Cartilage extraction processes and products

A biologically active cartilage product is prepared by heating a mixture of crude animal or fish cartilage in water under pressure to form an aqueous cartilage extract, removing suspended matter from the extract and concentrating the extract under vacuum. The product may be dried to form a fine granular material.

This invention pertains to cartilage extraction processes and products. 
More specifically, the invention relates to a process for preparing a 
biologically active cartilage product using raw animal or fish cartilage 
together with adhering tissue as the starting material. 
The preparation of powdered cartilage products for various therapeutic 
applications is discussed in the prior art. Thus, U.S. Pat. Nos. 3,400,199 
and 3,772,432 teach the preparation of pharmaceutical materials from raw 
cartilage. In the preparation of these prior art products it was 
considered important to remove the adhering tissue (mainly proteinaceous 
and fatty tissues) from the raw cartilage material by pre-treatment with a 
suitable proteolytic enzyme solution. Thus, the raw cartilage material 
might be treated with a solution of pepsin and an acid (e.g., acetic or 
hydrochloric) at a temperature of about 50.degree. C. for about five 
hours. After the adhering tissues had been removed the clean cartilage was 
vacuum dried, de-fatted with a suitable solvent (e.g., hexane), the 
solvent evaporated and the cartilage mechanically comminuted to a fine 
powder of between about 5 to 40 microns average particle size. The 
cartilage powder so obtained could be utilized in powder form or, 
extracted at a temperature between about 3.degree. to 4.degree. C. with 
distilled water or an aqueous salt solution which facilitated 
solubilization or peptizing of the cartilage material at low temperature. 
The solubilized cartilage obtained in this manner is not a true solution 
but rather consists of a colloidal dispersion containing between about 1 
and 10% cartilage solids. 
Because of the complexity and number of steps required for preparation, 
cartilage products prepared according to the prior art processes are 
relatively expensive materials. The resulting materials also suffer from 
several significant drawbacks. Solutions containing solubilized cartilage 
materials are generally opaque. Although it has been recognized that the 
opacity is attributable to a small quantity of suspended particles (of 
larger than colloidal size) they are difficult to remove from cold 
solutions (either by centrifuging or filtration) without simultaneously 
incurring substantial losses of active material. Since it was found that 
the presence of oxygen during the extraction process tended to degrade the 
resulting extract, and lower its biological activity, it was believed 
necessary to maintain a low temperature oxygen-free environment during 
cartilage processing steps in order to avoid degradation and loss of 
biological activity in the finished product. An important element of prior 
art processing operations involved grinding the cartilage material to a 
predetermined average particle size, an expensive and time-consuming 
operation. 
It has now been surprisingly discovered that cartilage preparations having 
high biological activity can be obtained by extracting crude, mechanically 
trimmed cartilage (i.e., cartilage which still retains a portion of the 
adhering tissues--primarily proteinaceous and fatty tissues) together with 
water, under heat and pressure, for a predetermined time period. In 
contrast with prior processes, it has been unexpectedly discovered that 
cartilage materials need not be processed at low temperatures or in a 
completely inert, oxygen free atmosphere, but can be processed at high 
temperatures (from about 50.degree. C. up to about 150.degree. C.) and 
pressures (between about 10 and about 60 PSI) to yield extracts possessing 
high biological activity. As a further advantage the process of the 
present invention dispenses with the requirement of a pre-extraction 
grinding operation thereby substantially reducing processing costs. 
However, pre-extraction grinding can be optionally employed under certain 
processing conditions to yield a desirable product. 
According to the present invention, cartilage products having high 
biological activity are prepared by heating a mixture of crude cartilage 
and water under pressure and substantially non-oxidizing conditions for a 
predetermined period of time to form an aqueous cartilage extract, 
removing suspended solid matter from the extract and concentrating the 
extract under vacuum. As employed herein the term "substantially 
non-oxidizing conditions" refers to controlled environments in which small 
quantities, on the order of a few percent or less (by volume) of air or 
oxygen may be present. The concentrated extract, which may be in gel or 
liquid form, can be dehydrated using any of several techniques, to yield a 
friable granular material which forms clear or slightly hazy solutions 
with water. The solid and liquid cartilage products of the present 
invention are useful as ingredients in various pharmaceutical and cosmetic 
compositions. 
It is accordingly an object of the present invention to provide a process 
for preparing cartilage products from raw cartilage. 
Another aspect of the present invention relates to the preparation of a new 
cartilage products that are useful in various pharmaceutical and cosmetic 
applications. 
A still further aspect of the present invention involves a method for using 
cartilage products prepared according to the present invention in 
pharmaceutical formulations for the treatment of pruritus ani and 
hemmorhoidal conditions. 
These and other objects of the present invention will become apparent upon 
consideration of the following detailed description of the invention. 
The present invention involves a process for the preparation of cartilage 
products from raw animal or fish cartilage. As used herein, the term "raw 
cartilage" refers to cartilage from which the adhering tissues (primarily 
proteinaceous and fat) has not been separated or removed. Bovine cartilage 
(especially bovine tracheal cartilage) is the preferred raw material for 
use in the invention, however cartilage taken from other vertebrate 
animals including procine and canine cartilage as well as cartilage from 
the partly calcified skeleton, including fetal skeleton, of very young or 
newly born animals will also provide suitable results. Cartilage from 
young animals or young or newly regenerated cartilage from older animals 
has also been found satisfactory for use in the present invention. 
