Pharmacological Evaluation of an Asterosaponin Extract from Asterias Forbesi (Desor)

An a ste rosaponin extracted £rom. the Atlantic starfish _A. ste r~~ forbesi (Desor) has teen evaluated for pharmacological activity. The investigation has revealed that while the asterosaponin does not exi1ibit all the activities that would be expected, such as hernotoxicity to blood cells, choline rgic blocking activity, initiation 0£ a.voidance .i.'ebEJOnse reaction or production of autotomy, it does exhibit soLJ.e unique propert: ,o: s not demonstrated by other a sterosaponins. These activities include an z. lgesia, anti-inflarnrna tion, a.nd transient hypotension produced by a direct effect on the vascular

The transient hypotensive activity of the asterosaponin was demonstrated in rats, cats, and dogs. Utilizing the cat it was demonstrated that very low doses of the asterosaponin (0. 5 mg/kg) could significantly transiently lower the blood pressure, but had no effect on the nictitating membrane or on somatic muscle contra ction.
The rat was less sensitive to the intravenously administered asterosaponin then Lhe cat, eliciting a hypote nsive effect only when concentrations oi 4 . 0 rng/kg or higher were administered. The sensitivity of the d og to asterosaponin was intermedia t e . The dog exhibi.te d a drop in blood pr essu re when a dose of 2. 0 mg/kg of asterosaponin was adn1inistered, and similar to the cat and rat showed a dose response relation ship ~o the asteros aponin.
The rnechanisrn by which the asterosaponin caused the transi ent d1·op in blood pressure was investigated by an analy sis of vagal influence, the possibility of histamine release, direct action on the vasculature and th e possible complex involvement of some type of adrenergic blockade or stimulation using an experimental set up rnonitoring blood pressure, heart rate and ECG. The data showed that th e asterosaponin continued to exhibit its hyp otens ive ac tion even when the vagus nerves were severed, the animai was pretreated with antihistarnine, the a l pha recepto rs were blocked, or when the b eta receptors wer e bi.ocked.

l l
The hind leg perfusion experim.ent perfonned on the rat proved that the asterosaponin was acting directly on the vasculature. The experiment showed that the asterosaponin was capable of causing vasodilation directly when all neural influences were removed by cervical dislocation and severing, and was even capable of acting in the presence of beta blockade created by the administration of propranolol to levels which blocked isoproterenol effects.

