Pharmacological Evaluation of an Extract from Eisenia Bicyclis

An extract from Eisenia bicyclis containing -principally a polyphenolic polymer was examined for pharmacological activity. The investi gat ion centered around the established activity of a model polyphenolic compound, phloroglucinol. Studies on inflammation and alteration of hepatic microsomal enzyme activity were also performed. Heat denat uration of a 0.15 % bovine serum albumin solution was inhibited 96% by the addition of the material at a final concentration of 0.32 mg/ml. Since stabilization of such solutions is often indicative 9f anti-inflammatory activity, direct me asurements of this. activity were made . The inhibition of formation of carageenin-induced rat paw edema was utilized as a test system. As determined by paw volume changes, administration of a dose of 75 mg/kg (ip) one hour prior to carrageenin injection inhibited edema production by 88%. The potent anti-inflammatory agent phenylbutazone, at a dose of 90 mg/kg (ip), provided 100% protection. The protection given by the Eisenian extract apparently was not due to pituitary-adrenal stimulation, since the protection is 4· fold better than that given by a dose of 50 mg/kg ( ip ) of cortisone. The effect cannot be attributed to changes in body temperature, which r emains unchanged . The effect can be partially but not completely attributed to the irr·itant

Studies on inflammation and alteration of hepatic microsomal enzyme activity were also performed.
Heat denat uration of a 0.15 % bovine serum al bumin solution was inhibited 96% by the addition of the material at a final concentration of 0.32 mg/ml. Since stabilization of such solutions is often indicative 9f anti-inflammatory activity, direct me asurements of this. activity were made . The inhibition of formation of carageenin-induced rat paw edema was utilized as a test system. As determined by paw volume changes, administration of a dose of 75 mg/kg (ip) one hour prior to carrageenin injection inhibited edema production by 88%. The potent anti-inflammatory agent phenylbutazone, at a dose of 90 mg/kg (ip), provided 100% protection. The protection given by the Eisenian extract apparently was not due to pituitary-adrenal stimulation, since the protection is 4-· fold better than that given by a dose of 50 mg/kg ( ip ) of cortisone. The effect cannot be attributed to changes in body temperature, which r emai ns unc hanged . The effect can be partially but not completely attributed to the irr·itant i properties of the material since buffering a dose of 100 mg/kg (ip) provided 42% protection. The effect is partially due to stabilization of the lysosomal membrane since in vitro 309 ug of the extract per milliliter of incubation media inhibited by 64 % the release of the marker enzyme 8-glucuronidase from lysosomes. The compound administered at a dose of 75 mg/kg (i p) daily for 17 days offered no protection against rat paw edema induced by Freund's complete adjuvant.
Capillary fragility was determined by mouse lung hemoglobin content following sudden decompression. No decrease in fra gility was observed 1.5 hours after pretreatment with the extract at a dose o f 300 mg/kg (ip).
The material administered to mice (3 00 mg/kg , ip) 30 minutes prior to administration of lethal doses of curare exhibited p1"otection when the curare was also given by the intraperitoneal route . No protection was exhibited when the curare was administered subcutaneously, This s uggested a chemical rather than a pharmacological antagonism. Th:l.s hypothesis was supported when no hlltagon1sm was found to the action of curare on the cat gastrocnemius muscle in vivo by jugular infusion of 180 mg of the extract, and when a dose of 300 mg/kg (ip) one hour pr~or to sacrifice was found to have no effect on the activity of rat brain cholinesterase.
The extract offe red no antagonism to the action of either stryc hnine or tetrodotoxin.
No chan ge in rat liver mi crosomal enzyme activity, as i i determined by the extent of metabolism of 0-ethyl 0-(4-nitrophenyl) phenylphosphonothioate (EPN1 was found after the administration of 300 mg/kg (ip) of the material daily for 3 days. Also, a single injection of 300 mg/kg (ip) 1 hour prior to sacrifice for measurement of activity was without effect.
Estrogenic activity was assayed by changes in uterine weight in the immature female rat. The administration of 300 mg/kg of the extract intraperitoneally daily for 3 days did not alter uterine weight .
Rat liver homogenates were assayed for tryptophan pyrrolase activity by measuring the formation of the endproduct kynurenine. The administration of 300 mg/kg (ip) of the extract 1.5 hours prior to sacrifice caused 33% inhibition of the enzyme activity . The presence of the extract at a concentration of 486 µg/ml resulted in an in vitro inhibition of 82%.
iii ACKNOWLEDGEMENTS The author wishes to express his gratitude to his wife Rosemary for her support and understanding.
Dr. Gary P. Carlson provided invaluable guidance throughout th is study . Mr . Steven Zelenski and Dr. Leonard Wo~th e n were kind enough to supply the Eisenian extract.  properties. More thorough screening programs would undoubtedly uncover more agents from just these four species.
In this study, an extract from the algae Eisenia bicyclis (Kjel lman ) Setchell, native to the shores of Korea, containing principally a polyphenolic polymer, has been examined for pharmacological activity. The investigation centered· around the established activity of a model polyphenolic compound, phloroglucinol, or 1,3,5-trihydroxybenzene.
Studies on inflammation and alteration of hepatic microsomal enzyme activity were also performed.

