Negative pressure cavitation extraction and antioxidant activity of genistein and genistin from the roots of pigeon pea [Cajanus cajan (L.) Millsp.]

Abstract

A new method—negative pressure cavitation extraction (NPCE) was proposed and investigated for the extraction of the main isoflavonoids, namely genistein and genistin from pigeon pea roots. The effects of extraction time and particle size on the extraction yields were firstly optimized, then a central composite design (CCD) combined with response surface methodology (RSM) was used to study the effects of negative pressure, ethanol concentration and liquid/solid ratio on the extraction yields. The maximum extraction yields of genistein and genistin reached 0.418 and 0.398 mg/g, respectively, under the optimal conditions: extraction time 45 min, particle size 50 mesh, negative pressure −0.05 MPa, ethanol concentration 70% and liquid/solid ratio 44:1. Furthermore, the antioxidant activity of NPCE extract was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay. NPCE extract possessed notable concentration-dependent antioxidant activity with IC₅₀ value of 0.062 mg/ml.

Introduction

Pigeon pea [Cajanus cajan (L.) Millsp.], also known as red gram, no-eye pea, Congo pea, Gungo pea, etc., is a famous and multi-usage grain legume crop widely distributed in semi-tropical and tropical developing countries. Nowadays, pigeon pea is cultivated as an annual for both forage and its edible beans [1]. Some medicinal usages of this plant have been recorded in worldwide. Most of the literatures are focused on pigeon pea leaves, which demonstrated notable anti-inflammatory, anti-bacterial and abirritative properties [2]. Especially, pigeon pea leaves are used as an excellent traditional Chinese medicine (TCM), which has been brought to the market for the therapy of ischemic necrosis of femoral head [3]. Comparatively, research on pigeon pea roots is scanty, the phytochemicals and possible medicinal uses are not fully explored. In fact, pigeon pea roots have many medicinal uses. They were used as an alexeritic, anthelminthic, expectorant, sedative, and vulnerary as a folk medicine in some local producing areas. However, most of them are usually discarded as agricultural wastes or used as firewood by farmers, the total amount is huge. Hence, innovative technology concepts for the utilization of pigeon pea roots to provide the raw material for the manufacture of future-oriented products are desirable.

Preliminary chemical investigations and pharmacological studies on pigeon pea roots in our group have demonstrated the presence of polyphenols, especially isoflavonoids, and they were the major beneficial compounds responsible for their bioactivities. Genistein and genistin are a pair of isoflavonoid compounds found as the main constituents present in pigeon pea roots, their structures are shown in Fig. 1. They represent important members of the flavonoid family and possess a wide spectrum of pharmacological activities [4], [5], [6], [7]. Therefore, genistein and genistin have great potentials to be used as clinical therapeutic agents, food additives or nutraceutical products. The application of low-cost technology to obtain these two compounds from pigeon pea roots is a rational strategy to increase the economic value and expand the utilization of this plant.

Cavitation is a general fluid mechanics phenomenon, which can occur whenever a liquid used in a machine inducing pressure and velocity fluctuations in the fluid (e.g. pumps, turbines, and propellers). Cavitation serves as a means of concentrating the diffused fluid energy locally in very short duration and creating a zone of intense energy dissipation. Acoustic cavitation and hydrodynamic cavitation are two types sorted by the cause of formation [8]. The studies on acoustic cavitation such as ultrasonic cavitation have been widespread carried out in the fields of phytochemistry, biochemistry, physics and metallurgy [9], [10]. As an efficient extraction method, ultrasonic extraction has attracted growing interest for the extraction of various secondary metabolites from plant materials [11], [12]. However, some thermosensitive compounds such as genistin are easily degradable by ultrasonic cavitation due to its high temperature and intensity [13], [14], [15]. Therefore, a new kind of cavitation suitable for the extraction of thermosensitive secondary metabolites is warranted.

Negative pressure cavitation is another type in which the cavitation is generated by negative pressure. It is a cheap and energy efficient method. It can keep constant lower temperature and its intensity is not weaker than that of ultrasonic cavitation. Furthermore, during the extraction process, nitrogen is continuously introduced into a liquid–solid system to increase the turbulence, collision and mass transfer between the extraction solvent and matrix. Under the integrated action, it is efficient for mixing sample with the extraction solvent as well as migrating the target compounds out of the sample matrix [16]. Thus, this procedure has not only the advantages of ultrasonic cavitation but also is good for preventing the degradation of thermosensitive compounds.

In the present study, a NPCE method was proposed and applied for the extraction of genistein and genistin from pigeon pea roots. The effects of main operating parameters on the extraction yields were investigated. The extraction efficiency of two isoflavonoids with NPCE was compared with those obtained by three conventional extraction methods. The structural disruption to pigeon pea roots samples with different extraction methods was observed by scanning electron microscopy (SEM). Furthermore, the antioxidant activity of extracts, obtained using different extraction methods was determined by means of DPPH radical-scavenging assay.