Tissue fractions of sea cucumber for the treatment of inflammation

The present invention relates to fractions of sea cucumbers (Phylum Echinodermata, Class Holothuroidea), particularly those derived from the epithelial layers and the flower portion, that markedly inhibit inflammation in laboratory animals and show significant subjective benefit in humans with inflammation disorders such as arthritis, and methods for preparing the same.

FIELD OF THE INVENTION 
The present invention relates to compositions for treating warm-blooded 
animals afflicted with inflammation disorders. More particularly, the 
present invention relates to the treatment of certain inflammatory 
conditions in patients, by administering distinct fractions of the 
echinoderm sea cucumber which consists of: 
1. the isolated body wall of the sea cucumber, or its active derivatives; 
2. the epithelial layer of the sea cucumber body wall, or its active 
derivatives, 
3. the anterior flower or mouth portion of the sea cucumber, or its active 
derivatives, or 
4. combinations thereof. 
BACKGROUND OF THE INVENTION 
Mammals are known to suffer from various conditions involving inflammation 
with concomitant swelling, tenderness, decreased mobility, pain and fever. 
While a number of anti-inflammatory agents are effective in the 
symptomatic treatment of such inflammatory conditions as Rheumatoid 
Arthritis, Rheumatoid Spondylitis, Osteoarthritis, degenerative joint 
diseases, and the like, many such agents have a number of undesirable side 
effects, such as gastric irritation and the like. 
The etiology and pathogenesis of rheumatic and arthritic diseases remain 
obscure. Meanwhile, the need continues for safer, better calibrated drugs 
which will slow the process and alleviate the symptoms of inflammatory 
diseases. For example, in Rheumatoid Arthritis, any agent which reduces 
inflammation is important in lessening or delaying the development of 
crippling. 
It is the object of this invention to provide a new method for treating 
inflammatory conditions and slowing the development of arthritic 
conditions, without the side effects associated with previous treatments. 
SUMMARY OF THE INVENTION 
This invention provides a method of treating inflammation disorders in a 
warm-blooded animal in need of such treatment which comprises 
administering to said warm-blooded animal a therapeutically effective 
amount of a composition comprising the isolated body wall of a sea 
cucumber, the isolated epithelial layer of the body-wall of the sea 
cucumber, the flower of the sea cucumber, their active derivatives or 
mixtures thereof. 
It has been found that these portions of the sea cucumber dramatically 
inhibit inflammation in laboratory animals in assays which are known in 
the medical field as being acceptable in providing relevant data and valid 
indications of therapeutic efficacy for subsequent development and medical 
use of active compositions.

DETAILED DESCRIPTION OF THE INVENTION 
The sea cucumbers constitute the taxonomic Class Holothuroidea, Phylum 
Echinodermata. They possess an elongated body comprising a thick, leathery 
body wall of epithelial and collagenous layers surrounding the internal 
organs or viscera, an anterior mouth surrounded by numerous retractile 
tentacles (herein referred to as the "flower"), and a posterior portion 
comprising cloaca and anus. Muscle bands are found along the length of the 
interior surface of the body wall. 
Sea cucumbers are a well-known Chinese delicacy harvested from many areas 
of the world and are a valuable trading resource in Chinese-speaking 
countries. There are a number of patent applications by Chinese groups 
relating to sea cucumbers as nutritional supplements (e.g., Chinese 
application CN 1065019) and patents or applications from Japanese groups 
relating to various carbohydrate moieties from sea cucumber as 
anticoagulants (JP 94070085 B2; WO 9008784) and as active components for 
treating AIDS (WO 9202231; WO 9009181). Historically, sea cucumbers for 
the worldwide market have been harvested, boiled with the muscles intact, 
and then salted and dried over an open flame. Salting and drying are the 
traditional methods of obtaining a product that is safe for storage and 
transportation. Nutritional supplements have been prepared by finely 
dividing these salted and fire-dried sea cucumber body walls for use in 
encapsulated products. 
Pharmaceutical companies are expanding efforts to screen and assay 
biologically active compounds from natural sources. The term that has been 
applied to this discovery process is "bio-prospecting." When 
bio-prospecting is successful in finding and identifying promising 
compounds, efforts are then made to determine and perfect the process by 
which the compound is produced in its active form. Useful processes 
develop from these bio-prospecting discoveries, as well as useful 
compositions of matter and methods of using the same. 
