*Review* **Ricin: An Ancient Story for a Timeless Plant Toxin**

#### **Letizia Polito** †**,\*, Massimo Bortolotti** †**, Maria Giulia Battelli** †**, Giulia Calafato and Andrea Bolognesi \***

Department of Experimental, Diagnostic and Specialty Medicine—DIMES, General Pathology Section, Alma Mater Studiorum—University of Bologna, Via S. Giacomo 14, 40126 Bologna, Italy; massimo.bortolotti2@unibo.it (M.B.); mariagiulia.battelli@unibo.it (M.G.B.); giulia.calafato2@unibo.it (G.C.)

	- Tel.: +39-051-209-4700 (L.P. & A.B.)

† These authors contribute equally to this work.

Received: 15 April 2019; Accepted: 5 June 2019; Published: 6 June 2019

**Abstract:** The castor plant (*Ricinus communis* L.) has been known since time immemorial in traditional medicine in the pharmacopeia of Mediterranean and eastern ancient cultures. Moreover, it is still used in folk medicine worldwide. Castor bean has been mainly recommended as anti-inflammatory, anthelmintic, anti-bacterial, laxative, abortifacient, for wounds, ulcers, and many other indications. Many cases of human intoxication occurred accidentally or voluntarily with the ingestion of castor seeds or derivatives. Ricinus toxicity depends on several molecules, among them the most important is ricin, a protein belonging to the family of ribosome-inactivating proteins. Ricin is the most studied of this category of proteins and it is also known to the general public, having been used for several biocrimes. This manuscript intends to give the reader an overview of ricin, focusing on the historical path to the current knowledge on this protein. The main steps of ricin research are here reported, with particular regard to its enzymatic activity, structure, and cytotoxicity. Moreover, we discuss ricin toxicity for animals and humans, as well as the relation between bioterrorism and ricin and its impact on environmental toxicity. Ricin has also been used to develop immunotoxins for the elimination of unwanted cells, mainly cancer cells; some of these immunoconjugates gave promising results in clinical trials but also showed critical limitation.

**Keywords:** bioterrorism; cancer therapy; castor bean; folk medicine; immunotoxins; plant toxins; ribosome-inactivating proteins; ricin; rRNA N-glycosylase activity; traditional medicine

**Key Contribution:** Despite the large number of papers on ricin, it is often di fficult for inexpert readers to have a global view on this toxin. This manuscript intends to give an overview of ricin. Starting from the use of Ricinus plant in traditional and folk medicine, we highlight the milestones of research on ricin, with particular regard to its enzymatic activity, structure, cytotoxicity, toxicity for animals and humans and the double face of its employ, for biocrimes and medicine.

#### **1. Castor Bean in Traditional and Folk Medicine**

Ricin derives from *Ricinus communis* L. (Euphorbiaceae family), also known as castor bean or *palma Christi*. The genus Ricinus has only one known species: the castor oil plant. The plant possibly originates from Africa and Asia and now is widespread throughout temperate, subtropical, and tropical areas, growing as an invasive plant or being cultivated for di fferent purposes.

The castor plant has been known since time immemorial and its use in the prehistoric era has been evidenced by archaeological findings such as that of the Border Cave in South Africa. Traces of wax containing ricinoleic and ricinelaidic acids were found on a thin wooden stick, which was suggested to be a poison applicator, dating back to about 24,000 years ago [1]. The castor seeds and other parts of the castor plant were certainly utilized in ancient Egypt for pharmacological purposes. In the Ebers Papyrus, an Egyptian medical treatise dating back to before 1500 BCE, an entire chapter is dedicated to the castor bean that is indicated as an abortifacient, a laxative, a remedy for abscessual illness, baldness, and so on [2]. In the Hearst Papyrus, written approximately in the same period, various castor plant parts are included as ingredients in some prescriptions for internal use, with the aim of expelling fluid accumulation or promoting diuresis, as well as for external use as poultices for bandaging [3]. Ancient Egyptians knew the toxicity of castor bean and the use of seed pulp, included in drug preparations for oral ingestion, was recommended only in small amounts. In addition, a castor seed-containing concoction was prescribed to cure the urinary disease of a possibly diabetic child [4]. Around 400 BCE, the father of western medicine Hippocrates prescribed castor bean oil for laxative and detoxifying action [5]. The Greek herbalist and physician Pedanius Dioscorides (40 to 90 CE) in *De Materia Medica* wrote that castor seeds could be used as expectorant, diuretic, emetic, laxative, anti-inflammatory, to cure erysipelas, burns, varicose veins, etc. [6]. In the same period, Pliny the Elder (23 to 79 CE) wrote *Naturalis historia*, comprising the whole area of antique knowledge. In this encyclopedic work, also castor bean found a place [7].

