Citrullus colocynthis (L.) Schrad (bitter apple fruit): a review of its phytochemistry, pharmacology, traditional uses and nutritional potential.

Journal of Ethnopharmacology 155 (2014) 54–66

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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Review

Citrullus colocynthis (L.) Schrad (bitter apple fruit): A review of its phytochemistry, pharmacology, traditional uses and nutritional potential Abdullah I. Hussain a,b,n, Hassaan A. Rathore b, Munavvar Z.A. Sattar b, Shahzad A.S. Chatha a, Satyajit D. Sarker c, Anwar H. Gilani d,e,nn a

Department of Applied Chemistry & Biochemistry, Government College University, Faisalabad, Pakistan School of Pharmaceutical Sciences, University Sains Malaysia, Penang, Malaysia Medicinal Chemistry and Natural Products Research Group, School of Pharmacy and Biomolecular Sciences, Faculty of Science, Liverpool John Moores University, James Parsons Building, Byrom Street, Liverpool L3 3AF, England, UK d Department of Biological and Biomedical Sciences, Aga Khan University Medical College, Karachi 74800, Pakistan e Department of Pharmacy, College of Health Sciences, Mekelle University, PO Box 1871, Mekelle, Ethiopia. b c

art ic l e i nf o

a b s t r a c t

Article history: Received 28 December 2013 Received in revised form 2 June 2014 Accepted 3 June 2014 Available online 14 June 2014

Ethnopharmacological relevance: Citrullus colocynthis (L.) Schrad is a valuable cucurbit plant, widely distributed in the desert areas of the world. Citrullus colocynthis fruits are usually recognized for its wide range of medicinal uses as well as pharmaceutical and nutraceutical potential. This review aims to appraise the published information on the ethnobotanical knowledge, phytochemistry, ethnopharmacology, nutraceutical potential and safety studies of Citrullus colocynthis (bitter apple) fruit, with critical analysis on the gaps and potential for future studies. Material and methods: A literature survey was performed by searching the scientific databases including PubMed, Scopus, SciFinder, Google Scholar, Web of Science, ACS as well as published books. Results: The plant has been reported to possess a wide range of traditional medicinal uses including in diabetes, leprosy, common cold, cough, asthma, bronchitis, jaundice, joint pain, cancer, toothache, wound, mastitis, and in gastrointestinal disorders such as indigestion, constipation, dysentery, gastroenteritis, colic pain and different microbial infections. Several bioactive chemical constituents from fruits were recorded, such as, glycosides, flavonoids, alkaloids, fatty acids and essential oils. The isolation and identification of curcurbitacins A, B, C, D, E, I, J, K, and L and Colocynthosides A, and B were also reported. The fruit of Citrullus colocynthis has been studied extensively for its wide range of biological activities, which include antioxidant, cytotoxic, antidiabetic, antilipidemic, insecticide, antimicrobial and anti-inflammatory. The plant was also shown to be rich in nutritional value with high protein contents and important minerals as well as edible quality of seed oil. Conclusion: It is evident from the literature that Citrullus colocynthis possesses a wide range of medicinal uses and has been well studied for its antidiabetic, anticancer, antioxidant, antimicrobial and anti-inflammatory activities, while its therapeutic potential for gut, airways and cardiovascular disorders remains to be explored. Critical analysis revealed that the plant has the huge potential for pharmaceutical and nutraceutical application, with some indications for the presence of synergistic and /or side effects neutralizing combinations of activities. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Bitter apple Cucurbitacins Antioxidants Antidiabetics Anticancer Medicinal uses

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Botanical descriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Traditional uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

n Corresponding author at: Department of Applied Chemistry & Biochemistry, Government College University Faisalabad, Pakistan. Tel.: þ 92 41 9200037; H/P: þ 92 300 7631058. nn Corresponding author at: Department of Biological and Biomedical Sciences, Aga Khan University Medical College, Karachi-74800, Pakistan. Fax: þ92 21 34934294. E-mail addresses: [email protected], [email protected] (A.I. Hussain), [email protected] (A.H. Gilani).

http://dx.doi.org/10.1016/j.jep.2014.06.011 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

A.I. Hussain et al. / Journal of Ethnopharmacology 155 (2014) 54–66

4.

Phytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Cucurbitacins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Glycosides, phenolic acids and flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Fatty acids and tocopherols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Volatile compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Nutritional properties and potential as functional foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Amino Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. Minerals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Pharmacological Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Antidiabetic activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Antilipidemic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Anti-inflammatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5. Antioxidant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6. Anticancer activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7. Other pharmacological properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Conclusions and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The Cucurbitaceae family is one of the most genetically diverse groups of food plants (Zaini et al., 2011). Plants of this family are generally drought-tolerant, intolerant to wet and poorly drained soils and frost-sensitive. Some well-known members of this family are bitter apple, gourd, cucumber, melon, and pumpkin (Robinson and Decker-Walters, 1997). Due to consumer awareness on the health benefits of cucurbit plants and fruits, their production seems to have increased over the time. Over the last two decades, India and China have been the largest cucurbit producers followed by Russia, United States of America, Egypt and Republic of Iran (Zaini et al., 2011). Citrullus colocynthis (L.) Schrad, a valuable cucurbit plant, widely distributed in the desert areas of the world, including Pakistan, has medicinal and nutraceutical values (Sawaya et al., 1983; Asyaz et al., 2010). The fruit of Citrullus colocynthis is commonly called Colocynth/ Bitter Apple in English, Hanjal in Urdu, Indrayan in Hindi, Kattu Kattuvellari in Malayalam, Anedri in Sanskrit, Rakhal in Bengali, and Pcitummatti in Tamil (Kumar et al., 2009; Amamou et al., 2011). Data shown on the Australian New Crop website clearly demonstrate the increasing trend in number of research papers produced per year on Citrullus colocynthis (Australian New Crops Website, 2009). This plant is a traditional medicine, and a well-known remedy for the treatment of diabetes, jaundice and asthma (Baquar and Tasnif, 1984; Kirtikar et al., 1984; Qureshi et al., 2010). Recently, a number of studies have been conducted on the phytochemistry, toxicology and pharmacology (Salama, 2012; Ali et al., 2013). To date, there is no updated review available, which focuses on all aspects of this valuable fruit. The versatile utility of this plant as a nutritious fruit, folk medicine and as a functional food ingredient has prompted us to compile up to date and scattered data published on this plant in the form of a comprehensive review. This review presents an overview on the ethnobotanical uses along with recent studies concerning the ethnopharmacology, phytochemistry, pharmacological activities and toxicology of the Citrullus colocynthis fruit. 2. Botanical descriptions Citrullus colocynthis (L.) Schrad belongs to the Cucurbitaceae family. The plant is widely available in the Sahara and Arabian