II REVIEW OF THE LI TERATURE
The pharmacological activity of phloroglucinol (1,3,5trihydroxybenzene) has been investi gated by several groups. Okanishi and Shimaoka (1952) determined by gross observation that phloroglucinol pretreatment could decrease mouse lung capillary fra gil ity, utilizing a decompression hemorrhaging technique devel oped by Majovski et al. (1944). To induce lung hemorrhage, mice were placed in a. chamber which was decompressed to 40 mm Hg. The animals were removed from the chamber after two minutes and the lungs were excised and judged for extent of hemorrhage according to an arbitrary ratin g system. A capillary stabilizing effect uncovered by this fragility screen might find application in hypobaric environments or in the treatment of hypertension. Frieden et al. (1961) reported that phloroglucinol non-comp etitively blocks the convers ion of L-tryptophan to kynurenine by the liver enzyme tryptophan pyrrolase. Eightyone percent inhibition was achieved at a concentration of 3.0 mM. Such an inhibition in vlvo could result in nicotinic acid deficiency. Mogey and Young (1949)  for phenol was found to be 1.52 mg. The relative activity, on a weight for weight basis, for phloroglucinol was found to be one-tenth that of phenol. Thus the authors concluded that phloroglucinol acted as a very weak curare antagonist.
No inhibition of rat brain acetylcholinesterase or pseudo (horse serum) cholinesterase was exhibited, nor was the pH of the medium affected enough to alter the action of the . curare. Thus the mechanism of the antagonism was unexplained.
It has been demonstrated that a variety of phenolic substances such as genistein and coume strol possess estrogenic activity (Biggers and Curnow, 1954;Bickoff et al., 1958).
The administration of such agents could result in infertility due to suppressed hypophyseal gbnadotropin resulting in a disturbed estrous cycle. Conney (1967) discussed extensively the pharmacologi cal implications of the alteration of the activity of the drugmetabolizing liver microsomal enzyme system. Specifically, the administration of certain drugs, such as phenobarbital and 3-methylcholanthrene, results in enhanced metabolism of other agents due to induction of the enzyme system. Because of this important source of drug interaction, it appears important to test all drugs and chemical agents for the ability to induce or inhibit drug metabo lizin g activity .
According to Neal and DuBois ( 1965 ), metabolism of the pesticide 0-ethyl 0-(4-nitrophenyl) phenylphosphonothioate (EPN) by rat liver microsomal enzymes provides an indication of the activity of that system, thus providing a method to investigate the effect of drugs on this metabolic pathway.
Measurement of the anti-inflammatory activity of drugs is a complex problem and many methods and models have been proposed and utilized. Grant et al. (1970) demonstrated that anti-inflammatory drugs such as aspirin and indomethacin are often capable of inhibiting the heat denaturation of bovine serum albumin solutions. Winter et al.
(1 962 ) concluded that the polysaccharide carrageenin was highly suited as a phlogistin in the rat paw edema model of inflammation. Pearson (1956) showed that Freund's com- Many mechanisms have been proposed for the action of anti-inflammatory agents. Labilization of the lysoso mal membrane and subsequent tissue dissolution due to the release of degradative lysosomal enzymes has been implicated as a mechanism underlying several types of inflammation, ranging from heat and chemical burns to arthritis (Houck and Forscher, 1968). Ignarro (1971), utilizing the release of the marker enzymes $-glucuronidase and acid phosphatase from the lysosome, has demonstrated that anti-inflammatory drugs are often capable of stabilizing the membrane of that organelle . Such stabilization may therefore partially explain the mechanism of action of many anti-inflammatory agents. Garattini et al. (1965) indicate that many chemical agents, such as hypertonic saline, acetic acid , and formaldehyde , when injected by a general route, possess antiinflammatory activity by virtue of ~heir irritant properties, which allow for a minimization of local edema formation. determined to be about 10,000. The extract will be referred to as 40SA , preceded by letters which indicate the extraction batch.
Recent evidence sugges ts that an integral part of the polyphenolic polymer is polysaccharide (Zelenski, 1973).

