Recovery and purification of limonin from pummelo [Citrus grandis] peel using water extraction, ammonium sulfate precipitation and resin adsorption
Introduction
Limonoids are a unique class of highly oxygenated tetracyclic triterpenoids that exist in plants of the order Rutales, particularly in the families of Rutaceae, Meliaceae, and Cneoraceae [1], [2]. Members of the class limonoids have wide health-promoting and disease-preventing activities [1], [2]. As a typical limonoid, limonin has shown a wide range of biological properties, including anticancer [3], [4], antibacterial [5], [6], antioxidant [7], [8], larvicidal [9], [10], antimalarial and antiviral [11] activities, and thus has potential applications in nutriceuticals, pharmaceuticals and agriculture.
Limonin widely exists in Citrus tissues [12]. Li et al. found that the limonin content in the flavedo (colored portion of the peel) and albedo (white and spongy portion of the peel) of the Satsuma mandarin (Citrus unshiu Marc.) reached 1.2 and 0.9 mg/g (dry weight) [13], respectively. Sun et al. reported that the flavedo and albedo of C. changshanensis have limonin contents of 0.79 and 0.578 mg/g (dry weight)[8], respectively. It has been illustrated that the content of the seed, segment membrane, albedo, and flavedo of the citron (Citrus medica L.) are 1600–3270 ppm, 310–350 ppm, 150–250 ppm, and 150–225 ppm, respectively [14]. In general, limonin is practically extracted from citrus seeds due to their having the highest limonin content among the Citrus tissues. However, most commercially processed Citrus fruits have few seeds, making it difficult to collect the seeds for the industrial preparation of limonin.
In general, limonin is extracted from plant tissues using organic solvents such as methanol, ethyl acetate, acetone and ethanol [15], [16], [17], which need extra instruments and complicated processing to evaporate the solvents so that the limonin can be recovered. Aqueous hydrotropic (Na-Sal or Na-CuS) and alkaline solutions have been proposed to extract limonin from sour orange seeds and Citrus reticulate blanco [1], [18]], but this can lead to a high concentration of alkaline or metal ions in the extracts. Supercritical carbon dioxide fluid extraction, which represents an eco-friendly form of extraction, has been attempted to extract limonin from Citrus aurantifolia swingle [19], but a scaled-up process has not been applied because of the difficulty of holding high-pressure carbon dioxide gas in a large container. Thus, it is of significance to develop more effective methods to extract limonin in environmentally friendly ways.
Pummelo is botanically named Citrus grandis or Citrus maxima (in Japan); it is a traditional Citrus variety of Southeast Asia. Although the fruit is commonly known as the pummelo, it is also called the pamplemousse, pomelo, Bali lemon, limau besar, and shaddock, depending on the region where it is grown. Pummelo fruits contain remarkable amounts of naringin, limonin and nomilin [20], which may enter the juice during juice extraction and consequently imbue it with a strong undesirable bitter taste. As the debittering process is becoming more and more effective [21], a few plants have been set up in the Fujian Province of China to extract pummelo juice. There are now approximately 10,000 tons of pummelo processed in the Fujian Province of China every year, generating approximately 2000 tons of peels. Although the pummelo peel has been determined to contain limonin [22], there have been few attempts to recover the limonin.
In our previous studies, enzymes (i.e., naringinase, α-l-rhamnosidase, and β-glucosidase) extracted from the fermented broth of Aspergillus niger have been investigated to eliminate the bitter taste of citrus juice. It was found that some enzyme extracts prepared by ammonium sulfate precipitation could significantly decrease the content of the bitter chemicals naringin and limonin (detailed data not shown). Further experiments have revealed that naringin is hydrolyzed to the non-bitter compound naringenin by the action of naringinase, while the limonin is precipitated from the juice as a result of the action of the ammonium sulfate that enters the juice in the process of the enzyme treatment. Thus, by chance, ammonium sulfate was discovered to precipitate the limonin in citrus juice. In our recent study, the limonin in the pummelo peel was observed to be able to be extracted by warm water, which might indicate a limonin resource that could be extracted without using organic solvent or base solutions. Hence, the limonin in the water extract could similarly be precipitated after the addition of ammonium sulfate. In this context, the aim of the present study was to characterize the limonin recovery from the pummelo peel using water extraction and ammonium sulfate precipitation, with the expectation of developing a simple and green process for limonin production. The specific content includes (1) the analysis of the content of limonin in the pummelo peel, (2) the development of a procedure for limonin recovery using water extraction and ammonium sulfate precipitation, and (3) the purification and identification of the limonin.
Section snippets
Raw material and chemicals
Pummelo [Citrus grandis (L.) Osbeck] peel was purchased from the Fujian GuoNong Agriculture Development Company (Pinghe County, Fujian Province, China). After drying at 60 °C for 3 h, it was ground to a powder over 40 meshes by a FW100 pulverizer that was bought from Hangzhou Huier Instruments Co., Ltd (Hangzhou, China).
Standard limonin was purchased from Xian Xiaocao Botanical Development Co., Ltd. (Xian, Shanxi Province, China). HPLC-grade methanol and acetonitrile were obtained from
Analysis of limonin content of pummelo peel
To evaluate the possibility of the practical utilization of the limonin in the pummelo peel, the pummelo peel was extracted with ethanol, and the limonene content in the resultant extract was analyzed by HPLC. The pummelo peel showed a limonin content of 4.7 mg/g (Table 1), which is consistent with a previous study that found that pummelo tissues contain noticeable limonin content [15], [16], [17]. Although the limonin content is lower than those of some citrus seeds as previously reported [23],
Conclusion
The pummelo peel had a limonin content of 4.7 mg/g. The limonin could be effectively recovered from the pummelo peel via water extraction and ammonium sulfate precipitation. The optimal process for recovering limonin was extracting the pummelo peel with 20 mL/g of water at 80 °C for 30 min and then precipitating it at 50 °C with ammonium sulfate at a saturation of 90% for 90 min. The extraction and precipitation yields were 3.2 and 2.4 mg/g, respectively. The limonin was easily purified by resin
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
This research was financially supported by grants from the National Natural Science Fund Project of China (31271914) and the Fund for Innovative Research Team of Jimei University (2010A006).
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