55

56 56 58 59 59 59 59 59 61 61 61 62 62 62 62 63 63 63 63 63 64

deserts, Sudan and Southern part of Asia including Pakistan, India and Southern Islands (Al-Ghaithi et al., 2004; Krishnaraju et al., 2005; Perveen et al., 2007; Gurudeeban et al., 2010; Marzouk et al., 2010b). The fruit was introduced by the Arabs in the middle ages to Spain and Cyprus (Bellakhdar et al., 1991; Wasfi et al., 1995; Abdel-Hassan et al., 2000). Citrullus colocynthis is a perennial herbaceous vine that produces small flowers. The stems are angular, rough and having rough hairs; leaves are alternately arranged on the petioles and rough to touch, 5–10 cm in length, 1.5–2 cm in width, deeply 3–7 lobed; solitary pale yellow blooms (Savithramma et al., 2007; Amamou et al., 2011). Flowers are yellow and seen on the axils of the leaves. It is monecious, single and pedunculated. Each plant produces 15–30 round fruits, about 7–10 cm in diameter, green with undulate yellow stripes, becoming yellow all over when dry. The fruit of Citrullus colocynthis is bitter and globular with smooth texture. It is hard and has a rind around it and contains 200–300 seeds/gourd (Fig. 1). Seeds are small (6 mm in length), ovoid, compressed, smooth and brownish when ripe (Schafferman et al., 1998). Seeds contained about 75% of the weight of fruit. Some physical properties of fruits such as mass of fruit, seed and pulp, volume, thickness of epicarp and mesocarp, seed-fruit ration and density are reported in the literature (Aviara et al., 2007). Generally average mass of Citrullus colocynthis fruit is 506 g and mass of pulp is almost 50% of the mass of fruit, while the seed contents is 71.8 g (Aviara et al., 2007).

3. Traditional uses Citrullus colocynthis is used widely in different parts of the world for the treatment of a number of diseases including diabetes, constipation, leprosy, asthma, bronchitis, jaundice, joint pain, cancer and mastitis (Chopra, 1958; Perveen et al., 2007; Abo et al., 2008; Asyaz et al., 2010). The medicinal uses of this plant have been reported in the indigenous system of medicines in Pakistan, India, China, Africa and Asia, which include its uses in gut disorders such as indigestion, dysentery, gastroenteritis and colic pain as well as common cold, cough, toothache, wounds, and diabetes (Qureshi et al., 2010; Amamou et al., 2011; Gurudeeban et al., 2011).

56


Fig. 1. Citrullus colocynthis (a) Un-ripened fruit, (b) fully ripened fruit and (c) seeds. Sources: (Wikimedia Commons, 2006; ARS, 2009; Advisory Services UVAS, 2009)

In the tropical and subtropical countries, Citrullus colocynthis is traditionally used as an antidiabetic medication (Nmila et al., 2000; Aburjai et al., 2007; Huseini et al., 2009; Rahbar and Nabipour, 2010). In Morocco, it is also used to treat diabetes and hypertension (Ziyyat et al., 1997; Eddouks et al., 2002; Tahraoui et al., 2007). In Pakistan and India, the fruits are used for the treatment of intestinal disorders, bacterial infections, diabetes and cancer in human as well as animals (Sharma, 1998; Katewa and Galav, 2005; Perveen et al., 2007; Asyaz et al., 2010; Sharma et al., 2010). In the United Arab Emirates, Citrullus colocynthis is one of the most popular folk medicines because of its antiinflammatory property (Wasfi et al., 1995). The fruit can stimulate intestinal peristalsis and soften bowel contents by an irritant action on the enteric mucosa (Goldfain et al., 1989; Al-Faraj, 1995; Barth et al., 2002). A decoction of the different parts of this plant is used to cure rheumatic pain and as an anticancer and hepatoprotective agents ( Barth et al., 2002; Daradka et al., 2007; Asyaz et al., 2010). In Tunisia, and other Mediterranean countries, different parts of this plant, especially fruits and seeds, are often used to treat urinary infections (Marzouk et al., 2010a), in addition to treating many other diseases such as, hypertension, rheumatism, dermatological problems and pulmonary and gynecological infections (Marzouk et al., 2009; Marzouk et al., 2010a). In Saudi Arabia, fruits of Citrullus colocynthis are used as a purgative, antirheumatic, anthelmintic, carminative and as a remedy for sore throat and skin infections (Adam et al., 2001). It is a potent and drastic hydragogue and catharsis producer in humans. The fruit is also a blood purifier and remedy for tumors and enlargement of spleen.

The seeds of Citrullus colocynthis are used for the treatment of diabetes, while the leaves are used for the treatment of jaundice and asthma (Baquar and Tasnif, 1984; Kirtikar et al., 1984; Qureshi et al., 2010). Citrullus colocynthis is also well-known in Israel as a source of seed oil and its fruit has been used as a laxative (Schafferman et al., 1998).

4. Phytochemistry Several bioactive compounds of Citrullus colocynthis fruit have been recorded in the literature. They are grouped as glycosides, flavonoids, alkaloids, carbohydrates, fatty acids, and essential oils (Jayaraman et al., 2009; Najafi et al., 2010; Salama, 2012). But there are only a few reports on the isolation, and identification of individual chemical constituents. Cucurbitacins have been reported as the main components of Citrullus colocynthis fruits. 4.1. Cucurbitacins The cucurbitacins are a group of bitter tasting, highly oxygenated, mainly tetracyclic, triterpenic plant substances derived from the cucurbitane skeleton [19-(10-9β)-abeo-10α-lanost-5-en]. They cannot be considered as steroidal since the methyl group from carbon 10 has moved to carbon 9 (Fig. 2a). The cucurbitacins are predominantly found in the Cucurbitaceae family. According to the characteristics of their structures, cucurbitacins are divided into 12 categories, but all are not present in Citrullus colocynthis. Due to the cytotoxic behavior, cucurbitacins appear to play an important


57

Fig. 2. Structure of some compounds (a–m) isolated from Citrullus colocynthis; a, cucurbitane skeleton; b, cucurbitacin E; c, colocynthoside A; d, colocynthoside B; e, cucurbitacin I 2-O-β-D-glucopyranosyl; f, cucurbitacin L 2-O-β-D-glucopyranosyl; g, cucurbitacin J 2-O-β-D-glucopyranosyl; h, cucurbitacin K 2-O-β-D-glucopyranosyl; i, hexanocucurbitacin I 2-O-β-D-glucopyranosyl; j, khekadaengoside E; k, isosaponarin; l, isovitexin; m, isoorientin 30 -O-methyl ether.

role in drug discovery particularly in anticancer drug development (Chen et al., 2005). Among various cucurbitacins, cucurbitacin E (Fig. 2, compound b) was found abundantly in Citrullus colocynthis

fruit pulp (Chen et al., 2005; Ali et al., 2013). Colocynthoside A (Fig. 2c) and colocynthoside B (Fig. 2d), were isolated from the methanolic extract of the fruits grown in Egypt. Other cucurbitacins

58


Table 1 A comprehensive list of the chemical compounds isolated from Citrullus colocynthis fruits, pulps and seeds. Sr. #

Compounds

Part of plant

Reference

1

Cucurbitacins Cucurbitacin E

Whole fruit

Curcubitacin B Curcubitacin I Curcubitacin A Curcubitacin B Curcubitacin C Curcubitacin D Curcubitacin J Curcubitacin K Curcubitacin L Colocynthosides A Colocynthosides B

Whole fruit Whole fruit Fruit Fruit Fruit Fruit Fruit Fruit Fruit Fruit Fruit

(Chen et al., 2005; Tannin-Spitz et al., 2007b; Torkey et al., 2009; Ali et al., 2013) (Tannin-Spitz et al., 2007b; Ali et al., 2013) (Yoshikawa et al., 2007; Ali et al., 2013) (Adam et al., 2001; Yoshikawa et al., 2007) (Adam et al., 2001; Yoshikawa et al., 2007) (Adam et al., 2001) (Adam et al., 2001) (Yoshikawa et al., 2007) (Yoshikawa et al., 2007) (Yoshikawa et al., 2007) (Yoshikawa et al., 2007) (Yoshikawa et al., 2007)