C. Analytical Procedures
All incubations except for determination of cholinesterase activity were carried out in a Dubnoff metabolic shaker under an atmosphere of air. All absorbance determinations were made utilizing a Beckman DB-G spectrophotometer . Unless otherwise indicated, all 40SA fractions were dissolved in water or saline.

Turbidime tric Determination of Heat Denaturation of Bovine Serum Albumin
The extent of heat-induced denatura ti on of bovine serum albumin (BSA) was quantitated by a method suggested by Grant et al. (1970). Equal amounts of 0.3 % BSA and 0 buffer or drug solution were heated at 70 C for 4 minutes, cooled in ice water for 3 minutes, maintained at room temperature for 15 minutes, and the resultant degree of turbidity was determined by the absorbance at 660 nm.
The buffer empl oyed was 0.05 M Tris acetate, pH 6.0, and was employed as the solvent for c40SA. Phloroglucinol was dissolved in buffer containing 2.5% dimethylformamide.

Plethysmographic Rat Paw Volume Determination
Rat paw volume was determined by a plethysmographic t e chnique described by Van Arman et al. (1965).
) A 30 cc capacity syringe was filled with mercury and connected by means of cannula tubing to a Statham blood pressure transducer which in turn was wired to a Grass polygraph amplifier. The introduction of objects into the mercury resulted in a linear recorder output response from the amplifier. The apparatus was calibrated by the introduction of objects of known volume. Rat paws were prepared for volume measurement by marking just above the topmost callus pad. Paws were im~ersed to this mark in the mercury during measurement .

Carrageenin-Induced Rat Paw Edema
Carrageenin was utilized as a phlogistin to induce rat paw edema as suggeste d by Winte~ et al . (1962).
Male rats were administered (ip) either saline, 90 mg/kg phenylbutazone, 50 mg/kg cortisone, or 75 mg/kg c40SA. One hour later, 0.1 ml of ~.0% carrageenin in saline was injected into the sub-plantar tissue of the hind paw, using a 26 gauge needle. The paw volume was determined immediately plethysmographically, and again three hours later, and edema formation was calculated in mrn3.

Determination of Thermogenic Activity
Thermo geni c activity was determined by monitoring the rectal temperature of rats using a Yellow Springs Instru-

9, Determinat ion of Acetylcholinesterase Activity
The activity of rat brain acetylcholinesterase was estimated by the manometric method suggested by DuBois and Mangun (1 947) employing the Warburg apparatus.
Male rats were administered (ip) either saline or 300 mg/kg ec40SA one hour prior to sacrifice for brain excision .
Ten percent brain homogenates were prepared in calcium- After another 5 minute equilibration, the manometer readings of the amount of co 2 produced by the enzymatic hydrolysis of the acetylcholine were recorded at 5 minute intervals for 30 minutes. The co 2 production was corrected for both tissue and acetylcholine blanks and for thermobarometer changes.