Cartilage from mature animals in either the form which would in maturity 
retain the cartilaginous form or which would in maturity ossify to bone 
may also be employed. Skeletal cartilage from fish, particularly the 
shark, has also been found to provide an especially satisfactory raw 
material. 
Cartilage from the skeletons of shark or other cartilaginous fish may be 
used to prepare aqueous cartilage extracts in the same manner as bovine 
cartilage. However, in the case of the shark, the spinal column is the 
most convenient tissue to harvest. Despite the fact that in most sharks 
the vertebrae are calcified to a considerable degree, they contain 
sufficient cartilage material to yield a useful extract. While bovine 
trachea is the preferred source of raw cartilage, as it is the most 
readily accessible cartilaginous tissue in mammals, hyaline or costal 
cartilage from other parts of the animal's body may be utilized to produce 
satisfactory extracts. 
The raw cartilage may be prepared by any satisfactory means but generally 
is obtained by removing substantially all of the skin, integument and 
organs of the animal or fish and separating the cartilage. The separated 
cartilage, to which some proteinaceous tissue and fat will generally still 
be adhered may be subdivided into chunks or used in whole form as removed 
from the animal. It is not necessary to remove all vestiges of adhering 
tissue, as in prior art cartilage processing techniques. The size of the 
raw cartilage to be employed in the invention is not critical and is 
primarily dependent upon the dimensions of the reaction vessel in which 
the cartilage is to be processed. 
The raw cartilage is mixed with water in the ratio from 1:100 to 100:1 and 
preferably 1:2 to 2:1 by weight (cartilage:water). Preferably the water is 
deionized or distilled. The mixture of raw cartilage and water is 
transferred to a suitable pressure vessel (e.g., a steam pressure vessel), 
fitted with a pressure relief valve and the vessel heated to a temperature 
of between 50.degree. C. and 150.degree. C. and preferably between about 
105.degree. C. and 125.degree. C. or until a pressure of 10 to about 60 
p.s.i.g., and preferably between about 10 to about 30 p.s.i.g. (pounds per 
square inch--gauge reading) of steam has been built up within the vessel. 
The preferred extraction condition is 20 p.s.i.g. pressure for two hours 
(at a temperature of about 110 degrees C.). However, reaction periods of 
between 5 minutes and 5 hours may be employed to yield satisfactory 
results. Under optimum conditions the extraction is continued to the point 
at which the cartilage substance has just become completely dissolved, but 
the aqueous extract is still a light tan color and has not acquired a dark 
brown color. The connective, fibrous and fatty tissues adhering to the raw 
cartilage are extracted, but not solubilized, during the heat and pressure 
treatment. 
Substantially all of the fat contained in the cartilage, and adhering 
tissues, is released during the extraction under heat and pressure, and 
floats to the surface of the extract. The insoluble fibrous tissues 
containing a minor portion of fat will generally sink to the bottom of the 
reaction vessel. 
At the conclusion of the heat and pressure treatment, the vessel is opened 
and the aqueous cartilage extract removed. The solid matter consisting 
primarily of fibrous tissues and fat is separated from the extract by 
centrifuging. In some instances, the solids may be removed by decanting 
the extraction liquid. A small portion of the fatty content of the raw 
cartilage remains emulsified in the extract and may be removed either by 
prolonged centrifuging at very high speeds (centrifugal pressures of 1,000 
G's or more). Alternatively, the emulsified fat is removed by filtering 
through a filter press coated with a diatomaceous earth filter aid. The 
filtrate (or the liquid recovered from the centrifuge) has a clear amber 
color. Analysis of the recovered extract shows it to be rich in 
proteinaceous material and low in calories. The extract is suitable for 
use as a dietary food supplement or a health food. 
The filtered or centrifuged extract is concentrated by removing a portion 
of the moisture content under vacuum in a mechanically agitated thin film 
evaporator. Thin film evaporator devices are well known in the art and 
utilizes rotating slotted wiper blades to generate a thin film of liquid 
on the heated wall of a sealed vessel. The slots in the rotating wiper 
blades provide a pumping action which creates and moves the thin liquid 
film along a heated wall with constant agitation. As the concentrating 
residue travels downward, it is in continuous contact with the evaporating 
surface from which vapor is continuously separated. The vapor travels 
through a rotating entrainment separator to the surface of an internal 
condenser where it is condensed and flows by gravity to an outlet valve. 
The action of the wiper blades in moving the residue down and off the 
heated wall eliminates thermal degradation by controlling the residence 
time at the distillation temperature. The concentration is conducted under 
a vacuum of between about 10 and 100 torr (millimeters of mercury) and a 
jacket temperature of between about 90 and 180 degrees C. 
A wiped film (or thin film) evaporator apparatus suitable for use in the 
present invention is available from the Pfaudler Company, 1000 West 
Avenue, Rochester, N.Y. as a 12 inch diameter model WFE with four square 
foot heating surface, Teflon wiper blades, Denison fluid motor MF05-014 
and an 11 square foot internal condenser. The aqueous cartilage filtrate 
(or centrifugate) is concentrated under vacuum, preferably of 40 tor 
(absolute) to yield a liquid of between about 45 to 65% solids content. 