Sea cucumber tissue has been found to contain numerous compounds having 
potential as biologically active agents in medical and veterinary 
applications. These include sulfated polysaccharides (e.g. fucosylated 
chondroitin sulfate, Viera & Mourao, JBC, vol. 263, pp. 18176-83 (1988)) 
sterol glycosides, saponins (e.g., frondogenin and its glycosides, Findlay 
et al., J. Natural Products, vol. 47, pp. 320-324 (1984)), lactones (e.g., 
triterpenoid lactones, their acetates and glycosides, Findlay et al., 
supra), peptides, protamines, glycogens, saccharides (e.g. fucose, 
galactosamine, glucuronic acid, quinovose, xylose or o-methylglucose, 
Findlay et al., supra), polysaccharides (e.g., polyfucose sulfate, WO 
9202231) and various amorphous compounds rich in saccharide moieties 
(Findlay et al., supra). Fractions derived from sea cucumber have now been 
found to possess strong anti-inflammatory activity. This anti-inflammatory 
property can be used in numerous applications in research and medicine, 
particularly those relating to rheumatic and arthritic conditions, serine 
protease-mediated inflammation and the classic complement cascade. 
As used herein, the term "sea cucumber" refers to any species of the Phylum 
Echinodermata, Class Holothuroidea, such as species of the genera 
Actinopyvga (e.g., A. lacanora, L. echinites), Cucumaria (e.g., C. 
frondosa, C. echinata, C. chronhjelmi), Eupentacta (e.g., E. 
quinquesemita), Halodeima (e.g., H. cinerascens), Holothuria (e.g., H. 
pervicax, H. atra, H. edulis, H. scabra, H. monoacaria, H. leucospilota), 
Leptosynapta (e.g., L. inhaerens), Ludwigothuria (e.g., L. grisea), 
Microthele (e.g., M. nobilis), Molpadia (e.g., M. musculus), Parastichopus 
(e.g., P. nigripunctatus), Paracaudina (e.g., P. chilensis), Pelagothuria, 
Pentacta (e.g., P. australis), Polycheira (e.g., P. rufescens), Psolus 
(e.g., P. chitonoides), Stichopus (e.g., S. japonicus, S. chloronovus, S. 
variegatus), Synapta (e.g., S. maculata), Thelenota (e.g., T. ananas) or 
Thyone (e.g., T. briareus); 
the term "flower" refers to the anterior portion of the sea cucumber 
comprising the mouth and retractile tentacles; 
the term "B1000" refers to the isolated epithelial layer of the sea 
cucumber, substantially free of the flower portion, muscle, collagenous 
tissues and viscera; 
the term "T2000" refers to the isolated flower portion of the sea cucumber, 
substantially free of other portions of the sea cucumber body; 
the term "active derivative" refers to any compound, fraction or 
combination thereof, derived from a sea cucumber fraction described herein 
that has anti-inflammatory activity; 
the term "inflammation disorder" refers to any condition or disease in a 
warm-blooded animal having inflammation as a symptom or proximate cause. 
Active anti-inflammatory compositions can be obtained from sea cucumber in 
a variety of ways. For example, sea cucumbers can first be cleaned of 
muscle bands and viscera, boiled (but not salted), preferably for about 
1/2 hour, and then dried, preferably in low-heat mechanical driers such as 
those employing "heat pump" technology. The dried tissue can further be 
ground or divided as needed for ultimate use. This process decreases the 
sodium content of the tissue and helps protect various active ingredients 
from degradation. This fraction can be formulated and used directly as an 
anti-inflammatory composition, either above or in combination with other 
sea cucumber fractions, or as a raw material for further purification of 
active derivatives. A commercial sea cucumber body wall preparation is 
available from Coastside Bio Resources under the tradename "Ginseng of the 
Sea.TM.." 
Another active fraction can be obtained from the flower portion of the sea 
cucumber. During the evisceration process described above, the anterior 
portion ("flower") of the sea cucumber is cut away from the viscera and 
body wall. The isolated flower is then heated, preferably for about 1/2 
hour, dried at low temperatures (e.g. between about 140.degree. F. and 
about 180.degree. F. using conventional drying apparatus and per se known 
techniques). This dried fraction, designated "T2000" by the inventor, can 
then be ground or divided as needed for formulation and use directly as an 
anti-inflammatory composition, either alone or in combination with other 
sea cucumber fractions, or as a raw material for purification of active 
derivatives. The method and extent of division of the material is not 
critical to the invention, and can readily be determined by those skilled 
in the art according to the manner in which the composition will be used. 
Still another anti-inflammatory fraction can be obtained from the 
epithelial layer of the sea cucumber body wall. Muscle, viscera and flower 
are removed as described above, followed by isolation of the epithelial 
layer of the sea cucumber body wall from the harder collagenous layers 
beneath, preferably by one or more of the following means: 
heating the body-wall in water at temperatures from about 140.degree. F. to 
about 180.degree. F., preferably at about 170.degree. F., followed by 
mechanical separation by hand or machine (e.g., using machines known in 
the art as mincers or de-boners, which detect tissue density and separate 
harder tissues from softer tissues); 
enzymatic hydrolytic separation, using, e.g., organism's own digestive 
tract enzymes, proteases from mammalian sources, proteases from 
nonmammalian sources or acidic hydrolazes, preferably Alcalase (NOVO 
Nordisk Bio Chem, North Carolina), the enzyme preferably being in a 
solution of about 1% to about 10% enzyme, most preferably in a solution of 
about 10% enzyme; 
scouring/scrubbing or de-boning processes known to those skilled in the 
potato or chicken processing arts. 
