Microvascular Research 54, 178–182 (1997)Article No. MR972036

BRIEF COMMUNICATION

Inhibition of Angiogenesis by Oral Ingestion ofPowdered Shark Cartilage in a Rat Model1

Paul F. Davis,* Yi He,* Richard H. Furneaux,† Peter S. Johnston,‡Beate M. Ruger,* and George C. Slim†,2

*Department of Medicine and ‡Department of Surgery, Wellington School of Medicine, P.O. Box 7343, Wellington,New Zealand; and †Industrial Research Ltd. P.O. Box 31-310, Lower Hutt, New Zealand

Received January 6, 1997

INTRODUCTION kawa et al., 1990). However, the links between oralingestion of cartilage and its anti-angiogenic andanti-tumour properties have yet to be convincingly

The oral consumption of dried powdered shark demonstrated.cartilage has been widely promoted as a natural Clinical trials of powdered shark cartilage as anhealth remedy for the treatment of cancer (Wilson, anti-cancer agent have been initiated in Mexico and1994; Lane and Comac, 1992, 1996). It is proposed to the United States but the results have yet to be pub-act by preventing the angiogenesis required by solid lished. In this study we have examined whether thetumours to grow larger than 2 –3 mm (Folkman, oral ingestion of powdered shark cartilage by rats1995). has any effect on the angiogenesis induced in mesen-

There is some evidence for the presence of anti- teric windows by mast cell stimulation.angiogenic factors in shark cartilage. Implantation ofpolymer pellets containing a shark cartilage extractalongside tumours in rabbit corneas inhibited tu-

MATERIALS AND METHODSmour neovascularization (Lee and Langer, 1983). In-jection of a suspension of shark cartilage reducedangiogenesis in tumours implanted in mice (Cataldi

Shark cartilage. Dried powdered samples of carti-and Osbourne, 1995) and anti-angiogenic factorslage from two commercial batches (designated A andcould be partially purified from shark cartilage (Oi-B) manufactured principally from blue shark weresupplied by McFarlane Laboratories Ltd. (Auckland,

1 Supported by the New Zealand Foundation of Science, Research New Zealand).and Technology, the New Zealand Lottery Grants Board and the Induced angiogenesis. A modification of the ratWellington Medical Research Foundation. We thank Mr. John Croft

mesenteric-window assay (Norrby et al., 1990) wasof McFarlane Laboratories (NZ) Ltd. for providing samples of sharkused. Sprague– Dawley rats (6 weeks old, equal num-cartilage.

2 To whom correspondence should be addressed. bers of male and female) were assigned to one of

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Brief Communication 179

TABLE 1

Percentage of Rat Mesenteric Window Area Occupied by Blood Vessels 16 and 25 Days after Stimulation with Compound 48/80

Diet

Normal / Shark cartilage A / Shark cartilage BDays after Animalinduction grouping n %Area n %Area P:2a n %Area P:2a

16 All 52 50.2 { 2.6 42 23.4 { 2.6 õ0.0001 45 32.3 { 2.1 õ0.000116 Male 26 49.7 { 4.2 21 25.5 { 2.4 õ0.0001 23 36.1 { 2.1 õ0.0416 Female 26 50.5 { 3.3 21 21.2 { 4.7 õ0.0007 22 28.3 { 3.7 õ0.000325 All rats 80 37.7 { 2.2 79 17.5 { 1.9 õ0.001 nd25 Male 37 32.9 { 3.0 38 13.0 { 2.3 õ0.0001 nd25 Female 43 41.8 { 3.1 41 21.6 { 2.9 õ0.0001 nd

Note. Each of the five groups comprised eight rats (four male and four female). Shark cartilage was administered at 6 g/kg of food. All dataare means { SEM. Statistical significance was assessed by Student’s t test. nd, not determined; n, number of windows.

three groups and fed ground rat food (Diet 86, cooled and the mesenteric windows were surgicallyremoved, mounted on glass slides, and viewed withSharpes Grain and Seeds Ltd., Lower Hutt, New

Zealand) which was freely available. The groups a light microscope. The images were digitised andthe vascularized area was outlined manually and cal-were fed an unsupplemented diet or a diet supple-

mented with either Shark Cartilage A or B (up to 15 culated using the programme Image 1.59 (NationalInstitutes of Health, Bethesda, MD).g/kg of food). Most frequently 6 g/kg of food was

employed. This equates to about 0.6 g of shark carti- Approval for these experiments was obtained fromthe Animal Ethics Committee of the Wellingtonlage/kg body weight if the rats consumed all their

food, which is less than that generally recommended School of Medicine.for human use by the manufacturers of powderedshark cartilage (1 g/kg body weight) (Lane andComac, 1992). There was õ2% difference in weight RESULTSgain amongst the three groups, demonstrating thatthe supplementations had no evident detrimental ef-fect. Angiogenesis was induced in the virtually avas- Sixteen days after the commencement of Com-

pound 48/80 administration, the percentage areacular mesenteric windows of rats by intraperitonealinjection of the mast cell secretagogue Compound that was vascularized in each mesenteric window

from rats fed shark cartilage was significantly less48/80 (Sigma Chem. Co., St. Louis, MO) twice dailyfor 4.5 days (Norrby et al., 1990). Feeding of shark than that in rats on an unsupplemented diet (Fig. 1,