Cat Sciatic Nerve-Gastrocnemius Muscle Preparation
The in vivo cat sciatic nerve-gastrocnemius muscle preparation was utilized to quant itate curare antagonism.
A 3.2 kg cat was anesthetized with 1.5 g/kg urethane

Determination of Estrogenic Activity
To determine estrogenic activity, immature female rats were injected daily for three days with either saline (ip), 308 . mg/kg f40SA (ip), or 25 µg/animal estradiol-17-B(sc). The estradiol was prepared by diluting 1.0 ml of a 25 mg/100 ml 95% ethanol stock solution wit h 4.0 ml saline . On the fourth day, the uterus was excised, weighed, and the uterine-to-body weight ratio calculated.

D. Statistical Methods
The 2-tailed Student's "t" test for independent means was used to examine experimental means for statistical significance. The level of significance (P) was determined by comparison of the computed"t" value with values from standard tables. All calculated "t" values were tested at both the 0.05 and 0.01 probability levels for rejection of the null hypothesis.

Inflammation
The extract was initially evaluated by means of the albumin denaturation test , which is used to screen new drugs for anti-inflammatory activity. The results of this screening proced~re are shown in Tables 1 and 2 . Table 1 sho;;s that the heat denaturation of a 0 . 15% bovine serum albumin solution was inhibited 96% by the addition of the extract at a final concentration of 0.32 mg/ml, and that there was a dose dependent relationship at lower concentrations. '1.'able 2 shows th::tt the mo.del compound, phloroglucinol, enhanced by at least 24% the denaturation process.
Since the extract exhibited activity in the in vitro anti-inflammatory test system, determination of its effect in an intact animal model of inflammation would be valuable. Several experiments were undertaken to determine the mechanism of action of the extract as an anti-inflammatory agent in the carrageenin paw edema system. The first of these, a study of the effect of the extract on rat body temperature, is summarized in Figure 1. The extract, at a dose of either 75 or 150 mg/kg, had a negligible effect on body temperature over a period of four hours.
Since some compounds have been shown to possess antiinflammatory activity due to irritant properties, an expe riment was conducted to determine whether the extract was an irritant by virtue of its acidity. The carrageenin paw edema model was again employed, and the extract, at a dose of 100 mg/kg, was either buffered at or neutralized to pH 7.4. Table 4 shows the results of this study. The neutralized extract did not offer statistically significant protection (76% of control) while the buffered material inhibited edema formation by 42% (P .< .05).

27
Stabilization of the lysosomal membrane has also been invoked as a me chanism of action of various anti-inflammatory agents. The degree of stabilization or labilization can be inferred from the extent of release of the lysosomal marker enzymes acid phosphatase and S-glucuronidase. The extract was so examined for it s effect on the lysosomal membrane.
The results of this study are shown in Tables 5 and 6.  Phloroglucinol and 0.031 mg/ml f40SA were ineffective. Table 6 shows that the extract retains its lysosomal membrane stabilizing ability even when further chemically purified. The refined material offered statistically si gnificant (P < .01) inhibition of release of B-glucuronidase (43 % of con trol ).
The extract was further tested for anti-inflammatory activity by means of the Freund 's adjuvant-induced rat paw ede ma model of arthritis . Table 7 summarizes the results of this study. The potent steroid triamcinolone inhibited ede ma formation by 77% (P < .05), iri contrast to the extract, which provided negligible protection against this type of inflammation.

Capillary fragility
The effect of the extract on capillary fragility as determined by lung hemoglobin content following sudden decompression was examined. Table 8 is a summary of this study. None of the drugs tested, which included e40SA,  phloroglucinol, and phenylbutazone, reduced capillary fragility in this test system.

Curare, strychnine, and tetrodotoxin poisoning
The extract was tested for its ability to protect against poisoning in mice induced by curare, strychnine, or tetrodotoxin. Table 9 shows that if both curare and the extract were administered intraperitoneally, there existed protection against curare lethality. As shown in Table 10, if the curare was administered subcutaneously and the extract intraperitoneally, no protection was exhibited by the extract.
Further experimentation was performed to investigate the mechanism of action of the apparent curare antagonism of the extract. Table 11 shows that the material had a negligible in vivo effect on rat brain cholinesterase activity, in contrast to an in vitro inhibition of 53% (P < .01). The extract was also tested for its in vivo effect on the curarized cat gastrocnemius muscle. Jugular infusion of 180 mg of e40SA over a period of nine minutes exhibited no effect on the curarized muscle, in contrast to the anticholinesterase agent physostigmine, wh~ch completely restored contractile force after infusion of 0.18 mg over a period of three minutes.
To determine whether the extract might protect against poisoning due to other agents, its effects on strychnine and tetrodotoxin lethality were examined. In both cases,  1.38 ± 0.16d 47 (5 mg/ml) aExpressed as µmoles ACh/50 mg brain/10 min. bNumber of animals. cAdministered one hour prior to sacrifice. the poison and the extract were administered by the intraperitoneal route. Tables 12 and 13 show that the extract does not protect against the lethality of either strychnine or tetrodotoxin, respectively .