ACKNOWLEDGMENTS
The author wishes to express his gratitude to his family and friends for their support, encouragement and und e rstanding.
Th e author is indebted and thanks Dr. Gary P. Carlson for his guidanc e thrcughout this study. The author would also like to thc.nk D:·. "' ".{11::curu Shimizu who su-pplied the orig in al asterosaponin and g uid ed the author during the ext raction of the ast e ro saponin.
Special thanks go to Dr. Eugene Jv1iller for the confid en ce h e had in the c..utho r.  The phannacological evaluation of comp oun ds extracted from plant or animal sources is basic to the science of pharmacology.
The employment of animals and plants from the marine environment as a. soucce for biologically active compounds has created the field of n.1.arine ph2.rrnacology.
Dr. Yuzuru Shimizu isolated an antiviral asterosaponin extract fro1n the .A.tlantic starfish Asterias forbesi in 1971. This asterosaponin ( Figure 1) is an aglycone with a sulfate group and sugars attached , That structure is characteristic of saponin cornpounds derived from starfish and is very similar to compounds extracted from I-Iolothurins .
The investigation of the pharmacological properties of this purified and identified asterosaponin from ~-ster~as £9rbesi was the basis for this thesis. The investigation was aimed at evaluating the asterosaponin for pharn1acological activities that had be e n exhibited by other asterosaponin or related compounds. The investigatorfl were a l so interested in evaluating the extract for properties not previously associated with a&terosaponins and to determine the mechanism by which these activities we re produced in the hope that the compound rnight exhibit activities that would be cf use as either a research tool or a possible drug.  Burnett,11 The rnechanisrn ernployed by the starfish Asterjas forbesi to gain access to the interior of the bivalve, Venus mercenaria, 11 Eculogy ±l_ (1960): 584 . Lavoie (1956) presented additional evidence that starfish e1nploy no toxic compound in their acquisition of food by using extracts of Asterias i_orbesi on the rn.ussel Mytilus edulis. Various extracts from the stornach and pyloric caeca were tested by a nw:nbe.r of techniques. The extracts were tested by injection into the rnantle cavity of the mussel, by injection into the adductor rnuscles, by addition to the sea water containing tile mussel and the extract was even per£uf>ed on the expo sed heart of the Mytilus. All of the experiments · ·with all the ext racts produced the san1 e results; there was no difference in the effect seen with the starfish extract from the response seen with sea water used as a control. Lavoie aiso de1nonstrated that the starfish could enter the clam through a gap as small as 0. 1 nun. However, the conclusion that was made by Lavoie was that, ' 1 The negative results of the experin1ents involv ing sea star extracts are not proof that asteroids do not secrete a toxin during predation, but they do indicate that no such substance can be separated from the sea star organs by the extraction methods used. 112 The conclusion indicates the dilemma these investigators face. It is irnpossible to prove that a toxin does not exist, and only possible to prove that one does exist when it is derr10nstrated un e quivocally. This has not be en done.
2," L _ .  Christensen (1957) also supported the mechanical theory of starfish access to mollusks, that is, that force was the 11 primary 11 method employed by seastars of the Asterias type to gain access to the tis sue of the bivalve prey. The hesitance on the pa rt of the researcher to completely dispel the idea of a toxin being present is evidenced here by the choice of the word 11 primary 11 • Nichols (1964) continues to include the idea that a toxic substance is b eing used by starfish in prey predation, anC. Aldrich presents observational data (Aldrich,195 4 ) in his Ph.D. dissertat .ion that extracts from Aste rias fo r~e si_ do indeed produce relaxation of the add uctor muscles r;f ]yiytilus edulis and 11odiolus demissus plicata. Aldrich 1 s data however i_s based on observations of the response of Mod_i.olus demissus to extracts of Asterias tissue where a gaping response was interpreted as being the result of a toxic substanc e present in extracts from. pyloric divertic;ilun-i. Unfortun.ately no reason was given by Aldrich as to why the response of Modiolus was inconsistant. With both the filtered and dialized extracts there appears to hav e been specimens which were not affected by the extract. Observations of an Asterias feeding on a Mytilus edulis is the second source for Aldrich 1 s belief that the starfish contains a toxin. The fact that the starfish stomach is evert-· ed at the begining of the attack supposedly suggests the role of a chemical rnediator prior to the mechanical phase of penetration. Hashimoto and Yasumoto (1960) in studying an extract from the starfish Asterjna pectinifera which had been shown to b e toxic to oyster hearts, could hinder the ecdysis of fly maggots, and was toxic to fish and warm blooded animals, noticed that the water in the aquarium which held the starfish oft en turn ed foamy when starfish died. I--lypotbes.izing that the extract that they had been studying might be respcnsihJe for this foaming, and knowing that one of the properties of saponins is their surfactant nature, they initiated a search for a saponin compound in the starfish. Inde ed , when isolation procedures were completed the compound was classifi e d as a saponin.
Continuing their work Yasurnoto a nd Hashimoto, together with Watanabe (Yasumoto et al., 1964) s h owe d that w ith better extraction proc edures u sing the starfish Asterias amurensis a highly purified toxic saponin fraction could be obta ined. This new asterosaponin proved to be hemolytic to rabbit blood and toxic to killifish, earthworms and fly maggots. When 80 rng of the starfish saponin was injected into the mantle cavity of three individual oysters through a hole in the edge of their shells, these shell fish, which were kept in an aquarium, showed no signs of relaxation of the adductor muscles for the six hour observation time.
These resul ts of Yasumoto ~__! ~· (1964) sugges t that the aste rosapo nin is not 1 esponsible .for facilitating the starfish 1 s ability to open the clam or oyster, but that it may be present for another purpose as y e t obscure. This corresponds with the opinion of F e der and Christensen (1966) who beli eve that there is no basis for the toxin theory. Howe'7er they do believe toxic substances a re pr e s e nt in starfish, but that they are not utilized for prey procurement. A toxic extra ct from Asterias £orbesi was demonstrated by Chaet in 1962. He showed that the scalded coeleo1nic fluid of Astei-i as contained a toxi n which when injected into the peritoneal cavity of the s tarfis h Asterias £or1~~si ca'...ised autoto1ny (the rejection of the star-fish1s own arms) 2. nd death. Toxic fractions from other species of Asteria s we re examined by Fange (1963) who showed that an extract from Asterias _E21bens produced the irreversible contraction o.f the isolated Buccinum radula muscle. This fraction was inactivated by b o iling . Rio et al. (1965) evaluated the biological activities of extracts from five starfish species . Althoug h one of the asterosaponins eva luated was extracted from Aste rias forbesi, it is not believed that -the c ornponnd is t he same as the compound isolated by Shimizu.
No structure for the compound was determined, but the extraction procedures are n ot the same. The Aste rias for be si toxin was shown to be toxic t o Fundulus heteroclitus down to a level of 2 pg/ml. However, no mark e d eosrnophil.ia or hernolytic activity was reported for the compound. The compo1-md was shown to imrnoboli z e sperm and cytolyz e unfertilized sea urchins eggs. Th e sulfate group was suggested to be the part of th e compound re sponsible fur tD.e toxicity and anionic character. Comp ound A co'1.tained only tl~e terminally reduced sugars fucose and quinovose but both asterosaponin A and B were 11 quite'' powerful in destruction of excitability of a cholinergic neuromuscular preparation.
Shimi zu in 1971 iso l ated an anti-viral substance from the starfish Asterias forbesi (Shimizu, 1971) . This compound later (Shimizu, 1972) was established to be of the class.ical steroidal aglycone, polysac cha ride moiety and sulfate derivative form, and is the compound that was used in this investigation.
Mention has already been made to the possibility of an avoidance respons e reaction being caused by the asterosaponin. The work of Fange (19 63 ) Feder andL asker (1964) Feder andArvidsson (1967) and Mackie et al. (1968) clearly shows that extracts from starfi sh could initiate an escape response in rnollusks . The observations of Pratt and Campbell (195 6 ) suggested that the mollusk Me rcenaria mercenaria n1ay be able to sense the presence of Asterias forbesi and initiate an avoidance or escape response. These beliefs were based on the observation that quahogs appear to burrow deeper when Asterias are in the vicinity of the bivalves.
There is evidence that asterosaponins can be cardioactive (Hashimoto and Yasumoto, 1960) and this is another side to the debate as to .,,-_,he~.he;_· the sta riis h opens the bivalve with shear force or through ihe use of a toxie. In order to facilitate an evaluation of the bivalve toxicity, and the cardioactive and cholinergic properties of the asterosaponin b ackgrcund information on the anatomy and physiology of !~e!cenaria rn e rc 811~ri a , its heart, and the chemicals it is sensjtive to was necessary. This information has be en given by Prosser (1940), Gre enberg and J ega l (1963), Greenberg (1965), Florey (1967) and Agar wa l and Greenberg (1969). The starfish (Asterias forbesi) that were us ed for the extraction procedure and for experimentation were collected from Narragansett Bay in the shallow areas (usually at low tide) off the rocks close to Hazzard Ave. in Narragansett, Rhode Island .
The snail L ittor~E_~ _!_itt o r ea was collected on the rocky shore 2, 2 of Narragansett Bay at lo w tide near the ''Towers" of Narragansett, R.hode Island.

B. Materials
Analytical grade chemicals or equivalent were used throughout the study. Carrageenin was obtained from Marine Colloids, (Rockland, Maine).
Two sc.n:pJ.i:, 5 of ast erosaponin were used in this investigation.

C. Analytical Procedur e s
The asterosaponin was dissolved in 0. 9% saline for intravenous and intraperitoneal administration, artificial sea water for marine preparations, distilled water for in vitro preparations and Ringer-Locke solut:i o n for hind l eg perfusion preparations. The asterosaponin was often used in doses of l. 5 x 10-powerg/ml because the estimated molecular weight of the compound was l, 500 (Shimizu, 1972) thus allowing for easy conversion to rnolar concentration.

Cholinergic Assay and the Effects of Asterosaponin on the Mercenaria mercenaria Heart
Large bivalves 1'.1ercenaria rn.ercen2.ria were kept in aerated artificial sea "''ater 15-20°C until u.scd. The dissection was carried out under sea water. A bivalve specimen was selected ci.nd the edge of the shell taped until a small crack appeared. The clam was then opened by cutting the adductor inuscles, and care was taken not to injure the portic)n of the clam close to the hinge, as this was the vicinity of the heart. After removal of extraneous tis sue, the dorsal half of the clam_ was transfered into the dissection dish with the dorsal side (containing the heart) up and a superficial sagital cut was made along the dorsal body wall. The heart was then isolated and prepared for removal by cutting the intestine which passed through the heart on both sides. Ligatures were tied around both ends of the ventricle, so that eventually one could be attached to a transducer and the other to a glass hook. The heart was severed distal to both lig atures and immediately transfered to a 10 ml muscle bath with a surrounding jacket through which cooled (18°C) wate1· was purnped to regulate the temperatur e of the heart and solutions. A constant but gentle stream of air bubbles continualJ.y oxygenated the bea rt preparation. The heart was held in the bath with one ligature connected to a glass hook at the botton~ of the bath while the other li gature was connected to the A-715 transducer attached to a Physiograph (E. M. Instrument Co., Houston, Texas).
Standard dilutions were nl.ade of acetylcholine in sea water -4 -12 to cover a range of 1. 0 x 10 g/ml to 1. 0 x 10 g/ml. One ml was added to 9 ml of sea \vater to reduce th e concentration by 1 power of 10. The effect of the acetylcholine, or test drug was recorded for 30 seconds and then the heart was flushed two times with sea water and a il o'>ve d to stabilize for o ne rninute or longer. Starting