Glycosides, flavonoids and phenolic acids 2-O-β-D-glucopyranosyl- Cucurbitacin I 2-O-β-D-glucopyranosyl-Cucurbitacin L Isosaponarin Isovitexin Isoorientin 3-o-methyl ether Catechin Myricetin Quercetin Kaempferol Gallic acid p-Hydroxy benzoic acid Chlorogenic acid Caffeic acid Vanillic acid p-Coumeric acid Sinapic acid Ferulic acid

Fruit Fruit Fruit, seeds Fruit, seeds Fruit, seeds Fruit pulp Fruit pulp Fruits Fruit pulp Fruit pulp Fruit pulp Fruit pulp Fruit pulp Fruit pulp Fruit pulp Fruit pulp Fruit pulp

(Delazar et al., 2006) (Delazar et al., 2006) (Delazar et al., 2006; Gurudeeban (Delazar et al., 2006; Gurudeeban (Delazar et al., 2006; Gurudeeban (Hussain et al., 2013) (Hussain et al., 2013) (Meena and Patni, 2008 ; Hussain (Hussain et al., 2013) (Hussain et al., 2013) (Hussain et al., 2013) (Hussain et al., 2013) (Hussain et al., 2013) (Hussain et al., 2013) (Hussain et al., 2013) (Hussain et al., 2013) (Hussain et al., 2013)

Whole fruit, pulp Fruit pulp

(Najafi et al., 2010; Mukherjee and Patil, 2012; Ali et al., 2013) (Sayed et al., 1973)

2

5

Alkaloids Choline

6

7

Volatiles/Terpenoids 4-(1-methyl) ethoxy-1-butanol; 5-methoxy, 2- methyl, 2-pentanol; 1-cyclopentyl, 2-propene-1-ol Fruit pulp and 2- furanmethanol, tetrahydro-5-methyl- cis and trans isomers, 3, 4-dimethyl, 2-hexanone; 2- methyl, 4-heptanone and 3-Methyl, 2-heptanone, 1-propoxy pentane and 2, 3-epoxy methyl propionate Fatty acids Palmitic acid, stearic acid, linoleic acid, oleic acids Myristic, palmitic, stearic, oleic, linoleic and linolenic acids Tocopherols and Carotenes α-tocopherol, γ-tocopherol, β-carotene

isolated from the butanol fraction were cucurbitacin I 2-O-β-D glucopyranoside (Fig. 2e), cucurbitacin L 2-O-β-D glucopyranoside (Fig. 2f), cucurbitacin J 2-O-β-D glucopyranoside (Fig. 2g), cucurbitacin K 2-O-β-D glucopyranoside (Fig. 2h), hexanocucurbitacin I 2O-β-D glucopyranoside (Fig. 2i), and khekadaengoside E (Fig. 2j), (Dahmén and Leander, 1976; Hatam et al., 1989; Kanchanapoom et al., 2002; Yoshikawa et al., 2007; Kumar et al., 2009). In another study (Torkey et al., 2009), 2-O-β-D-glucopyranosylcucurbitacin E was detected as one of the major cucurbitacins of Citrullus colocynthis fruits. Adams et al. (2001) also reported the cucurbitacins A, B, C, D and E from the fruits. Cucurbitacin E 2-O-β-D-glucopyranoside was isolated as a principal component from the EtOAc-soluble fraction of Citrullus colocynthis fruit, together with 3,4-dihydroxypropiophenone, and 3,4´-dihydroxy30 -methoxypropiophenone. From the n-BuOH-soluble fraction, colocynthosides A and B were isolated together with cucurbitacin I 2-O- βD-glucopyranoside, cucurbitacin J 2-O-β-D-glucopyranoside, cucurbitacin K 2-O-β-D-glucopyranoside, cucurbitacin L 2-O-β-D-glucopyranoside, khekadaengoside hexanocucurbitacin I 2-O-β-D-glucopyranoside,

Fruit seed oils

et al., 2010) et al., 2010) et al., 2010)

et al., 2013)

(Gurudeeban et al., 2011)

Seeds

(Sawaya et al., 1983; Sadou et al., 2007; Sebbagh et al., 2009) (Gurudeeban et al., 2010)

Fruit seed oil

(Kalhoro et al., 2002)

isovitexin, isoorientin 30 -methyl ether, isosaponarin, 4-(β-D-glucopyranosyloxy) benzaldehyde, 4-hydroxybenzyl β-D-glucopyranoside, benzyl β -D-glucopyranoside, and 4-(β –D-glucopyranosyloxy) benzyl alcohol (Yoshikawa et al., 2007). 4.2. Glycosides, phenolic acids and flavonoids Three flavonoid glycosides e.g. isosaponarin (Fig. 2k), isovitexin (Fig. 2l) and isoorientin 30 -O-methyl ether (Fig. 2m) and two cucurbitacin glucosides e.g. 2-O-β-D-glucopyranosylcucurbitacin I and 2-O-β-D-glucopyranosylcucurbitacin L were identified from the butanol fraction of the methanol extract of Citrullus colocynthis fruits (Delazar et al., 2006). Polyphenols are a class of natural compounds that exhibit antioxidant activity and act as free-radical terminators. Flavonoids are the secondary metabolites that exhibit antioxidant and radical-scavenging activities (Shahidi and Wanasundara, 1992). Preliminary phytochemical screening of the Citrullus colocynthis fruit showed the presence of phenolics and flavonoids. Subsequent quantification showed the 3.07 mg/g dry


