**2.2.** *Smilax bockii*

The rhizome, roots, stems and, occasionally, leaves of sarsaparilla are used as food and in traditional medicine. These plants are known to have immunomodulatory, antioxidant, antibacterial, antifungal and diuretic properties. Additionally, they are used for relief from climatery [1]. Also, the genus *Smilax* has pharmacological properties and is used to treat different types of cancer, diabetes, skin diseases, ulcers, as well as fever, gout and ophthalmic diseases [2].

**Figure 1.** (A) *S*. *bracteata*, (B) *S*. *china*, (C) *S*. *fluminensis*, (D) *S*. *glyciphylla*, images from https://www.inaturalist.org/taxa. (E) *S*. *campestris*, image courtesy of Mauricio Bonifacino Ph.D. (Universidad de la República, Montevideo, Uruguay).

In recent years, interest in the study of the genus *Smilax* has increased, mainly in Europe and Asia, due to the presence of phenolic compounds. Some species have also proven effective in the prevention and treatment of several cancers. In addition, extracts from the genus *Smilax*

There are reports about the antioxidant property expressed as DPPH• radical scavenging activity of species of the genus *Smilax*, as *Smilax bockii* [4], *Smilax campestris* [5], *Smilax glabra* [6], *Smilax lanceifolia* [7], *Smilax perfoliata* [8], *Smilax riparia* [9], *Smilax scobinicaulis* [10] and *Smilax sebeana* [11]. This property is attributed to phenolic compounds such as stilbenes, flavones,

exhibit pro-apoptotic activity and antioxidant activity [3].

234 Phenolic Compounds - Natural Sources, Importance and Applications

(F)–(I) *S*. *domingensis*.

*S. bockii* is a plant used in traditional Chinese medicine with antiinflammatory and antirheumatic properties. Xu *et al*. prepared a 70% aqueous ethanol extract from roots of *S*. *bockii* [4]. Then the extract was partitioned with chloroform, ethyl acetate and butanol successively. The butanol fraction was subjected to chromatographic purification leading to the separation of four flavonols (kaempferol (**5**), kaempferol-7-*O*-*β*-D-glucopyranoside (**6**), quercetin (**7**) and isorhamnetin (**8**), as well as three flavanone ((+)dihydrokaempferol (**9**), engeletin (**10**) and isoengeletin (**11**)), and a phenylpropanoid, caffeic acid *n*-butyl ester (**12**) (**Figure 3**). Additionally, the antiinflammatory activities of a 70% aqueous ethanol extract and chloroform, ethyl acetate and butanol fractions were evaluated and the results showed the butanol fraction had a

**Figure 2.** Anthocyanin glycosides isolated from *S*. *aspera* fruits.

relevant inhibitory activity against TNFαinduced NFκB activation with an IC50 value of 44.8 μg/mL. This activity can be attributed to phenolic compounds present in the butanol fraction.

### **2.3.** *Smilax bracteata*

*S. bracteata* is a littlestudied species. However, there are two representative chemical studies that describe the isolation and characterization of phenolic compounds. The first study was conducted by Li *et al*. who isolated and identified phenolic compounds from a methanol extract of *S*. *bracteata* rhizomes [15]. The air-dried and sliced rhizomes were extracted by maceration with methanol over 24 h. The extract was evaporated and re-dissolved in water and partitioned successively with dichloromethane, ethyl acetate and butanol. The butanol fraction was subjected to column chromatography and six new phenolic compounds were isolated and identified: two flavan3ol glucosides (**13**, **14**), one stilbene (**15**) (**Figure 4**) and three phenylpropanoid glycosides (**16**–**18**) (**Figure 5**). In the same study, Li *et al*. evaluated antioxidant activity of the six smilasides using the DPPH• radical scavenging activity. The smilasides J to L (**22**–**24**) showed an antiradical activity similar to αtocopherol [15].