·'
with the l. 0 x 10 --12 g/ml concentration of acetylcholine, the concentration was increased until the h ea rt beat stopped. ·Interme diate concentrations between the le vel of acetylcholine that conl.pletely stopp ed t:b.e contractions and the next more dilute concentration we1·e occasionally appli ed to exceptionally sensitive hearts to attain a finer comparison of activity.
The acetylcholine effect was then compared to the effect of different concentrations of the asterosaponin, following the same procedure. Sarnpl e s of acetylcholine, giving known activity were administered intermittently to the test preparation during asterosaponin testing to rnonitor the deterioration of the sensitivity of the preparation. The anticholinesterase neostigmine and the anticholinergic atropine were used to gain more info rmation £ro1n the preparation.

Effect of Asterosaponin on the 'Whol e Clam Mercenariamercenaria
Fiv e lar ge bivalves 1v1ercenaria mercenaria had s1nall hol es drilled in the ventral lateral section of their shell so that a small syringe needle could be inserted into the mantle cavity of the clam.
Four of the clams were injected with l rnl of 1. 5 x l0-4 g/rn_ l asterosaponin while the fifth received a 1 rnl injection of sea water. The c lams were all placed in a container of aerated sea water for 24 hours and observed for any type of reaction to the experimental pl'ocedure .
Expe rim en ts designed after the experiments of Mackie et al.
(1968), were performed to determine if there were any escape response reactions 0£ the local salt water species of gastropod, _Littorina littorea to the asterosaponin.
The snails were placed in individual beakers of sea water that were positioned over pieces of graph paper. When nonna1 exploratory activity of the snail had resurned, the asterosaponin was gently injected into th e sea water close to the head of the snail with a syringe.
T h e response of the snail was then recorded, using the graph paper squares to dete rmine the movement of the snail. Injections of sea water via syringe into the snail 1 s sea water environment at the pr ox--in1ity of his head were used as a control. A different snail was used for each dose of asterosaponin tested.
The following procedure \Vas used to determ ine if the Asterias forbesi asterosaponin exhibited blood cell lysing properties similar to other asterosaponins. Rat blood obtained from tail veins was treated with concentrations of asterosaponin ranging from 0. 3 to 10. 0 mg/nil, or witli the st3.ndard lvierck saponin used for blood count deterrni: ; .1ations. The bloo<l san1ples were placed in a Coulter Counter and the blood count determined.
Chact in i9(;2 had dernonstrated the ability of a co1npound from the sea lded coe lornic fluid of Asteria s forbesi to bring forth an au tonoewus loss of arms by that starfish, when the coin pound was injected into its peritoneal cavity. The procedure used in this res~arch to investigate this property being possessed by the asterosaponin from Asterias forb e si was similar. -----·----- Intermediate sized starfish 8-12 crn in diarneter were injected intraperitoneally with 1 ml of l. 5 x 10-4 g/ml asterosaponin and then placed in the aqua·riurn for observation. Control starfish received intraperitoneal injections of l ml of seav,1ater . The starfish were observed for .four hours and a bno nnal reactions were recorded.
The starfish were then observed periodically for the ne x t twenty hours.

Analg esia
Th e rnethod of Koster (1959) was the method used to evaluate the analgesic properties of the &sterosaponi;-i. Groups of 5 mice were pretr eated by ip injection with either; l) saline (0. 1 ml/g) 2) morphine (10 mg/kg) or 3) asterosaponin. The asterosaponin was dissolv ed in saline and was adrninis terpd at 6 dosage levels 27 ranging from 10 mg/kg to 50 mg/kg . Fifteen minutes after the pretreatment :injections the mice received ip injections of 0. 01 ml/g of 0. 6% fresh acetic aci d . Three minutes after the acetic acid injections the w ri thes exhibited by the group of mice were counted and to~aled fo _ r twelve minutes. In this procedure reduction in w ri thes is equated with analgesia.

_In Vitro Anti-inflammatory As~
To evaluate possible anti-inflarnmatory activity by the a ste ro-<::aponin the in vitro assay of Grant~ al. (1970) was e1nployed.
The assay en1ploys the use of bovine serum albumin. Many corn -poun ds that prevent inflarnmation also are capable of reducing ti1e heat denaturation of a l.bumin protein. The effect of the asterosaponin on blL)Od pressure, respiration, the autonon1ic nervous systen1, and skeletal in us cle re spans e to neuronal. stin1ulation were determined using a cat.
A cat was anesthetized wi.th pentobarbital. The cat's respiration The effects of the asterosaponin on the monitored parameters were eva l uated after intravenous administr ati on of the canpound.
Vfhenever th e asterosaponin or other drug was administered the cannula was immediately flushed with saline.
/ 11. Blood Pressure and ECG Resoo nses of the Rat to Ast erosaoonin The response of the cat to asterosaponin adrninistration initiated interest into the cardiovascular effec t s of the asterosaponin. These cardiovascular effects of intrav enous asterosaponin achninistration were further investi gated primarily in the rat.
A rat was anestheti7-ed with ur ethane (6 rn.l/kg of 20% solution, i p). A tracheal cannula (a 1. 5 11 piece of PE tubing , size 280 or smaller) was then inserted and tied securely. The femoral vein, as it passed into the iliac vein was exposed and cannulated with PE 10 tubing and conn ec ted to a l ml syringe that was filled with saline.
This cannula was used for intravenous drug ad1ninistration. Each of the con1mon carotids were exposed and separated from the surrounding tissues . Then the two nerves, the vagus and the cervical syrnpathetic which lie along the carotids, were carefully and gently separated from the section of the vesse l to be us ed for blood pressure catherterization usin g PE 90 tubing. Prior to its insertion the cannula was attached to the saline filled transducer system in the 1.isual .
rn.ann er and the tip filled with heparin solution. A blood pr e ssure bottle filled with 250 ml of saline and 2. 5 ml of heparin (1000 units /ml) had been previously attached to this transducer. This system was used to re co rd the blood pressure changes following drug administration .
To record the rat 1 s ECG three needle electrodes were placed subcutaneously. The ground electrode was connected to the leg of In a s e cond part 0£ the experiment, asterosaponin was adn1inistered after the vagus nerves had been severed, and the results recorded to compare them to the normal effects of the asterosaponin or; a rat with intact vagus nerves.
14. Evaluation of the Histamine ReleasiE_g Potential of A stero saponin The prJssibility that the hypotensive effect seen with the asterosapo nin adrnini8 tration could be caused by endogenous histamin e release was evaluated. Rats were prepared for ECG monitoring and blood pres s ure determination. The antihistamine diphenhydran1ine w.a s adrninistered intravenous ly to the r a t until su££icient amounts were present to prevent the hypotensive activity of intravenously adrninistered histamine (0. 5 mg/kg). When a protective level of antihistamine was attained asterosaponin (15 mg/kg) was administered intravenously to d eter rr1ine ii the hyp oten sive action of the asterosaponin could be blocked by pr etreatment with the antihistamine.