Liver microsomal enzymes
The possible effect of the extract on the activity of the drug-metabolizing hepatic microsomal enzyme system was determined by measuring the effect of the extract on the metabolism of the pesticide EPN. Table 14 surrunarizes the result s of the experiment. The classical inducing agent phenobarbital significantly increased microsomal enzyme activity by more than 4-fold (P < .01). The extract was ineffective in inducing this system after three days of treatment or in inhibiting the activity when given 60 minutes before sacrificing the animals. ~tophan pyrrolase Table 15 summarizes the effects of various drugs on rat liver tryptophan pyrrolase activity. In vi VO administration of hydrocortisone and f40SA resulted in increased enzyme activity (322 % control, P < .01) and inhibition (67% control, P < .01), respectively. In vivo administration of phloroglucinol had no effect on the enzyme act1.vity, while the in vitro effect of phloroglucinol was that of inhibition (~8% control, P < .01). The presence of the Eisenian extract resulted in an in vitro inhibition of 82% (P < .01).    Rat uterus The extract was tested for possible estrogenic activity by examining its ability to alter the uterine to body weight ratio in immature female rats. Table 16 shows t he results of the study. Estradiol-17-8 increased the ratio more than 2-fold (P < .01), while the extract had no effect on the ratio.  ( Grant ~al., 1970). Heat denaturation is able to produce albumin a ggregates which are i mmunolo gicall y distinct fr om the parent protein ( Maurer , 1959). Such aggregates are known to occur spontaneou sly ( F oste r~ al., 1965); these spontaneous a ggregates behave as anti gens in the etiology of at least one i mmuno-inflammat ory disease, serum sickness nephritis (Lirenman et al., 1967).