Castor bean was used also in the pharmacopeia of eastern ancient cultures. In Chinese traditional medicine, castor seeds were recommended for their anthelmintic activity; seed poultice and leaf juice were prescribed for external use to treat ulcers and chronic wounds, whereas the latex was instilled in the ear for rhinitis treatment (reviewed in [8]). In Ayurveda, castor plant is used for rheumatic conditions, as well as for gastropathy, constipation, inflammation, fever, ascites, bronchitis, cough, skin diseases, colic, and lumbago. In Yunani medicine, castor root is used as a purgative and for skin diseases, the leaves are used to increase breastmilk production and are applied to skin for burns, the seeds and the oil act as a purgative, useful in liver troubles, pains, lumbago, boils, piles, ringworm, inflammation, ascites, asthma, rheumatism, dropsy, and amenorrhea (reviewed in [9]). Ground castor seeds or leaf paste have been applied in veterinary medicine to heal sprains, swelling, and wounds [10].

Castor bean has been used in folk medicine throughout the world and has been reported: (i) As a galactogogue on the Mediterranean coasts of Europe, where fresh leaves or leaf juice are applied on the puerperal breast to promote lactation; (ii) as a remedy for various articular, cutaneous, or ocular diseases in Africa, where crushed seeds or oil, sometimes in combination with other plants, are spread or rubbed on the part of the body in need, or a root decoction is drunk to induce uterine contraction as an abortive; (iii) as a medicament to cure erysipelas, flu, inflammation of the womb, and stomach aches in the Caribbean, where a leaf poultice is recommended; (iv) as an anthelmintic or a purgative in Brazil where the seed oil is orally consumed, or locally applied with the purpose of stopping hair loss, healing wounds, or burns (reviewed in [11]).

The laxative and abortifacient activities of castor seeds have been attributed to the activation of intestinal and uterine smooth-muscle cells via prostaglandin EP3 receptors induced by ricinoleic acid [12]. Castor oil-induced diarrhea can be antagonized by hexane extract of *Citrus limon* peel that activates antisecretory and antimotility mechanisms through the β adrenergic system [13]. The purgative and anthelmintic actions of the oral ingestion of castor seeds, at least in part, have been ascribed to the irritating effect caused to the intestine by ricin, as reported in toxicological studies (reviewed in [14]). In addition, the antiflogistic action of castor bean could be related to the high toxicity of ricin to macrophagic cells, which are responsible for producing inflammatory cytokines (reviewed in [15]). This effect, together with the anti-pathogen activity of ricin, could promote healing of the lesions, thus justifying its use in the treatment of various skin conditions.

## **2. The Ricin Story**

Castor seed toxicity began to be investigated at the end of nineteenth century at Schmiedeberg's laboratory in Strasbourg. The toxic component of Ricinus could be extracted with water and precipitated with alcohol, but it lost its toxic activity through heating, treatment with strong acid, or repeated precipitation with alcohol. In 1887, Dixson supposed that the toxicity of Ricinus was due to an albumen-like toxic body [16]. However, it was still unknown whether the seed toxicity was due to a protein or a glycoside (reviewed in [17]). The problem was solved at the Medical Faculty of Dorpat (now Tartu) where an extremely toxic protein was partially purified from castor seed or press cake and named ricin. This finding was published in the doctoral thesis written by Hermann Stillmark under the supervision of Prof. Rudolf Kobert [18]. Stillmark noticed the agglutinating activity of ricin on red blood cells, that had mistakenly been believed to be the cause of ricin toxicity until the agglutinin was separated from the toxin [19].

Paul Ehrlich began his experiments in immunology by feeding mice with small amounts of ricin or abrin, another similar plant toxin, until they were accustomed and became resistant to the toxin used, ye<sup>t</sup> still remaining sensitive to the other toxin. The immunization was strictly specific, started after a few days, and persisted at least for several months [20,21]. He was successful in the production of antisera against abrin and ricin and in the determination of antibody titer in serum and milk. Ehrlich drew animal experiments that clarified the transmission of passive immunity from mother to o ffspring through the transplacental transfer of antibodies and through breastfeeding. He investigated the dynamics of the antibody response and was the first to envisage the presence of binding sites on the cell surface (reviewed in [22]). These studies, together with those on the immunity to bacterial toxins, led him to formulate his side-chain theory of antibody formation and to win, in 1908, the Nobel Prize [23].

**Figure 1.** The main milestones of ricin research.

Interest in ricin was rekindled when the anticancer activity of this toxin on Ehrlich ascites cells in a mouse model was published [24]. A strong inhibition of protein synthesis by ricin was observed in cultures of both Ehrlich ascites tumour cells and Yoshida ascites hepatoma cells. The inhibition of protein synthesis by ricin requires more time in rat liver than in neoplastic cells [25]. The prospect of a

possible use in cancer therapy highlighted the need to investigate which part of the proteosynthetic machinery was damaged and how the toxin managed to enter the cell to reach its target. Hereinafter, we highlight the milestones of research on ricin, with particular regard to its enzymatic activity, structure, cytotoxicity, toxicity for animals and humans, and its use as an immunotoxin, used in experimental models and in clinical trials. The main milestones are shown in Figure 1.