}\ ste ros_aponin
An analysis of the in£luence of the asterosaponin on the alpha receptors of the adrenergi.c nervous system was possibl e from the cat nictitating membrane exp e riment. To gain more direct eviden ce for the possibility of this type of activity, a specific experirnent was designed, incorpor2.ting the rat blood pr e ssure pr eparation .
Rats were prepared for the usual ECG and blood pressure d eterminations. Phenoxybenzamine was then administered to the rat to the point where no further drop in blood pressure was seen.
Then asterosaponin (15 rng/kg) was adrninistered intravenously to deterrnine if the asterosaponin could produce further hypotension even th ough the alpha receptors we re blocked. The normal proce -<lu re of flushing each dose of a.dministe red drug with saline before the next drug was administered was maintained and ample tirne was a.llowed for each drug to have an effect.

Evaluation of Beta S!imulating Properties of .Asterosaponin
The stirnulation of the vascular beta receptors by adrenergic agonists can significantly alter peripheral resistance. The stimulation of these beta receptors leads to a drop in blood prei;)sure through vasodilation. The determination of the possibility of asterosaponin acting as a peripheral vascular beta stimulant was carried out using the rat blood pressure technique. The possibility of b eta influence was also inv est i gated with the rat hind leg perfusion experiment (see below).
A rat was pr e pared for blood p ressure and heart rat e rnonitoring following the standard procedure for the blo od pressure and ECG A rat was anesthetized and attached to a rat board that was mounted on approximately a 50° angle so that the scrotal sac of the rat was positioned at the lower end of the board. The abdorninal cavity was opened and the skin pulled away from the musculature surrounding the scrotal sac and along one leg. The vena cava and decending aorta were carefully isolated below the level 0£ the kidney and anterior to where they bifurcate into the lower app endage s.
The vena cava was tied just below the lev el of the kidney and the aorta was cannulated. The cannu la to the aorta was attached to a three way stopcock permitting aerated body temperature Ringer-Lockc solution t o be p erfused at a constant pressure into the artery.
The stopcock could also be turned to allow for the administration of drugs via a syringe. The veins of the le g that had the skin pulled away were severed so that the perfnsate could travel through the arterioles and vascular bed and ernerge through these severed veins.  EXPLANATION OF TABLE 1 a This recording was r ecorde d as a slight drop. However this was probably due to the instability of the preparation since after sea water replacen1ent the contractions were larg e r then with the toxin for the first 3. 5 minutes but then the amplitude of the beat was com-· parable to that exhibited with the toxin.

b -7
The dose of 1. 5 x 10 g/ml was r e peated later in the expedment, after trials with both to xin and acetylcholine, with the same effect.
c The 1. 5 x 10-7 g/ml dose of toxin by itself had no effect, and with ' neostigmine (0. 25 mg) pretreatment it continued to show no effect.
d Atropine was used to assure that the preparation was respondin g correctly.
-6 When atropine was pr esent at 1. 0 x 10 M the heart stopped contracting.  Table 1 show that the hearts -9 were all sensitive to acetylchol.ine at the concentration of 1. 0 x 10 g/ml (5. 5 x l0-9 M) and rnany of the hearts were sensitive to concen--13 -13 trations as low as 1. 0 x 10 g/ml (5. 5 x 10 M). Table 1 contains -7 data that also show that when atropine was present at l. 0 x 10 M it protected the preparation against the effect of l. 0 x l0-8 g/ml Lvi ac e y c .o ine. At doses of the asterosaponin ranging -18 -7 from 1. 5 x JO g/ml to 1. 5 x 10 g/ml there was no effect produc ed on the heart. The adrninistration of neostigmine (0. 25 mg) to the test preparat.i.on did not alter the lack of effect on the preparation produced by l. 5 x 10-7 g/ml of asterosaponin.
B. Effect of Asterosaponin on the Whole Clam Mer~e~·ia_mer -

cena ria
Neither the asterosaponin injec tio n nor the sea water injection into the intact clan1 caused any abnormal reac tio n by the cla:;:n.
-4 Clams that were injected with 1. 0 ml of 1. 5 x 10 g / ml of asterosaponin did n ot appear to differ in any way fro1n the clan1 receiving l. 0 ml of sea water.
Since many asterosaponins reviewed in the literature and saponins in general exh ibjt hemolytic a ctivity it was detern.l.ined that it \.Vould be nEcessary to evaluate this ::i.sterosaponin for l:ernolytic activity. Table 2 shows that the astero saponin possessed no cytotoxic activity towards rat l e ukocytes or erythrocytes at doses ranging up to 10 mg/ml. The ability of thf:: asterosaponin to initiate an avoidance response in Littorina littorea wa s evaluated (T a ble 4). The results demon--5 strate that wh::;n 1. 5 x 10 g of asterosaponin was i nje c ted into the water near the snails head no abnormal behavior was generated and no avoidance r e sponse observed.

F . Anti -inflammatory Acti v ity
Becaus e of the kno w n anti--inflammatory activity of a wide var1ety of steroids th e steroidal asterosaponin was tested for anti-infla1nmator y activity with two djfferent tests, the in vitE_£ bovine serum albumin denaturati::rn as say and the in vivo carrageenin induced rat bind paw edema a ssay.   The bovine serum album.in assay for anti-inflammatory activity showed (Table 5) that a concentration of 500pg/ml of asterosaponin caused an 85% inhibition of protein dena tu ration. Phenylbutazone, the well known anti-inflarnrna.tory agent, restricted the denaturat:ion by over 80% at a co:c.centration of l2Cpg/rnL Vvhen the data was plotted (Figure 2) both the aste rosaponin and phenylhutazone produced this restriction of c!enaturation in a log dose response rnan- The rat paw edema assay ( The evaluation of the LD 50 of any toxic co.r:npound is pararno 1 :rnt to any type of drug tes t ing procedure. The d2. t~, of the LD ,l.-, dete rm-:::i J ination (Table 7) for the asterosaponin dern.onstrate that the aste.::-osaponin has a . LDr ,-, v::t lne ln mice between 100 and !.'.CO mg/kg .      In a further evaluation of pharrnacological activity, the analgesic activity of the asterosaponin was evaluated, employing the Koster acetic acid assay (Koster, 1959). The asterosaponin was effective in reducing the writhes exhibited by mice due bJ acetic 50 acid injection (Table 8)