In a later study, Zhang *et al*. obtained a 95% aqueous ethanol extract from stems of *S*. *bracteata* [16]. The extract was concentrated and redissolved in water and successively extracted with hexane, dichloromethane and butanol. The dichloromethane fraction was purified with chromatography in several steps until smilasides G to L (**19**–**24**) were obtained (**Figure 5**).

### **2.4.** *S. campestris*

*S. campestris* is commonly called sarsaparilla blanca [5]. Its roots and rhizomes have been used in folk medicine to treat skin diseases. An infusion from the leaves and aerial stems of *S*.

relevant inhibitory activity against TNFαinduced NFκB activation with an IC50 value of 44.8 μg/mL. This activity can be attributed to phenolic compounds present in the butanol

*S. bracteata* is a littlestudied species. However, there are two representative chemical studies that describe the isolation and characterization of phenolic compounds. The first study was conducted by Li *et al*. who isolated and identified phenolic compounds from a methanol extract of *S*. *bracteata* rhizomes [15]. The air-dried and sliced rhizomes were extracted by maceration with methanol over 24 h. The extract was evaporated and re-dissolved in water and partitioned successively with dichloromethane, ethyl acetate and butanol. The butanol fraction was subjected to column chromatography and six new phenolic compounds were isolated and identified: two flavan3ol glucosides (**13**, **14**), one stilbene (**15**) (**Figure 4**) and three phenylpropanoid glycosides (**16**–**18**) (**Figure 5**). In the same study, Li *et al*. evaluated antioxidant activity of the six smilasides using the DPPH• radical scavenging activity. The

smilasides J to L (**22**–**24**) showed an antiradical activity similar to αtocopherol [15].

matography in several steps until smilasides G to L (**19**–**24**) were obtained (**Figure 5**).

In a later study, Zhang *et al*. obtained a 95% aqueous ethanol extract from stems of *S*. *bracteata* [16]. The extract was concentrated and redissolved in water and successively extracted with hexane, dichloromethane and butanol. The dichloromethane fraction was purified with chro-

*S. campestris* is commonly called sarsaparilla blanca [5]. Its roots and rhizomes have been used in folk medicine to treat skin diseases. An infusion from the leaves and aerial stems of *S*.

fraction.

**2.3.** *Smilax bracteata*

**Figure 2.** Anthocyanin glycosides isolated from *S*. *aspera* fruits.

236 Phenolic Compounds - Natural Sources, Importance and Applications

**2.4.** *S. campestris*

**Figure 3.** Four flavonols (three aglycones and one glucoside), three flavanones (one aglycone and two glycosides) and one phenylpropanoid ester isolated from *S. bockii* roots.

*campestris* is used to relax the digestive system [5]. Rugna *et al*. reported antioxidant activity from 50% aqueous methanol extract from *S*. *campestris* rhizomes; the activity was expressed as total reactive potential (TRAP) [5]. Morais *et al*. obtained an ethanol extract by maceration and fractions from fresh stems of *S*. *campestris* [17]. The ethanol extract was concentrated, and the dried extract was redissolved in aqueous ethanol (7:3) and partitioned with hexane, dichloromethane, ethyl acetate and butanol. The antioxidant activity as DPPH• radical scavenging was evaluated for all fractions. The ethanol extract and butanol fraction exhibited a strong

**Figure 4.** Two flavan3ol glycosides and one stilbene isolated from *S*. *bracteata* rhizomes.

antioxidant activity and was higher than Butylated hydroxytoluene (BHT), a commercial antioxidant. Also, Morais *et al*. reported that rutin (**25**) (**Figure 6**) and quercetin (**7**) (**Figure 3**) flavonol glycosides were the most abundant phenolic compounds present in ethanol extract and butanol fraction, respectively [17].