material total phenolics (measured as gallic acid) and 0.51 mg/g dry material total flavonoids (measured as catechin equivalents) (Hussain et al., 2013). Eight phenolic acids including gallic acid, chlorogenic acid, p-hydroxy-benzoic acid, caffeic acid, vanillic acid, p-coumeric acid, sinapic acid and ferulic acid, and four flavonoids including catechin, myricetin, quercetin and kaempferol were detected in the ethanol extract of Citrullus colocynthis fruits (Hussain et al., 2013). Meena and Patni (2008) reported quercetin in the Citrullus colocynthis fruit. 4.3. Fatty acids and tocopherols The major fatty acids of Citrullus colocynthis seed oil from different countries are summarized in Table 1. Palmitic (8.1–17.3%) and stearic acids (6.1–10.5%) constitute the two principal saturated fatty acids of this oil (Sawaya et al., 1983; Sadou et al., 2007; Sebbagh et al., 2009). Linoleic and oleic acids are the principal mono-unsaturated fatty acids (Table 1), and this high content of linoleic acid (50.6–60.1%) in seed oil, which is an essential fatty acid, makes this oil medicinally valuable. The fatty acid profile of the seed oil reveals that it falls in the category of linoleic-oleic acid oils and is analogous to several other vegetable oils (Sawaya et al., 1983; Sadou et al., 2007; Sebbagh et al., 2009). Therefore, the Citrullus colocynthis oil, like some other cucurbit seed oils, is likely to have a potential uses as a cooking oil. Schafferman et al. (1998) reported that the seed oil composition of this plant was similar to that of safflower oil, with a total of 80–85% unsaturated fatty acids. A study reporting the physico-chemical characterization and the fatty acid composition of the fixed oil of the seeds revealed that it is a good source of natural antioxidants like α-tocopherol, γ-tocopherol and β-carotene with respective composition of 45.1, 435 and 0.18 mg/kg (Table 3) (Kalhoro et al., 2002). 4.4. Alkaloids Many studies reported the presence of alkaloids in the Citrullus coloccynthis fruits, but only a few reports are available on the isolation and identification of individual alkaloids (Sayed et al., 1973; Najafi et al., 2010; Mukherjee and Patil, 2012; Ali et al., 2013). Sayed et al., 1973 isolated choline and two unidentified alkaloids from fruit pulp of Citrullus colocynthis. 4.5. Volatile compounds Table 1 presents the chemical composition of volatiles from the pulp of Citrullus colocynthis fruit (Gurudeeban et al., 2011). Seventeen volatile compounds from the fruit pulp were identified, ranging from 0.51% to 48.0% of peak area (Gurudeeban et al., 2011). The alcohols identified were 4-(1-methyl) ethoxy-1-butanol; 5-methoxy, 2- methyl, 2-pentanol; 1-cyclopentyl, 2-propene1-ol and 2-furanmethanol, tetrahydro-5-methyl- cis and trans isomers. The ketones characterized were 3, 4-dimethyl, 2-hexanone; 2-methyl, 4-heptanone and 3-methyl, 2-heptanone. Two epoxy compounds (1-propoxy pentane and 2, 3-epoxy methyl propionate) were identified (Gurudeeban et al., 2011). Four hydrocarbons (tridecane, tetradecane, pentadecane and hexadecane) were detected on the surface of the fruit in small quantities. The ketones characterized were 3, 4-dimethyl, 2-hexanone; 2- methyl- 4-heptanone and 3-methyl- 2-heptanone. Two epoxy compounds were 1-propoxy pentane and 2, 3-epoxy methyl propionate and palmitic acid. Four hydrocarbons might have been present on the surface the fruit in minimum quantities viz., tridecane, tetradecane, pentadecane and hexadecane. The two remaining compounds - one (viz., trimethyl silyl methanol) impurity component derived from silicone oil used in the isolation process and another impurity (viz., 1, 2- benzenedi carboxylic

59

acid, di-iso octylester) was stabilizer for plastics. Most of these compounds must have been derived by fatty acid pathway (Gurudeeban et al., 2011).

5. Nutritional properties and potential as functional foods The nutritional data are the key parameters in defining the quality of a food. Besides the medicinal uses, Citrullus colocynthis fruit is also employed as food for animals and humans (Sadou et al., 2007). For over a decade, the scientists of the United States Department of Agriculture (USDA), have investigated the nutritional and functional properties of the seeds of this plant, and concluded that they have the potential to find a place in the food industry (National Research Council, 2006). 5.1. Nutrients In spite of the widespread importance of Citrullus colocynthis as food, little nutritional detail is readily available to an international readership. The proximate composition (moisture, ash, protein and fat) of the seeds and the fruits from different countries is presented in Table 2. Some variations in the values might be due to different agro-ecological conditions and agricultural practices which differ for each country. The seed kernels contain about 50% oil, 30% protein, 10% carbohydrate, 4% ash, and 3% fiber (National Research Council, 2006). Moisture contents of the mature fruit are quite high accounting for more than 90% of edible weight portion (Aviara et al., 2007). In another report, moisture contents of bitter apple seeds were found to be 4.91 g/100 g and the amount of seed protein and ash were 13.19 and 2.00 g/100 g, respectively (Sadou et al., 2007). The seeds have been investigated as a possible source of medicinal/edible oil. The yield of oil extracted from Citrullus colocynthis whole seeds was found to be 26.60 g/100 g and oil content from seed kernel was found to be 56.5 g/100 g (Sawaya et al., 1986; Sadou et al., 2007). This oil yield is comparable to the other crops such as safflower and sunflower and is rather higher than that of soybean and cotton (Swern, 1979). In one study from Pakistan, 19% oil yield was reported (Sawaya et al., 1986), whereas, higher oil yield (36%) was reported from the seeds obtained from India (Singh and Yadava, 1978). It has been reported in the literature that the oil extracted from Citrullus colocynthis seeds is potentially suitable for human and for animal consumption (Sawaya et al., 1983). Freshly extracted oil was found to be dark yellow in color with a greenish tint and had a mild flavor and odor (Sawaya et al., 1983). Apart from their application as a source of oil, Citrullus colocynthis seeds, also contained approximately 13% protein by Table 2 Proximate composition and mineral contents of Citrullus colocynthis seeds. Parameters

(Sadou et al., 2007) (Sawaya et al., 1986) (Sawaya et al., 1983)

Proximate composition (g/100 g) Moisture 4.91 Ash 2.00 Protein 13.19 Fat 18.59 Mineral composition (mg/100 g) Calcium 569 Copper 5.1 Iron 11.6 Magnesium 210 Phosphorous 30.0 Potassium 465 Sodium 11.9 Zinc 1.1

13.5 2.1 – 26.6

28.7 4.5 – 56.5

13 0.3 3.3 50 119 322 79 1.4

28 0.6 7.0 106 253 684 168 3.0

60


Table 3 Pharmacological effects of Citrullus colocynthis fruits. Pharmacological effects

Extracts/ compounds

Dose/concentration

Results In vitro/ in vivo

Reference

Antidiabetic

Petroleum ether extract of fruit Hydro-ethanol extract of fruit

Dose: 300 mg/kg

in vivo

(Jayaraman et al., 2009)

Dose: 300 g/kg

in

(Dallak, 2011)

Aqueous extract of fruits Alkaloid rich fraction of fruit Glycosides rich fraction of fruit Saponin rich fraction of fruit Saponin rich fraction

300 mg/kg

in

50 mg/kg

in

50 mg/kg

in

50 mg/kg

in

20 mg/kg

in

Fruit powder

100 mg capsule/ three time a day

in

Aqueous extract of seeds

300 mg/kg

in

Seed powder

300 mg seed powder/day

in

Aqueous ethanol extract

1.2 g/kg/day

in

Water extract, ethanol extract Ethanol extract of fruit Ethanol extract of fruit Ethanol extract of fruit Cucurbitacin E