I. Cat Blood Pressure and Nervous Syst e m
The effects of aste ro saponin injected intrav eno usly at diffe cent dosag es upon a number of physiological processes we re monitored in the cat. The effects of the toxin on blood pressure, respiration , prega nglionic sympathetic nerve stimulation, and stimulation of a sornatic motor nerve to a striated muscle were all recorded . Figure 3 shows the polygraph recordings that we r e taken of a cat prior to any drug administration. The chart shows an initial blood pr essure of 100 mm Hg, ll, 5 breaths per minute, and the     respiratory rate was 11. 5 breaths per minute. After the aste rosaponin the rate increased to 16 breaths per minute for approximately one minute then dropped to 15 per minute. Nine minutes after asterosaponin injection resp.i.ration was still at an elevated level 0£ 14 breaths per minute although at this time the a ste ro sapo nin had ceased to have an effect on the blood pressure.
The data for the response of the cat 1 s physiological para1neters ' for all of the do se s of asterosaponin adrninistered are compiled in Table 9. These data clearly show that the asterosaponin from As--!erias forbesi was capable of dose dependentl y causing a transi ent lowering of the blood pressure o f the anesthetized cat. These data also show that the asterosaponin had no eifect on autonomic ganglia as the response 0£ the feline nictitating membrane was not altered throughout the dose range administered when the preparation was stimulated preganglionically. The asterosaponin did not appreciably affect the functioning of the somatic peroneal nerve or the functioning of the skeletal muscle.
J. Rat Blood Pr essure , Heart Rate and ECG Response to Asterosaponii: Figure 6 shows the norn1al recording for blood pr es sure and th normal ECG pattern of a typical . rat before any drug vva.s injected.
The blood pressure was 75 mm Hg, there were 270 heart beats per miP..ute and the ECG was r egular. A control determination was   It rose fr om the 75 mrn Hg to a level of 82 rnm Hg in 20 seconds and the heightened blood pressure l as ted for more then 90 seconds.
The heart rate was 280 beats per :minute during the tirne of activity which vvas interpreted as being no t appreciably different from the initial value. The ECG did not change.
The intravenous injection of 1. 0 mg/kg of asterosaponin in the rat resulted in a rise in blood pressure from 75 m .m Hg to 90 mm Hg in 25 seconds. Heart rate was 300 beats per minute, and the shape of the ECG did not change. The data in Table 10 show the effects of 2 mg/kg of asterosaponin. There was an initial drop in the blood pres sure from 85 to 82 rnm Hg which lasted only eight seconds and the pressure returned to about the norrnal level. The heart rat e just prior to injection and 40 seconds after administration was 300 b e-'1 ts per minute, and the ECG pattern was not altered.
The adrn.inistration of 4. 0 mg/kg asterosaponin (Table 10) was the lowest dose in the rat where unequivocal alterations in the rat blood p ressure occured. Thr ee to £our seconds after the drug was administered there was a dr op in blood pressure from an initial value 0£ 87 mm Hg to 70 rnm Hg in 18 seconds. The blood pressure was back to its normal value 35 seconds afte r drug adrninistration. The heart rate was 312 beats per minute before and after drug administration and there was no change in the ECG. For the 8. 0 and 20. 0 mg/kg (Figure 7) doses of asterosaponin the initial blood pressures were 92 and 82 mrn Hg respectively and the initial hearts rates were 312 and 300 beats per minute (     isolated by Goldsmith rate was 360 beats per minute. Four seconds after the aste ro saponin administration a reduction in the blood pressure began. Approximately 60 seconds aite r injection the lo\vest blood pres sure was reached, a value of 57 mm Hg, which lasted for about five minutes. The blood pressure did not return to normal £or m .ore then eight minutes. During these eight minutes the amplitude of the QRS complex of the ECG was dee reased and the beats per minute decreased from 360 to a low of 285 beats per rninute. Several replications were made for the rat receiving 10 mg/kg 0£ asterosaponin (Table 11). The first 10 mg/kg injection was of Dr. Shimizu' s extract. The heart rate at the initiation 0£ the experiment was 285 beats per rninute, the blood pressure was 62 mm Hg, and the ECG was normal. Three seconds after drug administration the blood pressure started to drop; 23 seconds aite r injection the pressure hit its lowest level of 24 mm Hg and the heart rate declined to 240 beats per minute. The effect only lasted one minute, and the rate and pressure returned to the initial levels. Similar alterations were seen following the administration of another 10 mg/kg dose (Figure 8). The blood pressure before the inj e ction was 107 mm Hg and the heart rate was 340 beats p e r minute, with a normal ECG. After the inj ection the pressure plumm eted to 15 mn1 Hg in less than 25 seconds, ren1ain ed at that level for two rn.inutes and then returr.ied to the 100 mn1 Hg level after another 30 seconds. Durin g this time the ECG became ' ' ...  Figure 9 is the recording of the effect of 20 mg/kg of the Les Goldsmith asterosaponin which was ad1ninistered following the second injection and affect of the 10 mg/kg 2.sterosaponin of Shirnizu. The blood pressure pri or to injection was 100 n1m Hg, aDd the heart rate was 340 beats per Seven seconds after the intravenous adrn.inistration of the asterosaponin, the blood pressure began to drop and 25 seconds after the injection the pressure st2.bilized at 55 mm Hg and remained at 55 mm Hg for inore then 3 minutes when the next dose of astero-saponin was administered. Two minutes after the injection the blood pressur e was still depressed (75 mm Hg). The ECG had obvious disturbances in its pattern, and the heart rate was depressed.

K. Aster osaponin and the Dog Blood Pressure and Heart Rate
Intravenous asterosaponin injections at various dosages were evaluate d in the dog. These data (Table 12) show that the dog was sensitive to asterosaponin at a dose of 2. 0 mg/kg and that the dog respond ed in the characteristic manner to the asterosaponin, i.e., followin g the asterosaponin injection a drop in blood pressure was seen which was only transient, and the response was dose depend e nt.
The drop 1n blood pressure was accompanied by an increase in pulse pressure at all dosage l eve ls (Fi gure 10). The increase rn pulse  (Table 13) indicate that asterosaponin at a dose of 5 mg/ kg was capable of reducing the blood pressure in rats who had been previously treated with atropine at a sufficient level, 0. 5 mg/kg to. suppress any vagal activity. The results also show that the a ste rosaponin was cap ab le of producing its effect when both vagi we re s eve red.