Heating in water, followed by mechanical separation using a de-boner is 
most preferred. 
The epithelial fraction so obtained (designated "B1000" by the inventor) is 
a dark, moist, viscous, carbohydrate-rich matter. B1000 can be dried as 
described above, formulated and used directly as an anti-inflammatory 
composition, either alone or in combination with other sea cucumber 
fractions, or used as a raw material for the purification of active 
derivatives. 
One embodiment of the present invention is the use of fractions derived 
from sea cucumber as inhibitors of detrimental aspects of mast cell 
products at a site of inflammation, based on their activity as serine 
protease inhibitors. Mast cells have been found to be implicated in 
diseases and events such as allergic and non-allergic Rhinitis, Atopic 
Dermatitis, including Psoriasis, Crohn's Disease, Cluster headaches, and 
Coronary Artery Spasm. It is now recognized that mast cells are implicated 
in the pathophysiology of inflammatory skin diseases as well as in other 
physiological disorders. Neutrophils as indirect mast cell products in an 
inflammatory response are a main source of serine proteases which are 
important in the tissue damage resulting from inflammation. Sea cucumber 
fractions, found to act as inhibitors of neutrophil rich granuloma in an 
antiphlogistic compound bio-assay such as that disclosed in Roberet, et 
al., Acta Endocrinologica, vol. 25, pp. 105-112 (1957) (incorporated 
herein by reference), can be considered to have specific activity for mast 
cell mediators or the proteases derived therefrom. 
Serine protease inhibitors have been found to play a major role in the 
direct inactivation of the mediators of inflammation so that the normal 
wound healing process can be accelerated without interference from the 
excess of materials released at the site of inflammation. The activity of 
sea cucumber fractions as inhibitors of serine protease would also make 
them useful deactivators of those mediators of inflammation which may not 
yet be recognized or are found in association with a particular disease. 
Another embodiment of the present invention is the use of sea cucumber 
fractions to inhibit the blood protein C1 of the Classical Complement 
portion of the immune system. This activity of Classical Complement 
inhibition is a component of a direct or indirect biological pathway of 
inflammation inasmuch as various portions of the Classical Complement 
cascade are being inhibited which would otherwise trigger inflammatory 
responses. 
Still another embodiment of the present invention is the use of sea 
cucumber fractions in humans to produce a subjective benefit in cases of 
decreased range of motion or arthritis inflammation. 
The sea cucumber fractions of the present invention may be in the form of 
powders, capsules, liquids, suspensions, ointments, or any other means of 
delivery which those skilled in the arts would deem appropriate. The 
formulation is dictated by the application, e.g., an application wherein a 
skin malignancy is treated might call for a topical formulation, whereas 
treatment of a liver malignancy might call for a formulation suitable for 
direct injection into the site of malignancy. It is well with the skill of 
the medical and veterinary arts to determine a suitable formulation for 
any particular application. For example, a topical ointment would be most 
appropriate for treatment of arthritic inflammation of the hands, while a 
rectal formulation might be most effective for treated inflammation of the 
colon. Furthermore, methods of making such formulations are well-known in 
the art (see, e.g. Remington's Pharmaceutical Sciences, 18th ed., Mack 
Publishing Co., Easton, Pa. (1990)). The anti-inflammatory compositions of 
the present invention may be administered orally, topically, rectally or 
via injection, alone or in mixture with an excipient or a carrier as set 
forth above and in accordance with the particular purpose of use. The 
active ingredient can be within a range of from about 0.01 to about 100 
w/w %, preferably from about 2 to about 80 w/w %. The dose per day 
thereof, also depending upon the particular use to which the composition 
is put, the frequency of administrations, the form of medicament, the 
symptoms, age and body weight of the recipient of the composition, can be 
within the range of about 0.1 to about 1,500 mg of the effective 
ingredient per kg of body weight, preferably from about 1 to about 1,000 
mg/kg, and most preferably about 10 mg of the effective ingredient per kg 
of body weight. The daily dosage of administration may be divided into two 
to four separate doses. 
The following examples are intended to illustrate, but in no way to limit, 
the invention set forth in the claims. 
EXAMPLE 1 
Preparation of Whole Body Wall From Sea Cucumber 
Muscle meat, viscera, anterior and posterior portions of the sea cucumber 
Cucumaria frondosa were removed in order to leave a sea cucumber body wall 
free of most, if not all of the above named portions. The thus obtained 
body wall was boiled for about 1/2 hour in fresh water and dried in a low 
heat utilizing a 40 hp "heat-pump" dryer (Southwind Mfg., Nova Scotia, 
Canada). The body wall fraction was dried to about 3% moisture and finely 
divided. 
