studies to isolate and characterize phenolic compounds from different parts of *S*. *glabra*. Chen *et al*. obtained a methanol extract of *S*. *glabra* rhizome. After solvent removal, the dry extract was suspended in water and partitioned with ether petroleum and ethyl acetate. The ethyl acetate extract was purified by chromatographic column to separate the phenolic compounds. The compounds isolated were five flavonoids (engeletin (**10**), astilbin (**41**), smitilbin (**48**), taxifolin or dihydroquercetin (**49**) and eucryphin (**50**), **Figure 13**), and two phenylpropanoids, resveratrol (**40**) and 5-*O*caffeoylshikimic acid (**45**) [33]. Cheng *et al*. isolated new five phenylpropanoid glycosides, containing a sucrose core, smiglasides A–E (**35**, **51**–**54**, (**Figures 8** and **13**) from *S*. *glabra* rhizomes, [46]. The extraction and isolation procedures were followed exactly as was described by Chen *et al*. [33].

Xu *et al*. conducted a comprehensive chemical study of *S*. *glabra* rhizomes [47]. The air-dried and powdered rhizomes of *S*. *glabra* were extracted with 95% aqueous ethanol and 50% aqueous ethanol under reflux, consecutively. The extracts were combined and evaporated until to dryness and the residue was suspended in water and partitioned with petroleum ether, ethyl acetate and butanol. The ethyl acetate and butanol fractions were subjected to chromatographic separation. The purification allowed to the isolation 13 flavanones (dihydrokaempferol (**9**), engeletin (**10**), astilbin (**41**), isoastilbin (**42**), neoastilbin (**43**), neoisoastilbin

maogenic [42] and anti-arthritis [43] activities.

246 Phenolic Compounds - Natural Sources, Importance and Applications

(**44**), taxifolin (**49**), naringenin (**55**), sakuranetin (**56**), arthromerin B (**57**), sinesin (**58**), (2*R*,3*R*) taxifolin3′*O*-*β*-D-glucopyranoside (**59**) and (2*S*,3*S*)-glucodistylin (**60**); 3 flavanes: (+)catechin (**26**), (−)epicatechin (**27**) and cinchonain 1b (**60**)); 2 flavanones (luteolin (**62**) and apigenin (**63**)); two flavonols (quercetin (**7**) and myricetin (**64**)); 1 chalcone, kukulkanin B (**65**); 3 stilbenes (piceid (**38**), piceatannol (**39**), and resveratrol (**40**)); 6 phenylpropanoids (5-*O*caffeoylshikimic acid (**45**), caffeic acid (**66**), 3-*O*-*p*-coumaroylshikimic acid (**67**), smiglycerol (**68**), juncusyl ester B (**69**) and 1-*O*-p-coumarylglycerol (**70**), **Figure 14**). It is noteworthy that in this study no smiglasides were detected, despite an intensive separation of phenolic compounds having conducted. One explanation for these results is that possibly the high temperatures used for extraction caused smiglasides degradation.