The viscosity of the liquid will vary depending upon the temperature and 
solids content. Thus the liquid may be readily flowable and of low 
viscosity at higher temperatures and low solids content, and conversely 
may be in gel form at low temperatures and with a higher solids content. 
The liquid concentrate (which is partially dehydrated product) may be used 
as such in various cosmetic, pharmaceutic and food products or it can be 
completely dehydrated to a dry granular substance. Suitable dehydrating 
techniques include freeze drying, vacuum spray drying, dehydration with a 
liquid (e.g., ethanol, isopropanol); azeotropic distillation with a 
hydrocarbon azeotrope (e.g., hexane). In making a product to be used in 
the manufacture of an injectable cartilage extract it is preferable to 
combine the liquid concentrate with an excess of isopropanol to 
precipitate the active ingredients of the cartilage extract as a solid. 
The completely dehydrated extract is a friable granular or lumpy material 
which is generally of a light tan color. The dry material can be readily 
ground to yield a fine powder and dissolves readily to form clear or 
slightly hazy solutions with water. 
The cartilage extracts of the present invention in either the dry or liquid 
form have been found to be especially useful in the treatment of 
hemmorhoidal conditions and pruritus ani. The liquid extracts are also 
useful as geriatric and dietary food supplements because of their high 
protein and low fat content. The extracts may be prepared in liquid or dry 
form for use as an ingredient (e.g., skin conditioner) in cosmetic 
formulations. 
The biologically active agent of the invention may be administered in the 
form of a liquid, as a suspension or solution, or alternatively in solid 
form as a tablet pellet or capsule. The tablet may be prepared using 
conventional tabletting procedures in which the active ingredient is 
combined with well known pharmaceutical excipients such as starch, sugar, 
bentonite clays and other commonly used carriers. Satisfactory 
pharmaceutically acceptable liquids include water, sugar solutions, and 
aqueous glycols which may be compounded with coloring agents and synthetic 
or natural flavors. In another embodiment, the dry active ingredient may 
be incorporated onto silica gel or other gel forming materials which are 
capable of coating the stomach walls. The active ingredient may also be 
administered as a suppository, or a topically applied cream, or ointment. 
A preferred embodiment of the invention useful for treating hemorrhoidal 
conditions involves rectal administration of the active ingredient in the 
form of a shaped suppository. 
For the preparation of cartilage extracts that may be administered by 
parenteral injection, it is desirable to remove as much of the adhering 
tissue and fat as possible from the starting cartilage material prior to 
the high pressure extraction step. This is accomplished by mixing the raw 
cartilage in a digestive solution containing proteolytic enzymes 
(preferably acid-pepsin). Trypsin and pepsin exemplify the wide variety of 
proteolytic enzymes that are useful in this aspect of the invention. 
Following the enzyme pretreatment, the digested cartilage can then be 
extracted under high temperature and elevated pressure. Although the 
pretreated materials or cartilage extracts prepared can be used for food 
additives, cosmetic applications and in topical and oral preparations, it 
is not desirable to do so, as the enzyme pretreatment results in the loss 
of some biologically active material from the new cartilage. This loss 
occurs when the cartilage is leached in the enzyme bath and during the 
subsequent washing operations. Also, the pretreatment adds significantly 
to the cost of the finished product. 
In preparing a cosmetic composition in accordance with the present 
invention, any suitable cream, emulsion or oil cosmetic base, which will 
keep the cartilage material in solution or suspension may be used. The 
base may be in emulsified form including waxes, oils, emollients, 
preservatives and humectants, or may be in the form of a vegetable oil, or 
mixture of vegetable and mineral oils or mixture of oil and water based 
compositions.

The following examples illustrate certain preferred embodiments of the 
invention. However, it should be understood that these examples are 
non-limiting illustrations and that other methods and embodiments are 
envisioned by the present invention. Parts and ratios are by weight except 
as otherwise stated. 
EXAMPLE I 
Extraction of bovine trachea 
5 kilograms of deionized water was admixed with 5 kilograms of well trimmed 
beef trachea subdivided into pieces of about 3 inches in the largest 
dimension. The water/trachea mixture was loaded into a 20 liter aluminum 
pressure vessel equipped with a pressure gauge and a pressure relief 
valve. The vessel was sealed, the lid clamped shut, but the relief valve 
left open. The vessel was heated to about 100.degree. C. to bring the 
water to a boil and the relief valve left open until the steam generated 
by the boiling water had displaced substantially all of the air from 
within the vessel. The relief valve was then closed and the steam pressure 
allowed to rise to about 20 p.s.i.g. (temperature 110.degree. C.) with the 
actual pressure fluctuating between about 17 and 25 p.s.i.g. 