EXAMPLE 2 
Mechanical Extraction and Processing of Sea Cucumber Epithelium 
A fraction termed B1000, consisting of sea cucumber epithelium, was 
produced by the following method. The anterior, posterior, viscera and 
muscles were removed from sea cucumbers of the species Cucumaria frondosa 
to obtain an isolated body wall. Body wall portions thus obtained were 
heated for about 30 minutes in fresh 170.degree. F. water, then cooled on 
wire racks to room temperature. Next, the body wall portions were passed 
through an industrial machine known to those in the food processing arts 
as a deboner or mincer (Paoli Machine, Illinois). The deboner was adjusted 
to separate the softer outer epithelial layer from the harder collagenous 
portion of the body wall. The black viscous layer of the epithelium so 
separated, designated B1000 by the inventor, was dried by conventional 
means using a 40 hp "heat pump" dryer as in Example 1 to approximately 3% 
moisture content and finely divided to obtain a powder. 
EXAMPLE 3 
Enzymatic Extraction and Processing of Sea Cucumber Epithelium 
Enzymes were used to help separate the epithelial layer from the harder 
collagenous inner layer of body walls from sea cucumbers of the species 
Cucumaria frondosa. The body wall portions were isolated and heated in 
water as described in Examples 1 and 2. They were then soaked in a 
solution of 10% Alcalase enzyme (NOVO Nordisk Bio Chem, North Carolina) in 
fresh water at a temperature of 130.degree. F. (.+-.30.degree. F.). The 
time of soaking depended on the condition of the particular lot of body 
walls and their characteristics, and varied from about 15 min. to about 3 
hours. The average time soaking in the enzyme solution was about one half 
hour. The body walls were then removed from the enzyme solution and 
processed by hand to further isolate the black epithelial layer B1000 from 
the underlying collagenous tissues. The B1000 thus obtained was dried and 
powdered as in Examples 1 & 2. 
EXAMPLE 4 
Extraction and Processing of Sea Cucumber Flower 
A fraction termed T2000, derived from the sea cucumber flower, was obtained 
in the following manner. 
During the processing operation of removing viscera and muscle set forth in 
Examples 1 and 2, the anterior portion of the sea cucumber Cucumaria 
frondosa was removed, taking care to include the mouth portion of the head 
with surrounding tentacles, which is a tissue rich in calcium carbonate 
(among other compounds). This separated flower portion was then boiled for 
about 1/2 hour to obtain the fraction designated T2000 by the inventor. 
The T2000 was then dried in a conventional heat-pump dryer as in Examples 
1-3 and finely divided. 
EXAMPLE 5 
Preparation of Derivative Fractions of B1000 and T2000 
The finely divided powders of epithelial layer (B1000) and flower fraction 
(T2000) obtained in Examples 2 and 4, respectively, were further processed 
by mixing in an aqueous solution and rotating for 12 hours with a magnetic 
stirrer. The resultant solution was centrifuged at 30,000 RPM for one hour 
and the supernatant was removed and lyophilized. 
EXAMPLE 6 
Antiphlogistic Activity of B1000 and T2000 as Determined by the "Rat 
Granuloma Pouch Assay" 
Method: Roberet, A. and J. E. Nezamis, "The Granuloma Pouch as a Routine 
Assay for Antiphlogistic Compounds," Acta Endocrinologica: 25, p. 105-112 
(1957) (incorporated herein by reference). 
Procedure: Thirty-nine Sprague-Dawley male white rats, weighing about 
300-350 grams each, were used in the test. They were divided into 7 groups 
of 5 rats each and 1 group of 4 rats. 
The rats were confined to individual cages in the animal room. The 
temperature was maintained at 23.degree. C..+-.1.degree. C. The rats had 
free access to Rodent Teklad Laboratory Diet and fresh water at all times. 
The rats did not have any disease or exhibit any abnormalities. 
The rats were clipped over their dorsal surface with a pair of fine 
electric clippers. The rats were then anesthetized with Sodium 
Pentobarbital, given intraperitoneally (0.5 ml equivalent to 3 mg). Using 
a sterile syringe, 25 ml of air was injected under the skin to produce a 
pneumoderma. All the pouches were made just over the shoulder blades. This 
was followed by 0.5 ml of a sterile 1% solution of croton oil made up in 
corn oil, injected directly into the pouch. 
Samples were prepared by mixing powdered B1000, T2000, or sea cucumber body 
wall fraction with corn oil. All of the rats were treated for a total of 
five days with the respective compounds made up on a volume of 0.25 ml. 
All treatments were given by gavage. Dose was 10 mg/kg body weight. 