46
The extract inhibited albumin denaturation by 96 %, as shown in Table 1, in contrast to the denaturation enhancement afforded by the model comp ound, phloroglucinol ( Table   2). While the mechanism of such a stabilization is pres ently unknown , it is possible that hydrogen bonding occurs between the extract and the protein, thus increasing the stability of the molecular structure.
The in vitro anti-inflammatory screen was followed by in vivo anti-,inflammatory determinations utilizin g the carrageenin-induced rat paw e dema model of inflammation, which is least influenced by non-specific factors (Garattini et al., 1965). The mechanism by which carrageenin, a high molecular weight polysaccharide, induces paw edema is as controversial as the mechanisms of action of the agents which inhibit this edema formation. The following have been invoked as mediators of carrageenin-induced edema: kinins and 5-hydroxytryptamine (5-HT) ( Crunkhorn and Meacock, 1971), histamine (DiRos a et al., 1971), and activation of the complement system (DiRosa, 1972). It is possible that the apparent complement s ys tem activation reflects a mild antigen-antibody response to carrageenin, since high molecular weight polysaccharides as well as proteins are capable of eliciting such a response (Porter, 1967). Thus, 5-HT and histamine release may be secondary to comp lement activation.
Administration of the extract one hour prior to carrageenin injection inhibited the formation of edema by 88 %.
Accordingly, various experiments were undertaken to determine the mechanism of action of this protection. Since stimulation of the adrenal cortex, either directly or indirectly via the hypothalamus or pitui tary, can be related to antiinflammatory activity, the action of the extract was co mpared with that of a large dose of cortisone (50 mg/kg, ip), which inhibited ed ema production by 46 %. Since cortisone afforded only one-hal f the inhibition offered by the extract, it is unlikely that the extract could stimulate the production or release of sufficient corticosteroid to reduce inflammation by the extent exhibited.
The thermogenic activity of new anti-inflammatory compounds must be determined since the peripheral vasodilation which would accompany a rise in body temperature might either promote edema, due to increased capillary permeability, or reduce edema, by more rapid removal of phlogistin.
The extract was tested for its effect on body temperature, and it can be concluded from Figure 1 that the extract had a negligible effect on body temperature over a period of four hours. Thus the anti-inflammatory activity of the extract is not based on thermogenic effects.
48 Garattini et al., (1965) Table 4, and it is evident that although the anti-inflammatory potency of the extract has been decreased by neutralization, the buffered material retains statistically significant anti-inflammatory activity which is one-h alf that of non-adjusted, acidic extract solution. Thu s the mechanism of action of the extract can be partially, but not fully, explained on the basis of its irritancy.
Labi lization of t he lysosomal membrane is known to occur at site s of inj ury and has also been implicated as a major factor in the development of inflammation, since lysosomal enzymes possess the capacity to degrade co mpletely the components of the connective tissue, such as collagen , protein-mucopolysaccharide complexes, glycoprotein, and elast in (Houck and Forscher, 1968;Ignarro, 1971). Many anti.-inflammatory agents have been shown to stabilize the lysosomal membrane through their inhibition of the release of the marker enzymes B-glucuronidase and acid phosphatase from lysos omal preparations. The extract was examined in this manner. It can be concluded from the data in Table 5 that the extract is as effective as phenylbutazone as a lysosomal membrane stabilizing agent.
Since the crude extract might contain an impurity which would account for the lysosomal membrane stabilization, a refined sample of the extract was also tested for its ability to inhibit the release of 8-glucuronidase from lysosomal preparations. The results, s11mmarized in Table 6, show that the refined material is as effective as the crude in its ability to stabilize the lysosomal membrane .
The extract was further tested for anti-inflammatory activity by means of the adjuvant arthritis model, which is a delayed hypersensitivity reaction. Table 7 shows the results of the study, and it can be concluded that the extract exerted no anti-inflammatory effect against this type of inflammation . The mechanism by which effective agents, such as triamcinolone, combat this model of arthritis is obscure, but may be in part due to lysosomal stabilization (Pollock and Brown, 1971).
An hypothesis may be formulated to account for the contrasting effects of the extract on the two in vivo models of inflammation. 11 Prima1•y" membrane stabilization ( stabilization of membranes of whole cells, e.g., basophils, platelets, or mas t cells) and "secondary" membrane stabilization {stabilization of the membranes of cell organelles, e.g. lysosomes) apparently are involved in the etiology of both carrageenin and Freund's edeffia. The extract has been shown to stabilize at least one type of membrane, that of the lysosome. However, Freund's edema may be more difficult to suppress than carrageenin edema because of overwhelming participation of the immunity mechanism in the case of the former.
The extract, by virtue of its polysaccharide content, might also act as a counter-irritant.