(105.degree.-116.degree. C.). The pressure and temperature was held 
constant for two hours. The vessel was then allowed to cool to atmospheric 
pressure, the pressure valve opened and the lid of the vessel removed. The 
partially processed trachea were thoroughly mixed, the lid replaced, and 
the liquid again brought to a boil. Air was purged from the vessel by 
means of steam generation as outlined previously, the pressure brought to 
20 p.s.i.g. and held at that level for another two hour period. The vessel 
was again cooled to ambient temperature, the pressure valve opened, the 
lid removed and the liquid contents of the vessel strained through a 100 
mesh stainless steel strainer. The strained liquid was centrifuged in 
order to remove the major part of the suspended matter. The yield was as 
follows: 
______________________________________ 
Liquid Extract 7500 grams 
Fat 850 grams 
Fibrous protein 
matter with some fat 1600 grams 
______________________________________ 
The fibrous matter residue contains 50% liquid which was expressed to 
increase the yield of the liquid extract. The liquid extract contains 8% 
non-volatile dry extract which includes about 1% suspended fat 
(emulsified) and protein. The remaining 7% represents 525 grams dry 
weight, or 10.5% based on the weight of the trachea. The liquid extract 
was a cloudy light tan colored fluid at ambient temperature but 
consolidated to form a firm gel upon refrigeration to 10.degree. C. or 
lower. 
The liquid was found to be stable upon storage under sterile conditions or 
when protected with a suitable preservative (e.g., benzyl alcohol or a 
combination of sorbic acid and sodium benzoate). The extract had a 
pleasant taste, characteristic of meat extract and which could be enhanced 
by seasoning with salt, pepper, or other condiments customarily employed 
for seasoning soups or meat products. The extract was suitable for use as 
a dietary food supplement or could be administered as a pharmaceutical 
preparation. 
EXAMPLE II 
The liquid extract of Example I was concentrated to 45% non-volatile solids 
content under vacuum (40 mm of Hg) and mechanical agitation at a jacket 
temperature of 120.degree. C. in a thin film evaporator (Pfaudler model 
WFE). The liquid material formed a very firm gel when cooled to 15.degree. 
C. or lower. The material had a pleasant taste and was ingested as a 
dietary food supplement. The gel was also useful as a moisturizing 
component in conventional cosmetic cream and ointment formulations. 
EXAMPLE III 
The concentrated extract (45% solids) of Example II was evaporated to 
dryness in a laboratory oven (110.degree. C.) and under 40 millimeters of 
vacuum. The dried material, which was hard and brittle, was pulverized. 
The pulverized material was used to fill one thousand 50 milligram hard 
shell gelatin capsules. The capsules were suitable for oral 
administration. The powder was also used to make shaped rectal 
suppositories for administration to humans to alleviate pruritus ani and 
hemmorhoidal inflammation (see Example IX). 
EXAMPLE IV 
2200 pounds of bovine trachea was quartered and ground in a Weiler (meat) 
grinder using a one-half inch orifice plate. After grinding, the material 
was transferred to a 100 gallon glass lined reactor equipped with a 
mechanized agitator. The liquid-trachea mixture was processed in six 
batches. The weight of trachea for each batch varied between 300 and 425 
pounds. Deionized water was added to the reactor on a one-to-one weight 
ratio (1 pound of ground trachea to 1 part water). Internal pressure of 
the reactor was brought to between 15 and 24 p.s.i.g. (temperature 
110.degree. C.) after air purging and held for two hours with the agitator 
in operation (120 r.p.m.). The extracted mixture was removed from the 
pressure chamber and pumped-through a centrifuge type decanter (Flottweg 
Decanter Z-1L). The solids discharged from the decanter were collected, 
weighed and sampled. The extraction liquid that remained after the 
decanting operation was subjected to a three way separation in a Titan 
disc-type centrifuge--the separations yielded: 
A. Light Liquid--grease 
B. Heavy solids--sludge 
C. Water phase--product 
The product from centrifuge batch 1 and 2 was further processed through a 
filter press. The pH of the product obtained from centrifuge batches 3 
through 6 was adjusted with glacial acetic acid to between about pH 5 and 
pH 5.5. The Titan disc centrifuge had a bowl speed of 6200 r.p.m. and an 
effective G force of more than 1,000 G's. 
The product from batches 1 and 2, after filtration, was combined, the 
combined material decanted and centrifuged again in the disc centrifuge. 
Approximately 1/2% of benzyl alcohol was added to the combined product as 
a preservative. The products of batches 3 and 4 were combined as were the 
products of batches 5 and 6. The following analyses were performed: 
______________________________________ 
Raw Material (Material In) - in lbs. 
Batch # 1 2 3 4 5 6 
______________________________________ 
Trachea 317 350 350 347 425 425 
Water 317 382 350 347 425 457 
Total 634 732 700 694 850 882 
______________________________________ 
Material Out - in lbs. 
Batch # 1 & 2 3 4 5 6* 
______________________________________ 
Decanter Solids 
81.5 40 41 53 84 
Cent. Eff. - Product 
1034 523 543 613 686 
Grease 76 51 38 56 89 
Cent. Sludge 
19 None 21 None 20 
Total 1210.5 614 649 722 879 
______________________________________ 
*Between Batch 5 and 6 the product from batch 1 and 2, which had been 
filtered and contained filter earth, was reprocessed thru the decanter an 
centrifuge. Therefore, the decanter solids for Batch 6 contained some 
filter earth. 