5 mg/mL

in

500 mg/mL

500 mg/mL

Cucurbitacin E

500 mg/mL

Cucurbitacin E

500 mg/mL

Cucurbitacin B

500 mg/mL

Cucurbitacin B

500 mg/mL

Cucurbitacin B

500 mg/mL

Cucurbitacin I

500 mg/mL

Cucurbitacin I

500 mg/mL

Cucurbitacin I

500 mg/mL

Water extract

4 mg/kg

Aqueous ethanol extract gel (3%) Hydro-ethanol extract of fruits

2 g of gel

in vitro 18 mm Inhibition zone against Staphylococcus aureus, while amoxyciline showed 22 mm @ 200 mg/mL in vitro 15 mm inhibition zone against Bacillus cereus while amoxyciline showed 18 mm @ 200 mg/mL in vitro 9 mm inhibition zone against Klebsiella pneumonia while amoxyciline showed 10 mm @ 200 mg/mL in vitro 18 mm inhibition zone against Staphylococcus aureus while amoxyciline showed 20 mm @ 100 mg/mL in vitro 9 mm inhibition zone against Klebsiella pneumonia while amoxyciline showed 10 mm @ 100 mg/mL in vitro 16 mm inhibition zone against Bacillus cereus while amoxyciline showed 15 mm @ 100 mg/mL in vitro 16 mm inhibition zone against Staphylococcus aureus while amoxyciline showed 20 mm @ 100 mg/mL in vitro 8 mm inhibition zone against Klebsiella pneumonia while amoxyciline showed 10 mm @ 100 mg/mL in vitro 10 mm inhibition zone against Bacillus cereus while amoxyciline showed 15 mm @ 100 mg/mL in vitro 16 mm inhibition zone against Staphylococcus aureus while amoxyciline showed 20 mm @ 100 mg/mL in vitro 8 mm inhibition zone against Klebsiella pneumonia while amoxyciline showed 10 mm @ 100 mg/mL in vitro 10 mm inhibition zone against Bacillus cereus while amoxyciline showed 15 mm @ 200 mg/mL in vivo Model: Carrageenan-induced paw edema assay in rats. Results: After 6 h, 97.29% inhibition in swelling was observed. in vitro Model: Carrageenan-induced paw edema assay in rats Results: 45% reduction of edema in vivo Model: Alloxan induced diabetic rats Markers: lipid peroxidation level; Results: Significant antioxidant protective effect on the liver of treated rats; increase in the enzymatic and non enzymatic components of the oxidative system in the liver in vitro 88% DPPH radical scavenging activity was found against ascorbic acid (89.5% @ 50 μg/mL) in vitro 61.4% Nitric oxide scavenging activity against ascorbic acid (86.0% @ 50 μg/mL) in vitro DPPH radical scavenging activity in term of IC50 of aqueous extract of Citrullus colocynthis seeds was tested in vitro ABTS radical scavenging properties

Hypolipidemic

Antimicrobial

Antiinflammatory

Antioxidant

Methanol extract Methanol extract Aqueous extract Cucurbitacin glycosides combination

500 mg/mL 500 mg/mL

300 g/kg

Concentration 2500 μg/mL Concentration 2500 μg/mL IC50 ¼0.021 mg/mL IC

50

145 μM

Gradual, time dependant reduction in blood glucose levels in STZ induced diabetic rats vivo Significant decrease in the levels of total cholesterol, triglycerides, free fatty acids and phospholipids in serum and liver of treated diabetic rats vivo Decrease of blood glucose from 132 to 93 mg/100 mL after 24 h of normoglycaemic rabbits vivo Decrease of blood glucose from 132 to 120 mg/100 mL after 6 h of normoglycaemic rabbits vivo Decrease of blood glucose from 132 to 89 mg/100 mL after 6 h of normoglycaemic rabbits vivo Decrease of blood glucose from 132 to 84 mg/100 mL after 6 h of normoglycaemic rabbits vivo Decrease of blood glucose from 332 to 198 mg/100 mL after 24 h of Alloxan- induced diabetic rabbits; Decrease from 340 to 310 mg/100 mL in control (normal saline) group vivo Clinical trial for 2 months on 50 type II diabetic patients; significant decrease in HbA1c and fasting blood glucose levels were observed vivo Model: streptozotocin (STZ)-induced diabetic rats; Study time: 2 weeks; Response: Reduced the plasma level of AST and LDH significantly. vivo Clinical trial on patients; significant reduction in the triglyceride and cholesterol concentration in non-diabetic hyperlipidemic patients vivo Model: Hyperlipidaemic Rabbits; Results: Serum cholesterol levels dropped from 940.7 to 230.41 (75.55%) and further to 119.2 (87.32%); phospholipids and triglycerides levels were also reduced vitro Inhibition against Staphylococcus aureus

(Abdel-Hassan et al., 2000) (Abdel-Hassan et al., 2000) (Abdel-Hassan et al., 2000) (Abdel-Hassan et al., 2000) (Abdel-Hassan et al., 2000)

(Huseini et al., 2009)

(Al-Ghaithi et al., 2004)

(Rahbar and Nabipour, 2010)

(Daradka et al., 2007)

(Najafi et al., 2010) (Ali et al., 2013) (Ali et al., 2013) (Ali et al., 2013) (Ali et al., 2013) (Ali et al., 2013) (Ali et al., 2013) (Ali et al., 2013) (Ali et al., 2013) (Ali et al., 2013) (Ali et al., 2013) (Ali et al., 2013) (Ali et al., 2013) (Marzouk et al., 2010a) (Aly and Naddaf, 2006). (Dallak, 2011)

(Kumar et al., 2008) (Kumar et al., 2008) (Marzouk et al., 2010b) (Tannin-Spitz et al., 2007a)


61

Table 3 (continued ) Pharmacological effects

Extracts/ compounds

Dose/concentration

Results In vitro/ in vivo

Reference

IC50 ¼ 7.14 μg/mL

in vitro DPPH radical scavenging activity in term of was tested

(Hussain et al., 2013)

Concentration: 5 mg/100 mL 20 μM

in vitro 76.5% inhibition of linoleic acids peroxidation was observed

(Hussain et al., 2013)

Cytotoxic

Ethanol extract of fruits Ethanol extract of fruits Cucurbitacin B, Cucurbitacin E (1:1) Alkaloid rich fraction

in vitro ER þ MCF-7 and ER-MDA-MB-231 human breast cancer cell lines

(Tannin-Spitz et al., 2007b)

in vitro The alkaloid rich fraction of Citrullus colocynthis fruit demonstrated strong cytotoxicity towards Artemia salina naupli in vitro Against human cancerous cells (MCF-7 and HepG-2)

(Mukherjee and Patil, 2012)

IC50 3.30 μg/mL

IC50 against MCFAlkaloid rich fraction of Citrullus 7 ¼17.20 μg/mL; colocynthis fruits against HepG2 ¼12.54 μg/mL Cucurbitacin B 1–100 μM isolated from fruits

Insecticidal

Antiallergic

Cucurbitacin B isolated from fruits n-hexane extract Methylenechloride extract Chloroform extract Ethanol extract Methanol extract

LD50 ¼ 0.1 μM LC50 23,065 ppm LC50 19,497 ppm LC50 17,328 ppm LC50 11,003 ppm Dose 200 mg/kg

Cucurbitacin E 2-O- Dose 200 mg/kg β-Dglucopyranoside

(Mukherjee and Patil, 2012)

in vitro Cucurbitacin B inhibited cellular proliferation of human laryngeal cancer cell line (Hep-2); Minimum cell viability ¼was 28% at 100 μM concentration in vitro Tested on human GBM cell lines in liquid culture, Cucurbitacin B inhibited 50% growth at 0.1 μM in vitro Against Aphis craccivora in vitro Against Aphis craccivora

Liu et al., 2008b

in vitro Against Aphis craccivora in vitro Against Aphis craccivora in vivo 72.5% inhibition in ear passive cutaneous anaphylaxis (PCA) reaction in mice in vivo 49.7% inhibition in ear passive cutaneous anaphylaxis (PCA) reaction in mice

(Torkey et al., 2009) (Torkey et al., 2009) (Yoshikawa et al., 2007)

weight of decorticated seeds that have a adequate amino acids profile (Sadou et al., 2007). The protein contents compares favorably with that in the most renowned legume seeds (National Research Council, 2006). The exceptional level of essential amino acids makes the protein composition special. Citrullus colocynthis seed meal after partially removal of oil is used to make patties that serve as a meat substitute. The undefatted meal is used in several dietary preparations that vary with the food habits of the people. Whole Citrullus colocynthis seeds are also consumed as snack after dry roast.