Table 1). Although Cartilage A appeared to be morecartilage commenced 2 weeks prior to the inductionof angiogenesis and continued until sacrifice. Water effective than B, this difference was not statistically

significant. A similar effect was seen 25 days afterwas available ad libitum. The animals were sacrificedafter 16 or 25 days by exposure to CO2 and injected the commencement of stimulation (Table 1). At this

time, Compound 48/80 was more effective at stimu-with India ink through the superior mesenteric ar-tery (Mattsby-Baltzer et al., 1994). The carcasses were lating angiogenesis in female rats than in male rats,

FIG. 1. Vascularization of rat mesenteric windows; (a) without mast cell stimulation, (b, c, d) animals sacrificed 16 days after mast cellstimulation. (b) Control animal; (c) animal fed Shark Cartilage A (6 g/kg of food), commencing 2 weeks prior to stimulation; (d) animal fedShark Cartilage B (6 g/kg of food), commencing 2 weeks prior to stimulation.

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180 Brief Communication

FIG. 1—Continued

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Brief Communication 181

FIG. 1—Continued

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182 Brief Communication

cross the intestinal wall (Warshaw et al., 1974), butthe majority will be digested to smaller peptides inthe gut. However, GAGs, including chondroitin sul-phate (Conte et al., 1995; Ronca and Conte, 1993), themajor GAG present in cartilage, have been shown tobe absorbed through the intestine largely intact whentaken orally (Volpi, 1996). Work is continuing to de-termine which of the components of shark cartilageis responsible for the orally available antiangiogenicactivity observed here.

FIG. 2. Inhibition of mesenteric window angiogenesis induced bymast cell stimulation showing effectiveness of different doses of

REFERENCESShark Cartilage A. Dietary supplementation commenced 2 weeksprior stimulation and rats were sacrificed 16 days after the initia-tion of mast cell stimulation. The percentages are calculated from Cataldi, J. M., and Osbourne, D. L. (1995). Effects of shark cartilagethe means of the vascularised areas. For the control (no cartilage), on mammary neovascularisation in-vivo and cell proliferation in-118 windows (5 rats); for 1.5 g/kg of food, 107 windows (5 rats); vitro. FASEB J. 9, A135.for 3 g/kg of food, 106 windows (5 rats); for 6 g/kg of food, 105 Conte, A., Volpi, N., Palmieri, L., Bahous, I., and Ronca, G. (1995).windows (5 rats); for 9 g/kg of food, 83 windows (4 rats); for 12 Biochemical and pharmacokinetic aspects of oral treatment withg/kg of food, 40 windows (2 rats); for 15 g/kg of food, 39 windows chondroitin sulfate. Arzneimittelforschung 45, 918–925.(2 rats). Folkman, J. (1995). Seminars in medicine of the Beth Israel Hospital,

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Lane, I. W., and Comac, L. (1992). ‘‘Sharks Don’t Get Cancer: Howas previously reported (Norrby et al., 1990), but sharkShark Cartilage Could Save Your Life.’’ Avery, New York.cartilage was as effective at reducing angiogenesis in

Lane, I. W., and Comac, L. (1996). ‘‘Sharks Still Don’t Get Cancer.’’males as in females. Avery, New York.

There was a direct relationship between the inhibi- Lee, A., and Langer, R. (1983). Shark cartilage contains inhibitors oftumour angiogenesis. Science 221, 1185–1187.tion of angiogenesis and the dose of Shark Cartilage

Mattsby-Baltzer, I., Jakobsson, A., Sorbo, J., and Norrby, K. (1994).A included in the rats’ diet up to an optimal level ofEndotoxin is angiogenic. Int. J. Exp. Pathol. 75, 191–196.6 g/kg of food (Fig. 2).

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Ronca, G., and Conte, A. (1993). Metabolic fate of partially depolymer-oral ingestion of powdered shark cartilage has a po-ized shark chondroitin sulphate in man. Int. J. Clin. Pharm. Res. 13,

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factor must be stable in the gut and absorbed by the Volpi, N. (1996). Physico-chemical properties and the structure ofgastrointestinal tract in order for it to affect angio- dermatan sulfate fractions purified from plasma after oral adminis-

tration in healthy human volunteers. Thromb. Haemostasis 75, 491–genesis in the mesentery. This raises the question as496.to what is the likely identity of the inhibitory factor.

Warshaw, A. L., Walker, W. A., and Isselbacher, K. J. (1974). ProteinThe major components of powdered shark cartilageuptake by the intestine: Evidence for absorption of intact macro-

are protein (Ç40%) and glycosaminoglycans (GAGs, molecules. Gastroenterology 66, 987–992.Ç5–20%), with the remainder being principally cal- Wilson, J. L. (1994). Shark cartilage: A review of background literature

and research. Townsend Lett. Doctors 133, 864–872.cium salts. Small portions of ingested proteins can

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