After five days of treatment, the rats were sacrificed and the volume of 
exudate was measured with a hypodermic syringe. Scissors were used to open 
each pouch to be certain that all of the exudate had been removed. The 
results are summarized in Table I. 
TABLE I 
______________________________________ 
Comparison of treatment groups, average pouch 
exudates and body weight gains. Dose - 10 mg/kg body weight. 
Pouch Mean Body 
No. Rats Exudate 
Weight 
Group Treatment Per Group (ml) (g) 
______________________________________ 
I Neg. control - 
5 9.48 +21.2 
water 
II Pos. control - 
5 3.74 -32.4 
hydrocortisone 
III T2000 5 2.88 +12.0 
IV B1000 5 2.86 +5.4 
V sea cucumber 
5 6.84 -7.0 
body wall 
VI sea cucumber 
5 4.18 -15.0 
body wall 
VII sea cucumber 
5 6.14 +4.0 
body wall 
VIII sea cucumber 
4 6.70 +1.0 
body wall 
______________________________________ 
Pouch Exudate: The average pouch exudate varied from 9.48 ml in the water 
treated group (negative control) to 2.86 ml in the B1000 treated group. 
T2000 gave an exudate value of 2.88 ml. These were excellent values 
compared to the positive control, hydrocortisone 3.74 ml). In fact, they 
were better than 10 mg/kg of hydrocortisone but without the weight loss 
seen in the hydrocortisone treated group. 
The other sea cucumber preparations had average pouch exudate values 
4.18-6.84 ml per rat. These were better values than the negative control 
of 9.48 ml. It is difficult to correlate the weight loss or low weight 
gain with the anti-inflammatory activity. 
Body Weights: Body weight gain ranged from 21.2 for the negative control to 
a weight loss of 32.4 g. per rat in the hydrocortisone treated group. 
The T2000 group and the B1000 group had weight gains of 12 grams and 5.4 
grams, respectively. Both these groups showed excellent anti-inflammatory 
response. Groups treated with sea cucumber body wall had both weight gains 
and weight losses. 
It appears that the anti-inflammatory effect is unrelated to the weight 
loss in the sea cucumber products in the treated groups, sea cucumber 
products. There must be other growth inhibition present. 
Summary: The rat granuloma pouch test for anti-inflammatory activity of 
various sea cucumber fractions or extracts, when compared to a negative 
control (water) and a positive control (hydrocortisone) indicate the 
following: 
1. T2000 and B1000 have an anti-inflammatory activity better than 10 mg. 
hydrocortisone. 
2. The mean exudate for the water control was 9.48 ml and for the 
hydrocortisone treated group was 3.74 ml. 
3. Sea cucumber body wall caused pouch exudate up to 4.18 ml, which 
indicates it is almost as good as 10 mg hydrocortisone per kilogram body 
weight. 
EXAMPLE 7 
Anti-inflammation Activity of B1000 as Determined in a Mouse Model of P. 
aeruginosa Infection Inflammation 
Materials and Methods 
Bacterial strains and preparation of inocula: P. aeruginosa 6294 was kept 
frozen at -85.degree. C. as individual aliquots in trypticase soy broth 
with 15% glycerol. The bacteria from these frozen stocks were inoculated 
onto trypticase soy agar overlaid with a 12,000 MW cutoff dialysis 
membrane {836}. After overnight growth, the bacteria were harvested from 
the membranes with a sterile cotton swab and suspended in sterile 1% 
proteose peptone to an optical density at 650 nm of 0.92 to achieve a 
bacterial concentration of approximately 2.times.10.sup.9 cfu/ml. The 
bacteria were diluted 1:10 sterile 1% proteose peptone to achieve the 
desired final concentration. The actual bacterial concentration was 
confirmed by plating of appropriate dilutions, in duplicate, onto 
Maconkey's agar. The dose of 6294 required to establish infection in 50% 
of mice challenged with this strain (ID50 value) is 1.93.times.10.sup.5 
cfu (95% confidence interval; 3.25.times.10.sup.4 -16.6.times.10.sup.5). 
Thus, the challenge dose for this experiment is approximately 50 times the 
ID50 value. 
Infection of Mice: C3H/HeN female mice (8 weeks old) were obtained from 
Charles River Breeding Laboratories, Wilmington, Mass. Groups of 5 mice 
were given an intraperitoneal injection of 0.2 ml of a cocktail containing 
6.7 mg of ketamine hydrochloride (Parke Davis, Morris Plains, N.J.) and 
1.3 mg of xylazine (Haver, Shawnee, Kan.) per ml. After the animals were 
anesthetized, they were placed under a stereoscopic microscope, and three 
1-mm-long scratches were made in the corneal epithelium and superficial 
stroma with a 27-gauge needle. Care was taken not to penetrate the stroma 
to the anterior eye chamber. The bacteria were immediately inoculated onto 
the abraded cornea in a 5-.mu.L volume dispensed from a micropipette. The 
mice usually regained consciousness 15-25 minutes after inoculation. All 
procedures were in accordance with the Association for Research in Vision 
and Ophthalmology Resolution on the Use of Animals in Research and were 
approved by the Harvard Medical Area Standing Committee on Animals. 