5.2. Amino Acids Citrullus colocynthis is an excellent source of different amino acids as arginine, methionine, and tryptophan (National Research Council, 2006). The biological indices of its protein quality have been described as: “lower than soybean but comparable to or higher than most oilseeds.” Nutritionally, the limiting amino acids are lysine and threonine (National Research Council, 2006). Sawaya et al. (1986) reported the amino acid composition of Citrullus colocynthis protein and compared it with other oilseed proteins. Glutamic acid and arginine were the main amino acids identified with concentrations 19.8 and 15.9 g of amino acid/100 g of protein, respectively. Other major (4 5 g of amino acid /100 g of protein) amino acids found were aspartic acid, serine, glycine, alanine, leucine and phenylalanine. Only a few nutritional trials have been conducted to date. However, significant growth improvement was reported when Citrullus colocynthis flour supplemented in the West African diets. Moreover, the literature reports suggest that its seeds have the potential for fortifying both traditional and modern food formulations (National Research Council, 2006).

Yin et al., 2008 (Torkey et al., 2009) (Torkey et al., 2009)

(Yoshikawa et al., 2007)

5.3. Minerals Minerals are the essential nutrients that are required by the body for carrying out normal functions. Citrullus colocynthis fruits and seeds contain several micronutrients (vitamin and minerals) that could significantly contribute to the diet. The potential for Citrullus colocynthis seed as a source of calcium and niacin is encouraging to the low milk-consuming regions of lower West Africa. The minerals contents of seeds are summarized in Table 2. Potassium (K þ ) and Calcium (Ca þ þ ) are the major minerals present in the seeds, with concentrations of 569 mg and 465 mg/ 100 g of seeds, respectively. The seed is also a rich source of magnesium and phosphorous. Iron (Fe) and zinc (Zn) contents are at the lowest level. Both of these minerals elements (K þ and Ca þ þ ) are well known to play beneficial role in maintaining electrolytic balance of body fluid as well contribute to alkalinizing the body (Sadou et al., 2007; Zaini et al., 2011). In another report, the major mineral component from Citrullus colocynthis was found to be phosphorus, potassium, magnesium, manganese, sulfur, calcium, iron, and zinc (National Research Council, 2006).

6. Pharmacological Properties The traditional medicinal applications of Citrullus colocynthis have inspired many pharmacological investigations. Several extracts and isolated compounds have been evaluated for their biological activities, especially anticancer and antidiabetic activities. There seems to be an interest in developing new anticancer/ antitumor drugs from Citrullus colocynthis due to its high contents of cucurbitacins. Table 3 lists the available pharmacological studies with detailed study methods and conditions.

62


6.1. Antidiabetic activity Diabetes mellitus is one of the fastest growing metabolic diseases. The treatment is symptomatic and requires life-long use of chemical drugs, which produce multiple side-effects in addition to high cost, hence, the search for more effective and safer anti-diabetic continues. Citrullus colocynthis has been widely used as antidiabetic in different countries (Al-Ghaithi et al., 2004) and interestingly attracted a large number of studies both on animals and humans. The oral administration of the aqueous extract could ameliorate some of the toxic effects of streptozotocin and blood glucose level (Abdel-Hassan et al., 2000; Al-Ghaithi et al., 2004). Literature also revealed the insulinotropic effect of the fruit extracts (Nmila et al., 2000; Bnouham et al., 2006). Administration of different extracts from the seeds significantly induced insulin secretion in vitro in the isolated rat pancreas and isolated rat islets in the presence of 8.3 mM glucose. Oral administration of the aqueous extract (300 mg/kg) caused a significant reduction in glycemia in normoglycaemic rabbits. Oral administration of components (tertiary and quaternary alkaloids, glycosides and saponins) isolated from the Citrullus colocynthis fruits was tested in normoglycaemic rabbits at a dose of 50 mg/kg. The alkaloids did not show any hypoglycaemic effect. In contrast, the glycosidic component significantly decreased glycemia. The saponin component reduced glycemia at much lower doses (10–20 mg/kg) in alloxan-induced diabetic rabbits indicating that the saponin and glycosides components could be responsible for the glucose lowering effect of the rind of Citrullus colocynthis (Bnouham et al., 2006). Citrullus colocynthis fruit is widely used by herbalists for the treatment of diabetes in Iran (Huseini et al., 2009). A clinical trial using the Citrullus colocynthis fruit for 2 months was conducted to determine its efficacy in 50 type II diabetic patients. Two groups of 25 each under standard anti-diabetic therapy, received 100 mg Citrullus colocynthis fruit capsules or placebos three times a day, respectively. The patients were visited monthly and glycosylated hemoglobin (HbA1c), fasting blood glucose, total cholesterol, low density lipoprotein (LDL), high density lipoprotein (HDL), triglyceride, aspartate transaminase, alanine transaminase, alkaline phosphatase, urea and creatinine levels were determined at the beginning and after 2 months of treatment. The results showed a significant decrease in HbA1c and fasting blood glucose levels in Citrullus colocynthis treated patients. Other serological parameters levels in both groups did not change significantly. No notable gastrointestinal side effect was observed in either group (Huseini et al., 2009). In UAE, Citrullus colocynthis is also used as an antidiabetic remedy. The effect of the aqueous extracts from the seeds on the biochemical parameters of normal and streptozotocin (STZ)induced diabetic rats was determined (Al-Ghaithi et al., 2004). Diabetes mellitus was induced by a single intraperitoneal (60 mg/ kg body wt) injection of STZ. Oral administration of the plant extract (300 mg/kg, daily for 2 weeks) reduced the plasma level of AST and LDH significantly, while it failed to reduce the increased blood level of GGT and ALP in diabetic rats (Al-Ghaithi et al., 2004). The differential effects of diets enriched with Citrullus colocynthis, sunflower or olive oils on the pancreatic M-cell mass were studied in streptozotocin (STZ)-induced diabetes in rats (TanninSpitz et al., 2007a). STZ injection induced rapid hyperglycemia in all animals. However, 2 months later, hyperglycemia was significantly less pronounced in the rats fed a Citrullus colocynthis oilenriched diet compared with other rat groups. Assessment of insulin sensitivity using the homeostasis model assessment method also indicated less insulin resistance in the rats fed on a Citrullus colocynthis oil-enriched diet than the other rats. Two months after STZ injection, the pancreatic M-cell mass was similar