### **2.5.** *Smilax china*

*S. china* is the most studied species of genus *Smilax*. Lee *et al*. evaluated antioxidant activity of *S*. *china* root using DPPH• radical scavenging activity, cell viability, lipid peroxidation activity, superoxide dismutase (SOD) activity, catalase (CAT) activity and glutathione peroxidase (GPX) activity. These authors obtained a 70% aqueous methanol extract from the root of *S*. *china*. The extract was concentrated and partitioned with hexane, dichloromethane, ethyl acetate and butanol. The extract and its fractions were evaluated for antioxidant activity. The antiradical activity expressed as IC50 of the extract is about 8 μg/mL, while the ethyl acetate fraction exhibited the principal antiradical activity (IC50 approximately 5 μg/mL) [18]. Jeong *et al*. obtained several fractions with solvents of different polarity from *S*. *china* root and evaluated the antioxidant activity using DPPH• radical scavenging activity, ABTS•+ radical scavenging activity, reducing power, ferric reducing/antioxidant power, ferric thiocyanate assay, malondialdehyde assay using mouse brain homogenates methods, and finally determined total phenols and phenolic composition [19]. The extraction was carried out with methanol at 70°C for 2 h. The methanol extract was evaporated to dryness. The dried extract was re-dissolved in water and the solution was consecutively partitioned with chloroform, ethyl acetate

antioxidant activity and was higher than Butylated hydroxytoluene (BHT), a commercial antioxidant. Also, Morais *et al*. reported that rutin (**25**) (**Figure 6**) and quercetin (**7**) (**Figure 3**) flavonol glycosides were the most abundant phenolic compounds present in ethanol extract

**Figure 4.** Two flavan3ol glycosides and one stilbene isolated from *S*. *bracteata* rhizomes.

*S. china* is the most studied species of genus *Smilax*. Lee *et al*. evaluated antioxidant activity of *S*. *china* root using DPPH• radical scavenging activity, cell viability, lipid peroxidation activity, superoxide dismutase (SOD) activity, catalase (CAT) activity and glutathione peroxidase (GPX) activity. These authors obtained a 70% aqueous methanol extract from the root of *S*. *china*. The extract was concentrated and partitioned with hexane, dichloromethane, ethyl acetate and butanol. The extract and its fractions were evaluated for antioxidant activity. The antiradical activity expressed as IC50 of the extract is about 8 μg/mL, while the ethyl acetate fraction exhibited the principal antiradical activity (IC50 approximately 5 μg/mL) [18]. Jeong *et al*. obtained several fractions with solvents of different polarity from *S*. *china* root and evaluated the antioxidant activity using DPPH• radical scavenging activity, ABTS•+ radical scavenging activity, reducing power, ferric reducing/antioxidant power, ferric thiocyanate assay, malondialdehyde assay using mouse brain homogenates methods, and finally determined total phenols and phenolic composition [19]. The extraction was carried out with methanol at 70°C for 2 h. The methanol extract was evaporated to dryness. The dried extract was re-dissolved in water and the solution was consecutively partitioned with chloroform, ethyl acetate

and butanol fraction, respectively [17].

238 Phenolic Compounds - Natural Sources, Importance and Applications

**2.5.** *Smilax china*


**Figure 5.** Phenylpropanoid glycosides with a sucrose core isolated from *S*. *bracteata* aerial parts.

and butanol. The results obtained by Jeong *et al*. show that the ethyl acetate fraction had the highest total phenol concentration, 401.62 ± 3.13 mg GAE/g of extract and the most abundant phenols were (+)catechin (**26**) and (−)epicatechin (**27**) (**Figure 7**) with a concentration of 135.26 ± 10.08 and 58.10 ± 0.51 mg/100 g, respectively. Consequently, this extract showed the most important antioxidant activity.