Capillary fragility
The extract was examined for its ability to decrease capillary fragility. It should be noted that the method of Majovski (1944) was employed except in the comparison of different treatmen ts, for which a quantitative spectre-. photometric assay for hemoglobin was used in place of the arbitra ry qualitative assay originally described.
The method employed is non-specific in that the underlying mechanism of a stabilizing agent cannot be di.fferentiated from the known possibilities, which include effects on the 1) endothelial cell membrane, 2) endothelial cell junction, and 3) sources of possible chemical intermediates (such as basophils, and platelets, and plasma kinins).
None of the drugs tested enhanced capillary stability I (Table 8) in cont rast to the effect of phloroglucinol reported by Okanishi and Shimaoka (1952). Close examination of the results shows that the method is not sufficiently sensitive to quantitate the possible stabilizing effects of drugs, inasmuch as the chamber control animals exhibited only a 13% increase in lung hemoglobin compared to the nonchamber animals. This increase is not statis tically significant.
Curare, strychnine , and tetrodotoxin poisoning The extract was tested for its effecti veness as an antagonist against curare, strychnine, and tet rodotoxin, because of the report of Mogey and Young (1949) that phloroglucinol acted as a mild curare ant agon ist in vitro.
These three agents act on the nervous sys te m dissimilarly: 51 1) curare, the classical neuromuscular bl ccker, competitively blocks postjunctional acetylcholine recep or sites, 2) strychnine, a CNS stimulant, acts by bloc~i ng post-synaptic inhibition, and 3) tetrodotoxin selective ly blocks axonal conduction.
Since the extract was ineffective in pre venting curare poisoning when administered via a different route than that of the curare, in contrast to the protection exhibited when the routes of administration were identical (Tables 9-10), a true pharmacological antagonism would seem an unlikely mechanism of action of the extract. This was supported by demonstrating that 1) the extract was an ineffective in vivo cholinesterase inhibitor (Table 11) and 2) the extract exerted no antagonism to the action of curare on the in vivo cat gastrocnemius muscle-sciatic nerve preparation.
An alternative mechanism of action might be based on the occurrence of a chemical interaction of the negatively charged extract molecule with the positively charged quaternary nitrogen of the curare molecule. Further experimentation in this context failed to support such a mechanism: while no protection was offered against the lethality of strychnine, which contains an uncharged tertiary nitrogen (Table 12), neither was there protection a gai nst poisoning by tetrodotoxin which contains a positively charged guanidinium group (Table 13). An untested hypothesis for the curare protection might be an inhibition of absorption of the curare from the peritoneal cavity.

Liver mi cro somal enzymes
In order to more fully characterize the action of a new drug, its interaction with other drug s must be examined. One potential site of drug interaction is the liver microsomal electron transport system, an enzyme system which is responsible for the biotransformation of a wide variety of drugs and other chemical agents. Most important relative to drug interactions are the stimulation (induction) and inhibition of the system.

53
The extract was tested for possible microsomal induction or inhibition (Table 14) by measuring the effect on the metabolism of the insectide EPN. The extract was observed to have no effect on microsomal enzymes after administration daily for three days or one hour following a single dose.  Table 15, indicate that the extract, both in vivo and in vitro, caused enzyme inhibition.
Such inhibition could result in nicotinic acid deficiency or i.n increased synthesis of serotonin, which might result in stimulation of cerebral activity.

Rat uterus
A variety of naturally occurring phenolic materials have been shown to exhibit estrogenic activity (Biggers and Curnow, 1954); the widespread metabolic consequences of such activity se emed sufficient justification for determining ~1e ther the extract acted in a similar manner.
The uterus, target organ for estrogen, is most frequently employed to bioassay for estrogenic activity . The response of this organ to estrogenic stimulation is biphasic in nature: initially, hyperemia and imbibition of water occur, followed later by hypertrophy and hyperplasia; thus an increase in the uterine to body weight ratio can be demonstrated.
The ext ract was found to cause neither estrogenic stimulation nor retrogression of this organ (Table 16).

VI SUMMARY AND CONCLUSIONS
1. An eitract from Eisenia bicyclis containing principally a polyphenolic polymer inhibited the heat denaturation of a 0.15% bovine serum albumin by 96% when added to the i n cubation medium at a concentration of 0.32 mg/ml.
This was suggestive of possible anti-inflammatory activity.
2. As determined by paw volume changes , administration of the extract at a dose of 75 mg/kg one hour prior to carrageenin injection inhibited edema production by 88%. 8. The mechanism of action of the apparent curare antagonism of the extract appeared to be chemical rather than pharmacological, since the material had no effect on either the curarized cat gastrocnemius muscle in vivo or the activity of rat brain cholinesterase in vivo.
An exact chemical mechanism cannot be formulated because the material was ineffective in preventing poisoning by either another positively charged toxin, tetrodoto xin, or an uncharged poison, strychnine.
9. The extract was observed to have no effect on liver microsomal enzyme activity after administr~tion of 300 mg/kg daily for three days or one hour following a single dose of 300 mg/kg.
10. The administration of the extract at a dose of 300 mg/kg 1.5 hours prior to sacrifice resulted in 33% inhibition of the activity of tryptophan pyrrolase.
The presence of the extract at a concentration of 486 µg/ml resulted in an in vitro inhibition of 82%.
11. The extract, administered at a dose of 300 mg/kg daily for three days, did not alter the uterine to body weight ratio in immature female rats.