in both STZ-treated rats fed the colocynth oil-enriched diet and their controls fed the same diet. In contrast, the pancreatic M-cell mass remained lower in the STZ-induced diabetic rats fed with olive oil- and sunflower oil-enriched diets compared with the Citrullus colocynthis group. Citrullus colocynthis oil supplementation may have a beneficial effect by partly preserving or restoring pancreatic M-cell mass in the STZ-induced diabetes rat model (Sebbagh et al., 2009). The effect of hydro-ethanolic extract of Citrullus colocynthis pulp was studied on alloxan-induced hyperlipidemia in diabetic rats and the results showed significant reduction in the levels of total cholesterol, triglycerides, free fatty acids and phospholipids in serum and liver of diabetic rats treated with Citrullus colocynthis when compared to diabetic untreated rats (Dallak, 2011). All these reports supported the traditional use of Citrullus colocynthis in diabetes and suggested it to be a safe antidiabetic. 6.2. Antilipidemic activity The lipid lowering effect of Citrullus colocynthis was studied both in the animal model of hyperlipidemia and hyperlipidemic human subjects. The ethanol extract of the plant (1.2 g/kg/day) brought the serum cholesterol level to normal value in hyperlipidemic rabbits (Daradka et al., 2007). When studied for its lipid lowering effect on hyperlipidemic non-diabetic patients, a daily intake of 300 mg of powdered seeds of Citrullus colocynthis lowered the triglyceride and cholesterol concentration (Rahbar and Nabipour, 2010). 6.3. Antimicrobial activity Results from different studies demonstrated antimicrobial and anticandidal activities of Citrullus colocynthis extracts and the antimicrobial effect varied from population to population (Marzouk et al., 2009; Maezouk et al., 2010a, 2010b). The Citrullus colocynthis extracts were active against both Gram-positive and Gram-negative bacterial strains (Marzouk et al., 2010a, 2010b). Study from Pakistan (Memon et al., 2003) displayed inhibitory effect of the ethanol extract of Citrullus colocynthis fruit against Bacillus subtilis. The response against Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) was poor. Active response of the fruit extracts against the tested strains of bacteria might also be due to bioactive like flavonoids, glycosides and tannin (Memon et al., 2003). Antibacterial activity of Citrullus colocynthis extracts prepared in various solvents was studied against some pathogenic bacteria i.e. Bacillus cents, Salmonella typhimurium, Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Mycobacterium smegmatis and, Staphylococcus epidermidis (Sharma et al., 2010). Most of the extracts exhibited moderate MIC within the range of 20–100 μg/mL against all the bacterial pathogens. In another study from Iran, the antibacterial activity of Citrullus colocynthis fruits extracts (water and ethanolic) against standard (ATCC 25923) and hospital isolated strains of Staphylococcus aureus from novobiocin treatment patients was evaluated (Najafi et al., 2010). The results indicated that the inhibitory effects of both Citrullus colocynthis fruit extracts were comparable with that of standard antibiotic, novobiocin. 6.4. Anti-inflammatory activity The non-steroidal anti-inflammatory drugs (NSAIDs) used in modern therapeutics are known to cause gastrointestinal tract ulceration (Graham et al., 1988). The NSAIDS with selective COX-2 inhibitory action are, however, considered to be less ulcerogenic but they have been reported to cause fatal cardiac toxicity based on which, some of


them have been withdrawn from the market. Therefore, there is an increasing need for safer anti-inflammatory drugs. Citrullus colocynthis is one of the main plants used in folk medicine due to its antiinflammatory property (Wasfi et al., 1995; Marzouk et al., 2010a). The aqueous extract (4 mg/kg) of the fruit was screened for antiinflammatory activity using the carrageenan-induced paw edema assay in rats (Marzouk et al., 2010a) thus validated the medicinal use of this plant in rheumatoid arthritis, and as an analgesic and antiinflammatory agent. In another study, the in vivo anti-inflammatory activity of the different types of Citrullus colocynthis extracts was reported using the carrageenan-induced paw edema model in albino rats (Aly and Naddaf, 2006). The anti-inflammatory study on the crude extract of immature fruit from South Tunisia using the carrageenaninduced paw edema assay in rats showed anti-inflammatory potential of this plant (Marzouk et al., 2011). Though not yet studied for its antiulcer activity, plants are known to be rich in antiulcerogenic activity (Zaidi et al., 2012), and it is possible that Citrullus colocynthis is free from ulcerogenic activity and offers safer and effective anti-inflammatory option.

63

the inhibitory rates on laryngeal squamous carcinoma xenograft model were 32.43%, 43.24% and 70.27% for lower, moderate and higher dosage group, respectively. Cucurbitacin B inhibited cell proliferation and induced apoptosis of Hep-2 cells by suppressing STAT3 signal pathway, down regulating the expression of cyclin B1 and Bcl-2 proteins (Liu et al., 2008b). Glioblastoma Multiforme (GBM) is almost inevitably a fatal tumour of the brain and it is the most frequent brain tumour in adults (Yin et al., 2008). When tested on human GBM cell lines in liquid culture, cucurbitacin B inhibited 50% growth at 0.1 μ M (ED50). Soft-gel assays demonstrated that nearly all of the GBM clonogenic cells were inhibited at 10 8 M of cucurbitacin B indicating that this drug might be a good candidate for clinical trial (Yin et al., 2008). Cucurbitacin E has also received considerable attention because of its cytotoxic and anticancer effects. Cucurbitacin L was also cytotoxic against KB and HeLa cell lines, but was less potent than cucurbitacin I, which was isolated from Citrullus colocynthis (Lavie et al., 1964; Cassady and Suffness, 1990; Chen et al., 2005). 6.7. Other pharmacological properties

6.5. Antioxidant activity The therapeutic effects of several medicinal and food plants, used in traditional medicine, are usually attributed to their polyphenolic compounds (Shahidi and Wanasundara, 1992; Hussain et al., 2008; Hussain et al., 2013). Citrullus colocynthis extracts are rich source of antioxidants (e.g. polyphenol and plant sterol) (Sebbagh et al., 2009). Some in vitro studies conducted on the fruit extracts revealed its excellent antioxidant potential (Kumar et al., 2008). Free-radical scavenging effect of its fruit increases with increasing extract concentration and maximum antioxidant activity was observed with 2.5 g/mL (Kumar et al., 2008). The antioxidant activity of three flavonoids (isosaponarin, isovitexin and isoorientin 3-O-methyl ether), isolated from Citrullus colocynthis was determined by the 2, 2-diphenyl-1picrylhydrazyl (DPPH) assay and the RC50 (concentration that scavenging 50% DPPH radical) values were found in the range of 5.62 10 4 to 7.13 10 2 mg/mL. Whereas, the RC50 value of the positive control, quercetin, was 2.78 10 5 mg/mL in that study (Kumar et al., 2008). Marzouk et al. (2010b) reported that the DPPH radical scavenging activity of the aqueous extract of Citrullus colocynthis seeds collected from Tunisia was very potent with IC50 of 0.021 mg/mL. Cucurbitacin glycoside from Citrullus colocynthis exhibited ABTS radical scavenging properties (IC50, 145 μM), probably through the involvement of a direct scavenging effect on several free-radicals (Tannin-Spitz et al., 2007a). The results strongly supported the use of Citrullus colocynthis as a source of natural antioxidant agents.