**Figure 6.** Chemical structure of rutin, a flavonol glycoside isolated from *S*. *campestris* rhizomes.

**Figure 7.** Two flavan3ol aglycones isolated from *S*. *china* roots.

Kuo *et al*. obtained a 70% aqueous ethanol extract from dried stems of *S*. *china*. The extracts were concentrated and suspended in water. The resulting suspension was partitioned with hexane and chloroform [20]. The chloroform fraction was purified by silica gel column chromatography. The purification conducted to isolate smilasides A to F (**Figures 5** and **8**), heloniosides A (**33**) and B (**34**) and smiglaside E (**35**) (**Figure 8**). The anticancer activity of smilasides A to F was evaluated *in vitro* and showed cytotoxic activity against cervical cancer cells (KB and HELA) and colon cancer cells (DLD-1).

Li *et al*. performed another study related to the evaluation of anticancer activity of *S*. *china* extracts with a high content of phenolic compounds [21]. Researchers in this study performed a bioassayguided separation and purification of kaempferol7*O*-*β*-D-glucoside (**6**) from *S*. *china* rhizome. First, a 70% aqueous ethanol extract was obtained under reflux. Then the solvent was removed and residue was extracted with ethyl acetate, and butanol, sequentially, in a Soxhlet apparatus. Both ethyl acetate and butanol fractions were subjected to column chromatography separately. Several fractions with large amounts of flavonoid were obtained and each fraction was evaluated for *in vitro* anticancer activity. The human cells used in this study included liver cancer BEL-7402, cervical epithelial carcinoma HeLa, high metastatic lung

#### Phenolic Compounds in Genus *Smilax* (Sarsaparilla) http://dx.doi.org/10.5772/66896 241

Kuo *et al*. obtained a 70% aqueous ethanol extract from dried stems of *S*. *china*. The extracts were concentrated and suspended in water. The resulting suspension was partitioned with hexane and chloroform [20]. The chloroform fraction was purified by silica gel column chromatography. The purification conducted to isolate smilasides A to F (**Figures 5** and **8**), heloniosides A (**33**) and B (**34**) and smiglaside E (**35**) (**Figure 8**). The anticancer activity of smilasides A to F was evaluated *in vitro* and showed cytotoxic activity against cervical cancer cells (KB

**Figure 6.** Chemical structure of rutin, a flavonol glycoside isolated from *S*. *campestris* rhizomes.

Li *et al*. performed another study related to the evaluation of anticancer activity of *S*. *china* extracts with a high content of phenolic compounds [21]. Researchers in this study performed a bioassayguided separation and purification of kaempferol7*O*-*β*-D-glucoside (**6**) from *S*. *china* rhizome. First, a 70% aqueous ethanol extract was obtained under reflux. Then the solvent was removed and residue was extracted with ethyl acetate, and butanol, sequentially, in a Soxhlet apparatus. Both ethyl acetate and butanol fractions were subjected to column chromatography separately. Several fractions with large amounts of flavonoid were obtained and each fraction was evaluated for *in vitro* anticancer activity. The human cells used in this study included liver cancer BEL-7402, cervical epithelial carcinoma HeLa, high metastatic lung

and HELA) and colon cancer cells (DLD-1).

**Figure 7.** Two flavan3ol aglycones isolated from *S*. *china* roots.

240 Phenolic Compounds - Natural Sources, Importance and Applications


**Figure 8.** Phenylpropanoid glycosides with a sucrose core (five smilasides, two helionoside and one smiglaside) isolated from *S*. *china* stems.

carcinoma 95-D, melanoma A375, gastric cancer MKN-45, epithelial carcinoma A431, human acute leukemia HL60, normal embryonic kidney HEK293 and normal embryonic liver L-O2. Li *et al*. found eight extracts of *S*. *china* tubers with anticancer activity against HeLa cells. Also, a bioassay-guided isolation of the polled extract lead to the detection of kaempferol-7-*O*-*β*-Dglucoside (**6**). This flavonoid induces apoptosis as an antiproliferative action related to radical scavenging activity [21]. Shao *et al*. developed a specific HPLC method for determination 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

anti-breast tumor cells MCF-7 and MDA-MB-231. The results showed that resveratrol and oxyresveratrol had the highest apoptosis rates [12].