______________________________________ 
Material Balance 
1 & 2 3 thru 6 1 thru 6 
Batch Lbs. % Lbs. % Lbs. % 
______________________________________ 
Material in (total) 
1366 -- 3126 -- 4492 -- 
Material out (total) 
1210.5 88.6 2864 91.6 4074.5 
90.7 
Assume lost as steam 
155.5 11.4 262 8.4 417.5 
9.3 
Decanter solids 
81.5 6.0 218 7.0 299.5 
6.7 
Cent. Eff. - Product 
1034 75.7 2365 75.6 3399 75.7 
Grease 76 5.6 234 7.5 310 6.9 
Cent. sludge 19 1.4 41 1.3 60 1.3 
______________________________________ 
ANALYSIS 
The following analyses were performed: 
Moisture: Volatile Material--The samples were dried in a forced air oven 
set at 105.degree. C. 
Fat Analysis--The dried samples were fat extracted with petroleum ether in 
a soxhlet extraction tube for a minimum of 2 hrs. 
Protein Analysis--Protein analysis on the dried, fat-free samples. The 
boric acid modification of the Kjeldahl method for the determination of 
nitrogen was used. The following factors were used: 
EQU % N.sub.2 =% NH.sub.3 /1.2158 
__________________________________________________________________________ 
Decanter Solids Centrifuge Extract 
1-2 3-4 5-6 1-2 3-4 5-6 
Batch Dry As Rec. 
Dry As Rec. 
Dry As Rec. 
Dry As Rec. 
Dry As Rec. 
Dry As 
__________________________________________________________________________ 
Rec. 
Moist. & Vol. 
0.0 73.8 0.0 72.3 0.0 72.3 0.0 83.7 0.0 83.3 0.0 84.6 
% Solids 
100.0 
26.2 100.0 
27.7 100.0 
27.7 100.0 
16.3 100.0 
16.7 100.0 
15.4 
% Fat 7.6 2.0 8.9 2.5 6.7 1.9 5.7 0.9 6.6 1.1 3.6 0.6 
% Protein 
-- -- 76.1 
19.2 76.6 
19.8 -- -- 54.5 
8.5 55.5 
8.2 
__________________________________________________________________________ 
Approximately 100 gallons of liquid from centrifuge batches 1 and 2 were 
fed into the feed tank of a standard P faudler WFE 4 square foot wiped 
film evaporator set up with a louvered rotor having spring mounted wiper 
blades in which the outer jacket temperature was 173.degree. C. The feed, 
a milky white, water-like liquid was heated by the outer jacket to 
40.5.degree. C. and the internal operating pressure of the evaporator 
reactor adjusted to 40 millimeters of mercury (absolute). The liquid was 
fed through the heated evaporator (173.degree. C.) at a feed rate of 225 
pounds per hour to yield a 70% distillate split (i.e. 70% of the water in 
the starting material was removed). 
EXAMPLE V 
______________________________________ 
Blue shark spinal column 
5305 grams 
Water, deionized 5400 grams 
Total charge 10,705 grams 
Yield 
Liquid Extract 7300 grams 
Insoluble calcified and 
proteinaceous residue 3000 grams 
______________________________________ 
Process: Water was charged into a pressure vessel (as in Example I), heated 
to about 90.degree. C. and sections of a blue shark spinal column (frozen) 
then added. The same heat and pressure extraction procedure was used in 
Example I, but the total heat and pressure treatment time was only two 
hours as the shark cartilage solubilized more quickly than the bovine 
cartilage. The liquid extract was centrifuged and then conducted through a 
filter press coated with a diatomaceous earth filter aid to remove 
suspended solid materials. The filtered extract had a light tan color and 
was almost transparent being essentially fat free. The extract is stable 
under sterile conditions or under ambient temperature conditions if 
suitable preservatives (e.g. benzyl alcohol 1/2-1%) are incorporated. 
EXAMPLE VI 
The process of Example V was repeated using sand shark spinal columns as 
the starting material. The yield and quality of the extract was 
substantially identical to that obtained in Example V. 
EXAMPLE VII 
Raw calf trachea was ground in a meat grinder to a 5.sup.3 mm average 
particle size and loaded into an extraction vessel. The extraction vessel 
used was a 4 liter jacketed stainless steel pressure vessel having a 
removable top and equipped with a steam inlet, a steam outlet valve, a 
pressure gauge, thermometer, a turbine mixer operating at 120 r.p.m., and 
a material inlet capable of introducing dry material against the 
prevailing pressure in the vessel. The vessel containing 500 ml of 
de-ionized water was preheated to the required temperature. The specified 
pressure was obtained by introducing into the vessel through the steam 
inlet valve. The air was substantially purged from the vessel with steam. 
The mixer was activated and the 500 grams of ground cartilage forced into 
the vessel with nitrogen. Satisfactory extracts were obtained at the 
pressures (in absolute psi), temperatures (in degrees Celsius), and 
extraction times (duration of treatment) given in the table below: 
______________________________________ 
PSI .degree.C. Extraction Time 
______________________________________ 
14.7 100 2 hours 
20 109 1.5 hours 
25 116 1 hour 
30 121 30 minutes 
35 126 20 minutes 
40 131 15 minutes 
50 138 10 minutes 
60 145 5 minutes 
______________________________________ 
EXAMPLE VIII 
The following tests were performed utilizing the aforementioned vessel (Ex. 
VII) but replacing the turbine mixer with a homogenizing type enclosed 
turbine mixer, e.g. Barinco, Model CJ4A (Arde-Barencolo, Mahwak, N.J.), 
operated alternately in the upward mode and in the downward mode at a 
transformer setting of 80 which results in a speed of about 5000 r.p.m. In 
these tests, the pressure in the vessel was not allowed to rise above 
atmospheric pressure (14.7 p.s.i.) during extraction. The extraction 
condition (temperature and time) were varied for each extraction. The 
vessel was blanketed with an inert gas or non-oxidizing gas (nitrogen). 