In view of its medicinal use as a hair tonic in Ayurveda, the effect of Citrullus colocynthis on hair growth was studied in albino rats (Roy et al., 2007). The results indicated that hair growth initiation time was reduced to half on treatment with the petroleum ether extract compared with untreated control animals. Moreover, the treatment was successful in bringing a greater number of hair follicles (470%) to anagenic phase than the standard drug, minoxidil (67%). The antiallergic constituent on the cucurbaticin E isolated from methanolic fruit extract of Citrullus colocynthis showed an inhibitory effect on ear passive cutaneous anaphylaxis as a type I allergic model in mice. The Cucurbitacin E and its aglycone exhibited anti-allergic activity at a dose of 100 and 125 mg/kg, p.o., respectively (Yoshikawa et al., 2007). Insecticidal activity of hexane, methylenechloride, chloroform and ethanol extracts of Citrullus colocynthis fruits against Aphis craccivora was observed (Torkey et al., 2009). The LD50 for respective extracts were 23,065, 19,497, 17,328 and 11,033 ppm. In another study, different Citrullus colocynthis extracts showed larvicidal effects against mosquito larvae (Rahuman et al., 2008). In that study, bioassay-guided fractionation of petroleum ether extract led to the separation and identification of fatty acids; oleic acid and linoleic acid were identified as mosquito larvicidal compounds, which were potent against fourth insect's larvae of Aedes aegypti, Anopheles stephensi Liston, and Culex quinquefasciatus. The results of this study clearly showed that the extract and fraction of Citrullus colocynthis that contain oleic and linoleic acid demonstrate a high larval mortality (Rahuman et al., 2008).

6.6. Anticancer activity Many reports are available in the literature on the antiproliferative activity of Citrullus colocynthis extract and its isolates (Chen et al., 2005; Tannin-Spitz et al., 2007a; Liu et al., 2008a, b; Yin et al., 2008). The cucurbitacin glucosides (20 μM) from Citrullus colocynthis leaves inhibited the growth of selected human breast cancer cell lines (Tannin-Spitz et al., 2007b). Cucurbitacin B (1–100 μM) when studied on human laryngeal cancer cell line (Hep-2) inhibited cellular proliferation in a dose and time dependent manner (Liu et al., 2008b). Flow cytometry analysis showed that the treatment with cucurbitacin B resulted in accumulation of cells at the G2/M phase of the cell cycle and cell apoptosis in a dose and time dependent manner. Marked morphological changes of cell apoptosis including condensation of chromatin, nuclear fragmentation and apoptotic bodies were observed clearly by Hoechst 33258 staining. Western blot analysis demonstrated that the expression of p-STAT3, cyclin B1 and Bcl-2 proteins was suppressed significantly. in vivo studies showed that

7. Toxicity Although Citrullus colocynthis fruit has long history as medicine, reports on systematic toxicity and safety evaluation have been rare. In the acute toxicity and histopathological effect of saponin (extracted from whole plant Citrullus colocynthis) on mice, Diwan et al. (2000) reported the median lethal dose (LD50) to be 200 mg/kg, which indicate that the studied plant constituent is not toxic when comparing the LD50 values of most bioactive pharmaceuticals currently used in therapeutics (Sharma, 1998).

8. Conclusions and future prospects In this review, we have documented the existing traditional uses of the Citrullus colocynthis fruit and summarized recent research into the phytochemistry and pharmacology of the fruit

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and nutritional value of seeds of Citrullus colocynthis. Some of the traditional uses have been validated by phytochemical and modern pharmacological studies but still many needs to be validated. The extracts and isolated compounds have been found to possess various biological activities, especially in the area of antidiabetic, anticancer, anti-inflammatory, antioxidant, insecticidal and antimicrobial. Interestingly, plant has been shown to be of high nutrition value being a rich source of protein, edible quality of seed oil and some important minerals such as calcium, potassium and magnesium, known to be of high medicinal value. Although increasing interest has prompted more studies on the phytochemistry and pharmacology of Citrullus colocynthis, there are still many areas where the current knowledge could be improved: 1) the detailed quantitative data about the alkaloids and other secondary metabolites are still needed, 2) the Citrullus colocynthis fruit is a rich source of cucurbitacins, many with same skeletal structure. Therefore, it would be interesting to investigate the structure-activity relationships of these compounds and 3) several traditional uses of the Citrullus colocynthis fruit have been validated in the recent pharmacological studies; however, some of these studies were only tested in vitro. Thus the effectiveness of Citrullus colocynthis fruit extracts and isolated compounds needs to be further investigated for their efficacy and safety using in vivo assays. The plant is known to be beneficial in multiple gut disorders such as, indigestion, gastroenteritis, constipation and colic pain, while there is no study available on any gut disorders. In our earlier studies (Ghayur and Gilani, 2005; Gilani et al., 2005a; 2005b; 2006; Mahmood et al., 2011), it was observed that the plants with multiple indications of gut disorders usually contain combination of gut stimulants (mainly acetylcholine like) and spasmolytic (mainly Ca þ þ antagonist like) constituents, which not only explain their medicinal use in constipation and colic pain/ diarrhea, but also offers side-effects neutralizing potential, thus not allowing gut stimulant component to go beyond certain limit, above which it could have been harmful (Gilani and Atta-ur-Rahman, 2005). Hence, there is sufficient potential to study this plant for the presence of such novel combinations of activities. The plant has been shown to possess anti-inflammatory activity, but, unlike NSAIDs, it may not cause gastric ulcer, because, it is traditionally used in gastroenteritis (yet to be scientifically validated). Thus, if proved to possess anti-ulcerogenic, this may offer a safer option for inflammatory conditions. The plant is also traditional used in airways disorders such as bronchitis, cough and asthma, and it would be interesting to study it for its effectiveness in airways disorders, particularly when currently available bronchodilator drugs render serious side-effect like cardiac stimulation. On the other hand, the plant remedies have been found to possess interesting combinations of bronchodilator pathways with “effect enhancing and sideeffect neutralizing potential (Gilani and Atta-ur-Rahman, 2005). For example, co-existence of Ca þ þ antagonist with antimuscarinic constituent(s) in Hyoscymus niger (Gillani et al., 2008), Carum roxburghianum (Khan et al., 2012) and Fumaria parviflora (Rehman et al., 2012), while Ca þ þ antagonist with phospodiesterase inhibitory constituent(s) in Nepeta cataria (Gilani et al., 2009), Acorus calamus (Shah and Gilani, 2010), Andropogon muricatus (Shah and Gilani, 2012) and Lepidium sativum (Rehman et al., 2012a) is likely to offset the cardiac stimulant effect usually seen with phosphodiesterase inhibitors and antimuscarinics when used alone orally. Citrullus colocynthis has been traditionally used in diabetes and hypertension, which are growing all over the world with a rapid pace. While a large number of studies exists showing its efficacy in diabetes as mentioned above in Section 6.1, the plant has not been studied for its effectiveness in cardiovascular disorders except a couple of preliminary reports showing antilipidemic effect

(Daradka et al., 2007; Rahbar and Nabipour, 2010). It is not uncommon that diabetes, dyslipidemia and hypertension coexist in many patients and the treatment in modern medicine is complex requiring life-long use of multiple drugs, which results in multiple side-effects, in addition to high cost. Thus, this plant with therapeutic potential in metabolic and cardiovascular disorders warrants immediate attention for preclinical and clinical studies. Based on the above mentioned facts and identified gaps in the literature, possibility exists for the presence of “effect enhancing and/or side effect neutralizing” combinations of activities for its use in multiple disorders like in GIT, airways, metabolic, cardiovascular and inflammatory disorders. It is evident that the fruit and seeds of this plant possessed a huge potential for further studies on the phytopharmaceutical and nutraceutical application.

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