In vitro and in vivo bioactivities of aqueous and ethanol extracts from Helicteres angustifolia L. root

Abstract

Ethnopharmacological relevance

Helicteres angustifolia L. (H. angustifolia L.) has been used as traditional medicine in the treatment of cancer in China and Laos. Its medical benefits, however, are still lacking of scientific evidence. Two extracts successively obtained from the root of H. angustifolia L., namely the aqueous root extract (ARE) and the ethanolic root extract (ERE), were used to evaluate the antioxidant and anticancer activities in vitro, and the antitumor efficacy of ARE was examined in vivo, respectively.

Materials and methods

ARE and ERE were extracted successively from H. angustifolia L. root with water and ethanol. In vitro antioxidant activities were assessed by radicals scavenging assay, ferrous chelating assay and reducing power assay. In vitro anticancer activities of ARE and ERE were evaluated by their cytotoxic effects against three human cancer cell lines. In addition, the anti-tumor activities of ARE in vivo were assessed by using Ht1080 (human fibrosarcoma cell line Ht1080) tumor xenografts mice. BALB/c nude mice were orally administrated with 200 mg/kg/d of ARE. The tumor inhibition rate was determined on day 42 after treatment by using histopathology analysis of the tumor tissues. Furthermore, relevant biochemical parameters in blood were analyzed to monitor their cytotoxic effect.

Results

In vitro assays indicated that ARE possessed relatively higher antioxidant and anticancer activities than ERE, with IC₅₀ values of 82.31±9.62, 62.50±6.99, and 127.49±2.9 μg/mL against DLD-1, A549, and HepG2 cells, respectively. In vivo tumor inhibition experiments suggested that ARE possessed significant antitumor efficacy in BALB/c nude mice with a tumor inhibition rate of 49.83±14.38% (p<0.05) and little toxicity was observed to the host.

Conclusion

ARE from H. angustifolia L. possessed high antioxidant activities is active against liver cancer HepG2, lung cancer A549 and colon cancer DLD-1 cells in vitro and tumor xenografts bearing BALB/c nude mice in vivo. Further studies on elucidation of the mechanisms involved and isolation of the active components may provide more valuable information for the development of functional products from H. angustifolia L. and their application in cancer treatment.

Introduction

Helicteres, a genus belongs to the plant family of Sterculiaceae, is distributed throughout both tropical Asia and America. Helicteres are shrubs or small trees and comprise about 60 species (Kubitzki and Bayer, 2003). Several species of this genus have been used in traditional medicine system for centuries. For instance, Helicteres isora has been used for the treatment of stomach affection, intestinal infection, and diabetes (Venkatesh et al., 2007). Helicteres ovata and Helicteres sacarolha have been used as a depurant and syphilis treatment (Truiti et al., 2005). Helicteres root has been used for the treatment of uterus pain (Libman et al., 2006). Biological studies have demonstrated that the extract of H. isora has antinociceptive, antidiabetic, hypolipidemic and hypoglycaemic activities (Chakrabarti et al., 2002, Kumar et al., 2006, Venkatesh et al., 2007, Bhavsar et al., 2009). The extract of Helicteres gardneriana leaf has been reported to possess antiprotozoal and anti-inflammatory activity (Truiti et al., 2005, Oliveira de Melo et al., 2011), and the stem extract of H. hisuta exhibits cytotoxic activity against cancer cell lines (Chin et al., 2006).

Helicteres angustifolia L. (H. angustifolia L.), widely distributed in sloping grasslands throughout southern China, Japan, Southeast Asia and Australia, is a small shrubby weed with gray green puberulent branchlets and small brown seeds. The root of this plant, known as “Shan-Zhi-Ma” in Chinese, is traditionally consumed by Chinese people as tea and medicinal liquor to promote general wellness, which is also widely used in daily tonics and various home remedy recipes for the treatment of influenza fever, headache, bacterial infections, inflammatory and cancer (Jiangsu New Medical College (JNMC), 1986, Chiu and Chang, 1995). Up to now, phytochemical studies are mainly focused on identification of sesquiterpenoid quinones (Chen et al., 1990), flavonoid glycosides (Chen et al., 1994), lignans (Chin et al., 2006), prename, coumarin and lupine derivatives (Chang et al., 2001), cucurbitacins (Chen et al., 2006a), triterpenoids (Pan et al., 2008), steroids (Chen et al., 2006b), and alkaloids (Wang et al., 2012). Currently, detailed information is still very limited with respect to H. angustifolia L. Chen et al. (2006b) discovered two cucurbitacin derivatives, cucurbitacin D and J, from aerial parts of H. angustifolia L. exhibited significantly inhibitory activities against the proliferation of hepatocellular carcinoma BEL-7402 cells and malignant melanoma SK-MEL-28 cells in vitro. After preparing the chloroform extract from root bark of H. angustifolia which was further fractioned, Pan et al. (2008) found that three triterpenoids exhibited remarkable cytotoxic activities against human colorectal cancer cells COLO 205 and gastric cancer cells AGS in vitro. More recently, Wang et al. (2012) isolated the ethanol extract of H. angustifolia root and discovered that cucurbitacin derivatives, cucurbitacin B, D, I and E along with 3β-acetoxybetulinic acid displayed significantly anti-proliferative activity against human colon cancer HT-29 cells and ovarian cancer OVCA429 cells in vitro. As it can be seen, however, the previous studies were mostly focused on the characterization of H. angustifolia L. and evaluation of bioactivity of isolated compounds against the growth of tumor cell lines. Little information could be found on its corresponding antioxidant and antitumor activities in vivo.

In this study, aqueous root extract (ARE) and ethanolic root extract (ERE) were obtained from H. angustifolia L. according to the designated extraction procedure (Fig. 1) and used for the evaluation of antioxidant and anticancer activities in vitro. Based on in vitro bioactivity assessment, tumor growth inhibitory experiments of ARE were followed by using BALB/c nude mice model. This work firstly explored the antioxidant and anticancer activities of ARE and ERE from H. angustifolia L. in vitro and also for the first time to disclose the antitumor activity of ARE in vivo.