M. Hista1nine Releasing Pr operties of Asterosaponin in the Rat
The possibility that the as te rosaponin was reacting through histamine release was investigated. The data in Table 14 show that when the antihistamine diphenhydramine wa s administered to a level when exogenously administered h istamine no lon ge r produced its hypotensive effects, aste rosaponin continued to exhibit its hypotensive action. Histamine when injected at 0. 5 mg/kg created a direct hypotensi on. Jn this experi1nent a drop in pressure frorn 100 to SS   Table 15. A rat was prepared for the usual blood pressure, ECG evaluation including the venous catheter iza tion. Phenoxybenzamine was then administered through the venous catheter in doses of 0. 4 mg/kg until sufficient drug was present so that further doses no longer lowered blood pressure. It was then considered that alpha receptor activity was blocked. Asterosaponin injected at 15 mg/kg produced its transient hypotensive effect dropping the already lowered blood pressure from 48 to 30 mm Hg.
In another rat the possible beta stimulating effect of asterosaponin was evaluated (Table 15). When the rat was pr etreate d with propranolol to the point where excess propranolol was present in the animal and b eta receptor activity was blocked, asterosaponin The pas sibility of the a ste rosaponin producing its hypotensive response through some centrally mediated mechanism was evaluated with the first rat hind leg perfusion preparation. The preparation also evaluated the ability of the aste rosaponin to produce hypotension via direct action on the vasculature. The data from Table 16 show that the asterosaponin at a dose of 6. 25 mg/kg was capable of dilating the blood vessels of the rat hind leg upon inj e ctior.. and thus increasing the flow of perfusate through that vascular system.
Spinal cord transection at the cervical level did not inhibit the vessel dilating properties of the asterosaponin.
The data presented in Table 17 were from a hind leg perfusion preparation that evaluated the reliability of the preparation, the direct effect of asterosaponin on the vasculature, and the beta stimulating effect of the drug. The injection of 4 pg of epinephrine, a pred ominately alpha stimulant caused a constriction of the vasculature reflecte d by a drop in the rate of perfusat e flow. Isoprcterenol (0. l mg iv) a pred ominate ly beta agonist, caused an incr ease in the flow of pe rfusate while the injection of l ml of perfusate solution had no effect on the flow. Asterosaponin when injected at 15 or 30 mg iv dos e s into the preparation caused a substantial increase in the rate of p e rfusate flow. The beta stimulating properties of the asterosaponin were eva luat ed with a two-fold experiment.
The preparation was pretreated with propanolol (0. 1 mg iv) a bet2. Why starfish contain complex asterosaponin corripounds has been considered from the tin1e that the first asterosaponins were extracted (Yasumoto et al., 1964). T he originaL researchers who worked with the asterosaponins considered the possibility that the asterosaponins could be compounds used by the starfish to facilitate food procur e ment via some toxic properties directed toward the starfish 1 s prey, supposedly acting primarily on the adductor muscles of the bivalves on which the starfish often feed (Aldrich, 1957;Nichols, 1964). Some investigators even thought that if the compound was toxic to starfish prey then it was possible that the prey species rnay be sensitive to the compound an d react to it through some type of avoidance response (Pratt and Campbell, 1956). This avoidance response reaction w as demonstrat ed for starfish extracts by Fange (1963), Lasker (1964), andMackie et al. (1968).
The work of Friess et al. (1968) and Friess (1970) demonstrated that asterosaponin compounds from starfish did react with cholinergic systems . His experiments showed that some asterosaponins could inhibit the response to stimulation of the cholinergic rat phrenic nerve-diaphram preparation. It was expected then that the asterosaponin from Asterias forbesi might possess some type of reaction on cholinergic systems. The clam heart bioassay (Florey, 1966) was used to eva mate the cholinergic properties of the asterosaponin.
The heart of Mercenaria mercenaria was used in these experiments to identify any toxic action by the asterosaponin that would give the starfish any advantage in the predator--prey relationship. The data showed that the assay preparation was very sensitive to acetylcholine, reacting to concentrations as low as 5. 5 x l0-13 M. However, the asterosaponin had no cholinergic activity for the dose range -18 -7 I of 1. 5 x 10 to l. 5 x 10 g ml and that there were no abnormal responses of the heart to the asterosaponin. These data support the contention that this asterosaponin from Asterias forb es i does not offe r the starfish aid in opening the elam through an action on the heart. Other experiments, discussed below, varify that the asterosaponin had n0 cholinergic a ctivity.
The investigation of th e toxicity of the aste rosaponin to clam tissues or the response oi mollusk ti ssu es to the asterosaponin was continu e d with an investigab.on of the effect of the asterosaponin on the whole clam using experiments modeled after those of Lavoie (1956) and Yasumoto et al. (1964) where holes were drilled into bivalve shells and the compound injected into the mantle cavity. The data from these experiments show that the asterosaponin did not cause the clam to open and that the compound was not toxic to clams, even at the high concentrations administered. These results correspond with th e work of Burnett (1955Burnett ( , 1960 who showed that Asterias forbesi possess sufficient strength to open the bivalve without the use of a toxin.
The possibility remained that even if the asterosaponin was not toxic to the cla1n that prey species could be sensitive to the compound and initiate an avoidance response to its pressence.
The experiments of Mackie et al. (1968) were used as a rnodel for the experiments performed in this research to test this possibility.
The data from Table 4 show that the asterosaponin did not initiate an avoidance response by Littorina littorea.
One of the important pharmacological prop erties reported for the asterosaponins is their blood cell lysing capability (Rio ~al. , 1963(Rio ~al. , , 1965Yasumoto et al. , 1964;Owellen ~t al. , 197 3 ). The work of Rio et al. (1965) is interesting in that they examined asterosaponins from five differ e nt species of starfish, one of which was derived from Asterias forbesi, but is not b e lieved to be the same compound that was us ed in this investigation. Rio reported h e molytic activity for four of the starfish species used but not for the asterosaponin from Asterias forbesi. The results show that the asterosaponin from Asterias forbesJ. used in this investigation was incapable of lysing either erythrocytes or leukocytes.
The aste rosaponin was moderately toxic to mice. When various doses of asterosaponin were intraperitoneally injected into mice the asterosaponin, was shown to produce 100 percent lethality -wii:h a dose of 200 mg/kg. These results show that the asterosaponin certainly had some pharmacological activity, even though it had not demonstrated avoidance response activity, clam toxicity, or hemolytic action.