Grading of corneal infection: Infected eyes were graded every 24 hours 
after inoculation for a total of six days by an investigator unaware of 
the treatments that the groups received. The following grading scheme was 
used: grade 0, eye macroscopically identical to the uninfected 
contralateral control eye; grade 1, faint opacity partially covering the 
pupil; grade 2, dense opacity covering the entire anterior segment; grade 
4, perforation of the cornea and/or phthisis bulbi (shrinkage of the 
eyeball following inflammatory disease). 
Preparation of derivative fraction of B1000 and administration to mice: 
Powdered B1000 was dissolved in sterile distilled water at a concentration 
of 10 g in 100 ml (10%). The sample was mixed by stirring overnight on a 
magnetic stirrer. The insoluble material was pelletted by centrifugation 
at 13000.times.g for 30 min. and the supernatant was harvested and 
lyophilized. Approximately 1.6 grams of material were recovered after 
lyophilization. 
Treatment of Mice: B1000 derivative was prepared as described above. The 
lyophilized powder was dissolved in the drinking water at a concentration 
of 200 .mu.g/ml, sterilized by filtration through a 0.45 .mu.m filter and 
put in sterilized water bottles. The bottles were given to the mice 
immediately after infection. The mice were given a fresh preparation of 
B1000 derivative in new water bottles after 3 days. 
Statistical analysis: Mann-Whitney U test for non-parametric data was used 
to determine the statistical significance of the corneal scores on the 
given days. 
Results: The results of the macroscopic grading of the infected eyes in the 
treated and untreated groups are shown in Table II. 
TABLE II 
______________________________________ 
Days post 
infection corneal scores 
corneal scores 
Significance 
______________________________________ 
1 1,1,1,1,1 2,2,2,2,2 p = .009 
2 1,1,2,2,2 2,3,3,3,3 p = .0216 
3 1,1,3,3,3 3,3,3,3,3 not significance 
4 1,1,3,3,3 3,3,3,3,3 not significance 
5 1,1,3,3,3 3,3,3,3,3 not significance 
6 1,1,3,3,3, 
3,3,3,3,3 not significance 
______________________________________ 
These data indicate that a derivative of B1000 significantly reduced 
inflammation as indicated by the reduced corneal scores on the first two 
days after infection with 10.sup.7 cfu per eye with P. aeruginosa 6294. 
After 48 hours, although there was a trend toward reduced inflammation 
these groups were not significantly different. 
EXAMPLE 8 
Inhibition of Tissue Plasminogen Activator by B1000 
B1000 prepared as in Example 5 was assayed for its ability to inhibit 
tissue plasminogen activator by performing a zymographic analysis of 
conditioned medium from C8161 human melanoma Cells treated with 1, 10 and 
100 .mu.g B1000/ml 
Twenty-four hour serum-free conditioned media from the upper wells of the 
Membrane Invasion Culture System containing C8161 human melanoma cells 
with and without different concentrations of B1000 (lanes -4) were mixed 2 
parts to 1 part Laemmli sample buffer minus reductant. Without prior 
boiling, 35 .mu.l of these samples were loaded onto a 10% polyacrylamide 
SDS-gel containing 0.1% gelatin plus plasminogen in the resolving gel and 
no gelatin/plasminogen in the stacking gel. After electrophoreses, the 
gels were incubated at room temperature in 50 mM Tris-Cl pH 7.5 plus 2.5% 
Triton X-100 for 30 minutes with gentle shaking, then incubated for 25 
hours in 100 mM glycine pH 8.3/10 mM EDTA at 37.degree. C. After staining 
with Coomassie Brilliant Blue R-250 (0.25%) in 25% isopropanol/10% acetic 
acid for 30 minutes, the gel was destained in 10% methanol/10% acetic acid 
until the was remained clear. A diminution in the tissue plasminogen 
activator (tPA) double was seen at approximately 65 kDa) with increased 
dosages of B1000. 
EXAMPLE 9 
Inhibition of Complement by a Derivative of B1000 
Preparation of B1000 derivative: B1000 was pulverized with a mortar and 
pestle and put into a tube containing 0.15M NaCl at a weight to volume 
ratio of 100 .mu.g/ml. The tube was rotated to mix at room temperature 
overnight and the following morning it was centrifuged to spin down the 
undissolved material. The supernatant was removed, and after final 
filtration through a 0.22 .mu.m filter, the B1000 derivative was put into 
a sterile tube, capped tightly and stored at 4.degree. C. 