The calf trachea was pre-ground to a 2.sup.3 mm average particle size to 
allow the mixer to operate at the closest rotor/stator setting thereby 
providing the highest possible whearing conditions. The following 
temperature/time schedules yielded satisfactory (biologically active) 
extracts under the above processing conditions. 
______________________________________ 
.degree.C. Time in Minutes 
______________________________________ 
50 60 
75 30 
90 20 
100 10 
______________________________________ 
By using a suitable homogenizing mixer, e.g. Barinco, Tekmar, Eli Dicon, 
Eppenbach etc., and ground cartilage, it is possible to extract the 
cartilage in a continuous manner at elevated temperatures and at 
atmospheric or only slightly elevated pressures. The resulting extracts 
are a pale amber color and show only minimal heat degradation. 
EXAMPLE IX 
Twenty male and 20 female patients ranging in age between 60 and 85 years 
received a dietic food supplement consisting of cartilage extract obtained 
from Example I herein. All of the patients suffered from joint pains and 
limited joint movement attributable to confirmed rheumatoid arthritic 
conditions. Each patient received a daily dose of the liquid cartilage 
extract of Example I at the equivalent rate of 8 grams per day or dry 
extract for a period of three months. The daily dosage was subdivided into 
three approximately equal size portions. 
Thereafter the patients each received the liquid extract at the equivalent 
of 6 grams per day of the dry extract (subdivided into two doses of 3 
grams) for three months. Thereafter each patient received the equivalent 
of 4 grams per day of the dry extract for a subsequent three month period. 
The cartilage material was self-administered via the oral route. 
The physical condition of each patient was checked prior to commencement of 
the trial, daily for the first week, and once a week thereafter. Blood and 
urinalysis tests were conducted on each patient prior to the test to 
determine a baseline, and thereafter on a weekly basis. All patients 
reported that movement in the affected joints became less painful and the 
range of motion had increased between 7 and 30 days after commencement of 
the cartilage regime. Patients who discontinued the cartilage food 
supplement experienced a gradual return of their previous arthritic pain 
level wherein three to eight months. The periodic physical examinations 
and laboratory tests confirmed that the cartilage diet had no adverse 
effects on any patients' vital functions. 
EXAMPLE X 
Rectal suppositories were prepared using a hydrogenated vegetable oil base 
having a melting point of about 37.degree. C. Two groups of one thousand 
two inch suppositories were prepared containing respectively 2%, and 5% 
vacuum dried cartilage extract powders obtained in Example III. The 
suppositories were administered to 40 patients in a double-blind study. 20 
patients with confirmed hemmorhoidal lesions received active suppositories 
in two groups. The first group received suppositories containing 2% by 
weight (40 mg) of dried cartilage extract and the second group received 
suppositories containing 5% (100 mg) active ingredient. The control group 
of 20 persons received only a placebo consisting of a hydrogenated 
vegetable oil base of the same shape, color, size and weight as the active 
suppositories. Each patient received three suppositories a day at 
approximately six hour intervals for a period of three weeks. The test 
results revealed that the cartilage extract containing (active) 
suppositories were highly effective in reducing the inflammation and pain 
of the hemmorhoidal lesion. Patients receiving the active suppositories 
experienced fewer bleeding episodes and experienced rapid healing of small 
anal fissures. Those patients receiving placebo suppositories reported 
negligible effects attributable to the lubricating effect of the vegetable 
oil base. 
EXAMPLE XI 
Skin cream with cartilage extract as the active ingredient. 
______________________________________ 
Composition: % by weight 
______________________________________ 
Lanolin-acetylated 2.50 
Petrolatum 4.00 
Beeswax, bleached 2.50 
Cetyl alcohol 1.50 
Isopropyl myristate 11.00 
Stearic acid 1.50 
Mineral oil 9.20 
Glycerol 3.00 
Borax 0.80 
Colloidal clay 2.00 
Triethanol-amine 1.40 
Sorbic acid 0.20 
Benzyl alcohol 0.90 
Cartilage extract, dry. Example III 
2.00 
Water, distilled 57.50 
Total 100.00 
______________________________________ 
Procedure: The water was heated to about 80.degree. C. The cartilage 
extract, borax, glycerol, triethanol-amine and sorbic acid were dissolved 
in the hot water. Using a high speed homogenizing mixer, the colloidal 
clay and stearic acid were dispersed. The balance of the ingredients were 
then dispersed in the aqueous medium. The resulting cream was then allowed 
to cool to ambient temperature. Cosmetic creams exhibiting high moisture 
retention qualities in the skin were obtained when 2 to 4% by weight of 
cartilage extract solids were incorporated into the cream. Similar results 
were obtained when the cartilage extract employed was a liquid extract of 
8% solids content (as in Example I) the 45% concentrate of Example II or 
the extracts, liquid or dried, obtained in Example III and IV. The shark 
cartilage of Example VII was used after it was deodorized by steaming. 