B. Activities of Asterosaponin not Previously Reported
The pharrnacological testing of the asterosaponin proceeded into an investigation of the compound for activities not previously reported to be present in asterosaponins. The method of Koster (1959) was used to evaluate the asterosaponin for analgesic activity.
The asterosaponin did, dose dependently, reduce the number of writhes exhibited by a group of mice that had received an intraperitoneal injection of dilute acetic acid. The presence of analgesic properties on the part of a drug is subjective by definition, and thus is difficult to confirm, but when a 25 mg/kg dose of asterosaponin caused a reduction in the expression of irritation by 99 percent it can at least b e said that there is a strong possibility that the asterosaponin ·is an analgesic. There is no reference to analgesic properties being exhibited or tested for by any of the other asterosaponins reviewed in the literature.
The initial test used to evaluate the anti-inflarnmatory capabilitie s of the aste rosaponin was the in vitro method of Grant et al. (1970). The Grant method evaluated the ability of a compound to inhibit the heat denaturation of bovine serum albumin. The ability oi a compound to inhibit the heat denaturation of protein may not be a direct indicator of anti-inflammatory activity but compounds ~hat are anti-inflammatory do exhibit this property (Grant~~·, 1970;Mizushina and Kobayashi, 1968).
The data show that the asterosaponin inhibited the denaturation of bovine serum albmnin as did phenylbutazone, and that the asterosaponin and phenylbutazone produced this inhibition of heat denaturation in a log dose response manner. These results indicate that the aste rosaponin has potential as an anti-inflammatory agent, and further investigation into the anti-inflam1nato ry potentia 1 of the , compound seemed desirable.
Following the positive results obtained for anti-inflarnmatory activity for the asterosa.ponin with the bovine serum albumin assay, a more stringent in vivo assay was used to evaluate the aste rosaponin.
The carrageenin induced rat hind paw edema assay of Winter was chosen for this purpose, (Winter et al., 1965) with the volume of the paw being measured plethysmographically following the proce -<lure of Van arman et al. (1965). While it is not clear why antiinflammatory agents inhibit the denaturation of protein it is equally obscure as to why the injection of carrageenin causes such marked inflammation. The carrageenin, which is a high molecular weight polysacchari d,~ is derived from either of two algae Chondrus crispus or 92:.g~rti n a st_!::llata, and has been hypothesized to cre ate inflammation by many different means. Crunkhorn and Meacock (1971) and DiRosa (1972) concluded that it may be due to: irritant properties, the release of histamine, 5-hydroxytryptamine, or bradykinin, prostaglan dins, and even reaction with the blood complement system_ as a 11 trigger 11 for non-immune inflammation. Nevertheless, the rat paw edema assay is a universally accepted method for the in vivo evaluation of anti-inflammatory activity. The assay results showed (Tal>le .6) that both a stero saponin and phenylbutazone re stricted the edema formed as a result of carrageenin injection. Ph enylbutazone ( 90 mg/kg) restricted th e edema by 5 2 percent and asterosaponin (7 5 mg /kg) restricted the edema by 53 percent. The pos ·itive results of the bovine serum albmnin assay and these results that show that the asterosaponin restricted the edema in rats to the same extent as phenylbutazon e , establishes the asterosaponin from l Asterias forbesi as an anti-in£la1nmatory agent. The combination of anti-inflammatory and analgesic activity is advantageous for possible drug compounds. Since the asterosaponin exhibits these properties it is an excellent candidate for further evaluation, however the potent hypotensive properties of the compound could restrict the use of the asterosaponin to certain specific situations. Nevertheless the analgesic and anti-inflammatory properties of the asterosaponin are deserving of more investigation.

C.-Cardiovascular Effects
The effect of asterosaponins on the mammalian cardiovascular 3ystem had not been investigated previously and the effects of the asterosaponin on respiration, skeletal muscle contraction, and autonomic nervous system functioning had only been studied using the isolated rat phrenic nerve-diaphram preparation (Friess et al., 1968;Friess, 1970 The intravenous injection of the aste rosaponin from Aste rias forbesi. caused significant results . In the cat dosages of 0. 5 and 1. 0 mg/kg (Table 9) of the asterosaponin caused a sudden, evanescent drop in blood pressure with little or no effect on respiration , the contraction of the nictitating m e mbrane or skeletal muscle.
The 2. 0 r.ng/kg iv injecti on of asterosaponin created an even greater drop in bl oo d pressure lasting much longer and caused an increase in the respirat ory rate along with a sli g ht decrease in the amplitude oi the contractions of the skeletal muscle. Obviously the asterosaponin from Ast~rias forbesi was not inhibiting the activity of th e phrenic nerve of the cat, as the respiratory rate rose and certainly the compound was not exerting any type of anticholinergic activity as the cholin ergic fibers of the pe ro neal nerve innervating the tibialis ante rior muscle were func.tioning almost normally. The lack of antich o lin ergic activity by the asterosaponin was also shown by the dr op in blood p1·essure, if the compound we re exerting anticholin ergic activity the blood pressure would be expected to rise, as a result of the loss of parasympathetic control. The compound did not appear to have any effect on the ganglia or the functioning of adrenergic nerves, as the responses to preganglionic stimulation of the nictitating membrane were normal. The hypotensive activity of the asterosaponin was investigated in rats and dogs after these effects were seen on the cat. The experiments performed on rats (Tables 10 and 11) and dogs ( No effect on heart rate or ECG in response to this asterosaponin injection were seen. These data correspond with the lack of activity the compound had demonstrated on the autonomic nervous system of the cat and suggested that the hypot en sive activity seen with the asterosaponin was not due to a depressor activity on the heart. In one rat the asterosaponin did appear to cause a depression in the heart rate. However, it is not believed that this was a direct effect of the drug, but rather a product of a unique rat preparatio n, or some type of a reflex. This is believed because injections of both the Goldsmith and Shimizu extracted asterosaponin into this rat precipitated ECG irr egula rities and a dr op in heart rate. On all other occasions the asterosaponin did not effect the ECG or the heart rate of the rat. The iv inj ection of asterosaponin i nto the dog created dose dependent hypotension and an increase in the heart rate, probably through a compensatory mechanism. The hypotension produced in the dog was unique in that the pulse pressure increased as a result of asterosaponin administration. Experiments performed by Lederis and Medakovic (1974) with extracts of teleost fish produced similar results in the rat. In the experiments by Lederis and Medakovic pulse pressure was increased following the administration of drug and the increase was due to a more extensive drop in diastolic pressure then in systolic pressure. They concluded that this j_ndicated dilation of resistanc~ vessels. Siinilar results were obtained with the dog preparation subjected to asterosaponin injections.
At all dosages of asterosaponin the pulse pressure increased due to a greater depression in diastolic rather than systolic pressure, implicating some type of vasodilai:ion as being produced by the asterosaponin. Lederis and Medakovic also observed a reflex mcrease in heart rate due to the hypotension caused by the teleost extract. The results of the dog heart rate and blood pressure experiments indicated that the asterosaponin rn.ight have created its hypotensive effect via a direct action on the vasculature . The production of hypot e nsion by the asterosaponin by working directly on the vasculature would also explain the effects of the asterosaponin on the respirato ry rate of the cat. Schoop e t~· (1957) demonstrate d that the hypotension produced by nitroglycerine caused a reflex hyperpnea in the rat. Thus if the asterosaponin was causing dir e ct vasodilation the respiratory stimuiation might be explained.
The transient hypotensive effect of asterosaponin injection was further investigated as to the mechanism of its action. The expe riments performed on the cat and dogs had not sufficiently established how the asterosaponin was creating its effe.:::t so fur·ther experim.ents \Vere performed on rats whose blood pressure and ECG were monitored, under a variety of conditions.
The possibility of the asterosaponin creating hypotension through vagal stimulation was investigated by testing the effect oi asterosap-.
onin on atropinized and vagotornized rats (Table 13). These results show that the asterosaponin continued to exhibit its hypot en sive effect even when the vagus nerves were sever e d. These results in ccnjunction with the results from the cat experi1nent that showed no cholinergic activity, and the negative results gained with the cla1n heart assay for cholinergic activity, discount the possibility 0£ the ast erosaponin from Asteria_::; forbesi acting through vagal stimulation.
The asterosaponin was examined £or histamine releasing properties in rats (Table 14). These data shovv· that antihistamine pretreatment did not interfere with a ste ro saponjn-induced hypo tension..
These resul ts correspond with the anti-inflamrn.atory activity of the asterosaponin so that it can be conclud ed that the hypotensive action of the asterosaponin is not due to endogenous histamine release.