The undissolved material was dried and weighted, and about half was found 
to have gone into solution. Culture of the unfiltered B1000 derivative on 
blood agar plates yielded several colonies of at least two different 
bacterial species, which were not further identified. 
Experiment with complement inhibition: Blood was collected from a healthy 
donor and allowed to clot at room temperature for 60 minutes. The serum 
was removed from the blood clot, and transferred to a clean tube. 
Complement activators with known activities were prepared as positive 
controls. These included heat-aggregated gamma globulin (63.degree. C., 30 
minutes) and zymosan. The former (HAGG) is a potent activator of the 
classical pathway, and also activates the alternative pathway weakly. The 
latter (Z) consists of boiled and washed bakers' yeast and is a strong 
activator of the alternative pathway. HAGG was used at 14 mg/ml and Z at 
10 mg/ml. These are relatively high doses. The B1000 extract was used 
straight. 
The experiment was done by mixing 8 parts of the normal human serum (NHS) 
with 2 parts saline, saline plus activator (or B1000 extract), or 
activator plus B1000 extract. These mixtures were incubated for 30 minutes 
at 37.degree. C. and the complement was examined by assaying total 
complement activity (CH50), C4d, Bb and iC3b split products. Results are 
shown in Table III. 
TABLE III 
______________________________________ 
Incubation mixture 
CH50 C4d Bb iC3b 
______________________________________ 
NHS + saline (neg 
318 5.33 2.3 119.0 
control) 
NHS + HAGG + saline 
0 25.75 32.67 
861.0 
NHS + Z + saline 
75 6.43 57.45 
585.0 
NHS + B1000 + saline 
0 4.38 17.32 
115.5 
NHS + HAGG + B1000 
0 5.92 36.84 
242.5 
NHS + Z + B1000 
0 4.52 74.17 
436.3 
______________________________________ 
FIGS. 1A-D are graphic representations of the data of Table 3. It can be 
seen in FIG. 1A that the B1000 by itself inhibited the classical pathway 
activation necessary for CH50 activity. In FIG. 1B, the C4d assay, the 
HAGG caused a good increase of C4d which it is supposed to do (classical 
pathway activation) but this increase in C4d was blocked by the B1000. In 
the Bb assay, there was some activation of the alternative pathway by 
B1000. This could be due to the polyanions or it could be due to 
endotoxins released from the contaminating bacteria (which were filtered 
out, but any soluble products from them would still have been present). 
There was no decrease in Bb produced by the HAGG or Z, and the increase 
seen was probably an additive effect of the B1000 activation by itself. In 
the iC3b assay, it appeared that the B1000 did nothing by itself and that 
it blocked most of the HAGG-, and some of the Z-mediated iC3b production. 
Dose-response of B1000 effect on CH50: In order to find a dose of B1000 
that didn't block 100% of the CH50 response (to determine when a slight 
change is happening in other assays) we mixed NHS (as above) with 
dilutions of B1000 made in saline. These were incubated at 37.degree. C. 
for 30 minutes, and then the CH50 assay was done. Results are shown in 
FIG. 2. 
EXAMPLE 10 
Nine subjects exhibiting moderately severe to severe arthritic pain were 
given Ginseng of the Sea.TM. sea cucumber body wall preparation for 
periods ranging from 8 to 16 weeks. All patients used a 500 mg capsule 
supplement orally in the following manner: 3 capsules in the morning and 3 
capsules in the evening for 3 weeks, then 2 capsules in the morning and 2 
in the evening subsequently. Occasionally, as noted, the patients reduced 
to 2 capsules per day after the target joint was significantly improved 
for maintenance supplementation. 
A rating scale of 1 to 10, with 1 being very mild and occasional pain which 
does not limit activity in any way, to 10 which is severe, continuous pain 
which prevents normal activity, is used as a subjective pain measurement 
tool in this study. 
An 81 year old gentleman presented with left shoulder adhesive capsulitis 
of one year duration. He rated the pain as a 7 with inability to abduct of 
forward flex the left shoulder. Dressing was very painful. After 8 weeks 
supplementation beginning in January, 1996 he rated the pain as a 1 and 
could fully abduct the shoulder and fully extend the shoulder to a full 
reach above his head with only minimal pulling feeling. No chiropractic 
treatments were given to the shoulder. 
A 50 year old female presented with cervical spine arthritis of 6 years 
duration. Radiographs revealed cervical degenerative disc and joint 
disease at C5, 6 and 7. Pain was rated at 5. She had loss of range of 
motion. She had been treated for 18 months with temporary resolution of 
cervical joint pain subsequent to adjustment. Following 12 weeks of 
supplementation beginning October, 1995 she rated the pain as 2 and had 
markedly increased ROM of the cervical spine. 