EXAMPLE XII 
Shaped lipsticks were prepared utilizing conventional techniques and 
standard cosmetic bases comprising hydrogenated vegetable oils, lanolin 
waxes, lanolin alcohols, beeswax and petrolatum. Into such bases cartilage 
extracts were incorporated in quantities of 2 to 10% by weight based on 
the dry extract (prepared as in Exh. III). The lipsticks were pleasant on 
the lips and had an effective emollient action. In use they effectively 
prevented development of chapped lips and hastened the healing of cracked 
lips. The therapeutic lipsticks also accelerated the healing of herpes 
simplex lesions ("cold sores") on the lips. 
EXAMPLE XIII 
The following cosmetic facial creams were prepared utilizing the cartilage 
extract of Example I. 
______________________________________ 
CREAM A 
% Gr. 
______________________________________ 
(I) Modulan (Acetylated Lanolin - 
Amerchol Corp.) 3.0 600 
White petrolatum 5.0 1000 
Beeswax 3.0 600 
Isopropyl Myristate 8.5 1700 
Cetyl alcohol 2.0 400 
White mineral oil 6.5 1300 
Stearic Acid 2.0 400 
Oil base 30.0 6000 
(II) Distilled water 35.0 7000 
Borax 0.7 140 
Veegum (colloidal clay - 
R.T. Vanderbilt Co.) 
1.5 300 
Titanium dioxide .2 40 
Triethanolamine .2 40 
Cartilage extract (Ex. I) 
1.2 240 
Benzyl Alcohol 25.0 5000 
Glycerol .9 180 
5.3 1060 
Water base 70.0 14000 
______________________________________ 
I and II were prepared separately. Both were heated to 70.degree. C. and I 
slowly added to II under constant mixing of II with a Barinco mixer 
(Arde-Barinco Corp., Mahwah, N.J.). 
The resulting material, a cosmetic cream of uniform consistency, was packed 
in 50 ml glass bottles and the bottles sealed with threaded covers. 
______________________________________ 
CREAM B 
______________________________________ 
(I) Modulan 300 
White Petrolatum 
500 
Beeswax 300 
Isopropyl myristate 
870 
White mineral oil 
700 
Stearic Acid 200 
Oil base 2870 
(II) Deionized Water 3500 
Titanium Dioxide 
80 
Sorbic Acid 20 
Carbopol 934 46 
Urea 300 
Triethanolamine 125 
Benzyl alcohol 80 
Cartilage extract (Ex. I) 
2500 
Water Base 6651 
______________________________________ 
II was placed in a vessel equipped with a Barinco Mixer, deionized water 
added and the mixture heated to 70.degree.-80.degree. C. followed by 
addition of TiO.sub.2, sorbic acid and Carbopol. The ingredients were 
mixed to obtain a uniform dispersion. Urea was then added and mixing 
continued while the Triethanolamine, cartilage extract and benzyl alcohol 
were added. Then under constant mixing molten (I) was added and the 
combined ingredients mixed until a smooth homogeneous emulsion was 
obtained. 
Cream B was packaged in 50 ml bottles, and the bottles sealed with threaded 
covers. 
Both creams (A and B) were topically applied on a daily basis to the facial 
area by a group of 20 women for a period of about 3 months. There was a 
general increase in turgor of the skin and all 40 participants (20A and 
20B) reported satisfactory skin toning and moisturization effects with the 
respective cosmetic formulations. 
EXAMPLE XIV 
Extraction of bovine trachea--Ratio of water to trachea 1/100 
50 grams of dionized water was admixed with 5 kilograms of well trimmed 
beef trachea that had been subdivided into pieces of about 1 inch in the 
largest dimension. The water/trachea mixture was loaded into a jacketed 
stainless steel pressure vessel having a 20 liter capacity. The vessel was 
equipped with a variable speed stainless steel turbine mixer, a steam 
inlet valve, theremometer, pressure gauge and pressure relief valve. The 
vessel was sealed, the lid clamped shut and the relief valve left open. 
Steam was introduced into the jacket and the mixer activated at a speed of 
about 20 RPM. When the temperature in the vessel reached 100.degree. C. 
the steam valve leading into the vessel was opened and a slow stream of 
moist steam introduced and kept on for about three minutes until 
substantially all of the air in the vessel had been purged. Both the steam 
valve and the pressure relief valve were then closed and the stem pressure 
in the jacket increased to 23 p.s.i.g. This brought the internal pressure 
in the vessel to 20 p.s.i.g. This pressure was maintained for 4 hours and 
the speed of the mixer increased to 60 RPM after the first 30 minutes. The 
vessel was cooled to ambient temperature by circulating cold water through 
the jacket. The pressure valve was opened, the lid removed and the 
contents of the vessel strained through a 100 mesh stainless steel 
strainer while they were still hot (90.degree. C.). The fibrous matter 
retained on the strainer was stripped of the major part of the absorbed 
liquor by compressing it in a Carver press at 5 p.s.i. pressure. The 
liquor obtained at this step was combined with the strained liquor and the 
combined liquors centrifuged to strip them of suspended matter. The solid 
residue from the centrifuge was combined with the residue from the Carver 
press. The yield was as follows: 
______________________________________ 
Liquid Extract 2,500 grams 
Fat 840 grams 
Fibrous protein matter 
some fat and moisture 1,550 grams 
Operation loss 160 grams 
______________________________________ 
The liquid extract contains 25% non-volatile dry matter which includes 
about 2% fat (emulsified and insoluble protein). The remaining 23% 
represents 575 grams dry weight, or 11.5% based on the weight of the dry 
trachea. The liquid extract was cloudy, had a light tan color which gelled 
upon refrigeration to about 12.degree. C. Except for its higher solids 
content and somewhat higher viscosity, the liquid extract was very similar 
to the extract of Example I. When freeze dried, the product was 
essentially identical to that obtained in Example I. In this Example the 
bound water, locked in the cartilage tissue was utilized in the 
extraction. The high solids content of the liquor obtained makes this 
process the most economical as less water has to be removed in the drying 
step. 