90
The data from the cat and dog blood pres sure experiments indicate that the aste rosaponin worked directly on the vasculature.
The lack of effect exhibited by the asterosaponin on the cat nictitating rn.ernbrane preparation indicated that the drug was not acting through the autonomic nervous system. The possibility that the hypotensive action was due to alpha adrenergic blockade or beta 2drenergic stimulation was further evaluated with the rat blood pressure and hind leg exp eriments . The experiments performed on rat Blood pressure and ECG (Table 15) established that the asterosap-· onin was fully capable of eliciting its characteristic hypotensive response when the alpha or beta receptors were blocked. These results thus add strength to the contention that the asterosaponin is not an alpha blocker or a beta stimulant.
The rat hind l eg perfusion preparation supplied much needed information on the mechanism of action of the asterosaponin. The first hind leg perfusiun preparation (Table 16) established that the asterosapoGin was not acting through any centrally rnedjated process by showjng that t~e: asterosaponin continued to be effective as a hypotensive agent even after cer v ical tr-an section. Th e second preparation ( -7 I ranging from 1. 5 x 10 g ml (1. 0 x 10 M) to 1. 5 x 10 g ml -7 (l. 0 x 10 M).

2.
The aste rosaponin produced no observable e££ects on the whole clam Merc enaria rnercenaria when a dose of l. 5 x 10-4 g/ml was injected into the mantle cavity.

3.
No blood ceil lysing activity was exhibited by the asterosaponin when tested from O. 3 mg /ml to 10. 0 mg /ml.

4.
The asterosaponin produced no autotomy in Asterias forbesi at a concentr ation of l. 5 x l0-4 g/ml when injected into the peritoneal cavity.

5.
No escape response was elicited in Littorina littorea by asterosaponin when 1 ml of 1. 5 x l0-5 g/ml was injected near the head of the snail that was in a dish of sea water. These results along with the results of the clam heart bioassay, and the whole clam assay indicate that the asterosaponin is probably not used by the starfish to facilitate prey procurement.

6.
The asterosaponin inhibited the heat denaturation of l percent bovine serum albumin by 89 percent when added to the incubation medium at a concentration of 1000 pg/ml. This inhibition of denaturation was dose dependent and suggestive of possible anti-inflamma tory activity.

7.
The asterosaponin at 75 m.g/kg inhibited rat paw edema formation due to subplanter injection of carrageenin by 53 percent. The results of the i~ vitro_ bovine serum albumin assay and those of the in ;:i.Yo carrageenin induced hind paw edema assay establish the asterosaponin as an anti-inflammatory agent.

3.
The LD 50 of the asterosaponin to mice was between 100 and 200 mg/kg.

9.
The blood pressure of the c at dropped in response to 0. 5 mg/kg of the intravenously administered asterosaponin. The respiratory rate rose and the blood pressure fell in response to 2. 0 m .g /kg of the asterosaponin, but there wa s no effect en the preganglion ic stimulation of the nictitating membrane and littl e effect on the neuronal stimulation of ske letal muscle at the 2. 0 mg/kg l evel.
10. Transient, dose dependent hypotension was produced in rats administered asterosaponin (4. 0 to 20. 0 mg/kg). The heart rate and ECG were not appreciably directly affected by the compound.
11. The iv administration of asterosaponin (2. 0 to 10. 0 mg/kg) caused transient hypotension in the dog. This hypotension was indicative of va sodilation as the pulse pressure rose because of a greater drop in diastolic rather than systolic pressure.
Heart rate rose following asterosaponin induced hypotension probably as a compensatory mechanism.
12. In the rat, the asterosaponin (5. 0 mg/kg) caused hypotension even afte1· tre yagus nerves were severed. This along with the lack of cholinergic activity exhibited by the clam heart bioassay for acetylcholine, the lack of effect the asterosaponin exhibited on the neuronaily stinrnlated skeletal muscle of the cat, and the increase in respiratory rate of the cat established that the asterosaponin was not causing hypotension through vagal stimulation or cholinergic activity .
13. The asterosaponin (15 mg/kg) continued to exhibit its characteristic evanescent hypotension following antihistamine (diphenhydramine 10 mg /kg) pretreatment. This indicated that the asterosaponin did not produce hypotension through endogenous histamine r e lease. These conclusions were supported by the anti-inilarnmatory properties oi the asterosaponin .
14. The aste r osaponin's (15 mg/kg) hypotensive properties rn the rat were not stopped by alpha adrenergic blockade by phenoxyb enzan1ine (0. 4 mg/kg). These results along with the lack of activity the asterosaponin had on the nictitating membrane of the cat established that the asterosaponin was not causing hypotension th.rough alpha blockade.
15. When the aste rosap oni n (15 mg/kg) was injected iv into the rats pretreated with the beta adrenergic blocking agent propranol o l (0. 9 mg/kg), the hypotensive activity was still present, indieating thac the as te rosaponin was not a beta stimulator. The absence of beta-stimulating properties by the asterosaponin was further established by the results of the rat hind leg perfusion experiments, where 30 mg of asterosaponin was effective in creating hypotension after established beta blockade.
16. The fa.ct th.at the asterosaponin was not causing hypotension through any cenLally mediat e d process was established when cervical t r ansection of the hind leg perfusion preparation failed to diminish the asterosaponin (6. 25 mg) induced vasodilation.