A 44 year old male presented with chronic thoracic spine pain of 13 years, 
status post lifting injury at work. Radiographs revealed degenerative 
joint disease with loss of disc height and osteophyte formation at T10, 11 
and 12. There was hyper-kyphosis at this level. He has been treated once 
per month for chronic mid-back pain and spasm for 10 years. He rated the 
pain as 3 after an adjustment but building up to 7 or 8 before the next 
treatment time. Following supplementation of 12 weeks beginning November, 
1995, he rated the pain as a 1 and it only occasionally built prior to 
adjustment. There was clinically improved intersegmental ROM at the above 
level and decreased para spinal myospasm which remains stable between 
treatments for the first time in 10 years. 
A 53 year old female presented with cervical spine pain and headache. She 
had osteoarthritis of the hands with joint enlargement, decreased ROM and 
mild ulnar deviation. Radiographs revealed cervical spine osteoarthritis. 
She rated the pain in the cervical spine as 5 and the hands as 7 with 
inability to knit or do fine work. Following 8 weeks supplementation she 
had decreased joint enlargement, increased ROM, and rated the pain as a 3 
in her hands. Her cervical spine was rated a 3 also. She stated that even 
her husband has commented on how good her hands look. She had cervical 
spine manipulation but no hand treatment other than Ginseng of the 
Sea.TM.. 
An 79 year old female presented with severe crippling bilateral hand pain 
of increasing severity over 1 year. She could only approximate the 
fingertips to within 3 centimeters of the palm. She had moderate joint 
edema. She rated the pain as 10. Following supplementation of 8 weeks she 
began to note improvement. Then in the following 8 weeks she observed 
significant improvement with improvement of pain and dysfunction. At 18 
weeks of supplementation she reported that her pain is 3-4, that she had 
no swelling in the finger joints, and that all the digits of her right 
hand touch the palm and all but one of the left fingers could touch the 
palm. 
A 65 year old female presented with chronic knee pain. She rated the pain 
as a 5 or 6. After six weeks of treatment she reported that the knees are 
not much improved, but that her chronic arthritis of the proximal 
interphalangeal joint of the right hand was much better. The edema of the 
joint was so reduced as to be 50% improved in size with no pain on joint 
flexion. Following 8 more weeks of supplementation she reported gradual 
improvement in the knees with pain rating at 3. 
A 60 year old male presented with generalized joint discomfort, primarily 
in the shoulders, neck and upper back. He rated the pain as 4. Following 9 
weeks supplementation he rated the pain as 2 and had improved ROM in the 
involved areas. 
A 40 year old male presented with left hip pain. Examination revealed 
moderate left hip osteoarthritis subsequent to a rollerblading accident 4 
years prior. He rated the pain as 5-6 and exacerbating to 8 with increased 
activity. He also had recurrent left shoulder and neck pain and left knee 
pain. Following 16 weeks of supplementation beginning in October, 1995 he 
reported a pain of 1 in his left hip with ability to play basketball 
without pain. The pain in the left knee was minimal and occasional and 
there was no left should pain. Remaining cervical spine pain was relieved 
by cervical manipulation. 
A 46 year old male presented with pain the midthoracic spine. He had 
suffered with this pain since an injury in 1975. Thoracic spine films 
revealed osteoarthritis of T4, 5, 6 with osteophyte formation in this 
region. He had been using shark cartilage for 2 years with some relief. He 
was advised to change to the sea cucumber in place of the shark cartilage 
and within 2 weeks he noted increased motion without pain. Within 8 weeks 
he noted that the mid-back did not bother with normal daily activity. He 
mentioned a significant improvement over the shark cartilage. 
As can be seen from the forgoing, all patents treated exhibited significant 
improvement in subjective evaluation of pain and range of movement after 
treatment. 
EXAMPLE 11 
Subjective Benefit from Treatment of Arthritic Inflammation with B1000 and 
T2000 
After testing B1000 in a standard LD50 assay ("FHSA/CPSC Design 16 CFR 1500 
Acute Oral Toxicity" (Feb. 3, 1995), Tox Monitor of Oak Park, Ill.) to 
determine existence of toxicity, finely divided B1000, as obtained by 
Examples 2 and 3, was mixed with sea cucumber collagen and administered in 
an average dose of 600 mg per day, corresponding to the inhibition of 
inflammation at 10 mg/kg that was effective in reducing inflammation in 
laboratory animals, adjusted for patient weight. 
Thirty persons were entered into an open label, non-controlled trial to 
determine presence or absence of subjective benefit in cases of arthritis 
inflammation and decreased range of motion. 
The trial was conducted by Dr. Michael Aker of Blue Hill, Me., and the 
results are summarized as follows: 
Of the 30 patients enrolled in the trial, 28 patients reported subjective 
benefit as either increased range of motion or lessened pain and 
inflammation. Twenty-one of those reporting subjective benefit reported 
that benefit to be an improvement of more than 75% and seven reported 
improvements at more than 90%.