EXAMPLE XV 
Extraction of bovine trachea. Ratio of water to trachea 100/1 
5 kilos of deionized water was admixed with 50 grams of well trimmed beef 
trachea subdivided into pieces of 1 inch in the largest dminesion. The 
pressure vessel and procedure were the same as in Example I. The yield of 
liquid extract was 4,950 grams. The extract was hazy, slightly opalescent, 
had a mild taste and did not gel, even after refrigeration at 3.degree. C. 
When freeze dried, the product was very similar in physical 
characteristics to that obtained in Examples I and I-A. 
EXAMPLE XVI 
Extraction of bovine trachea suitable for injectable preparations 
The following ingredients were used in an Enzyme pretreatment operation to 
remove adhering tissues prior to extraction of the cartilage tissues. 
______________________________________ 
Bovine trachea, well trimmed 
3,000 grams 
Water, deionized 6,790 
Acetic acid 180 
Pepsin 30 
______________________________________ 
The trachea and the water were loaded into a jacketed stainless steel 
vessel equipped with a variable speed stainless steel turbine mixer. The 
acetic acid and pepsin were added with the mixer operating at 60 RPM. Hot 
water was introduced into the jacket, the temperature of the mix in the 
vessel raised to 55.degree. C. and held at that temperature until the 
cartilage rings of the trachea were free from adhering tissues (about 5 
hours). The liquid, containing the hydrolyzed tissues and most of the fat, 
was then drained off. The residual trachea and cartilage rings, were 
washed twice with deionized water (80.degree. C.) and twice with cold 
deionized water until the pH of the last wash above pH 5.0. The yield was 
______________________________________ 
Trachea-cartilage rings 
1,500 grams 
Deionized water 1,500 grams 
______________________________________ 
The trachea cartilage rings and deionized water were loaded into a 
stainless steel pressure vessel equipped with a variable speed stainless 
steel turbine mixer, a steam inlet valve, theremometer, pressure gauge and 
pressure relief valve. The vessel was sealed, the lid clamped shut and the 
relief valve left open. Steam was introduced into the jacket and the mixer 
activated at 60 RPM. When the water in the vessel begain to boil, the 
steam generated was allowed to purge the air from the vessel; the relief 
valve was then closed. While the mixing continued, the internal pressure 
in the vessel was allowed to rise to 20 p.s.i.g. and maintained at about 
this pressure for 21/2 hours. The steam in the jacket was then replaced 
with cooling water and the temperature in the vessel brought down to 
ambient temperature (about 17.degree. C.). The material in the vessel was 
blanketed with nitrogen and 30 grams of a diatomaceous earth (acid washed 
Hyflo Supercel) filter aid was introduced into the vessel and the mixing 
continued for another 15 minutes. The batch was then filtered through a 
ceramic filter precoated with the same diatomaceous filter aid. 
The filtrate was concentrated to 25% of its volume under vacuum with 
maximum pot temperature of 75.degree. C. The concentrated filtrate was 
introduced, under vigorous agitation, into ten times its volume of 
isopropanol. The precipitated and partially dehydrated cartilage substance 
was filtered, washed with isopropanol and then with acetone and finally 
stripped of the solvents in a forced air dryer at 50.degree.-45.degree. C. 
until the loss on drying was less than 2% of the dry weight of the 
substance. 
The procedure yielded a product that was 5.5% by weight of the crude 
trachea or 12.4% by weight of the cartilage rings after the enzyme 
treatment. The material was light gray-tan in color and was readily 
soluble in water of ambient temperature, forming a light tan colored 
solution which had a slight haze. (When the solution was filtered through 
a Milipore filter a sparkling clear solution suitable for use in 
formulating injectable preparations for clinical administration to animals 
or humans. 
EXAMPLE XVII 
The extraction was carried out as in Example III, but the concentrated 
filtrate in this case was dehydrated azeotropically using heptane as the 
azeotrope. The dehydrated material was stripped of the solvent in a forced 
air dryer at 45.degree. C. until the weight loss on drying was less than 
2%. The dried material was a granular friable solid which had no 
perceptible solvent odor and which was soluble in water at ambient 
temperature forming a slightly hazy light amber colored solution. The 
solution could be clarified via filtration. 
EXAMPLE XVIII 
The extraction process as in Example XV, but the concentrated filtrate in 
this case was dehydrated by freeze drying in a VirTis 25-SRC sublimator at 
a shelf temperature of +30.degree. C. and a vacuum of 45 microns. The 
dried material was a flaky and very fluffy product of light amber color, 
soluble in water at ambient temperature forming a slightly hazy solution 
which could be readily clarified by filtration or by centrifuging.