THE AVA ROOT ***

                          Transcriber’s Notes

Obvious typographical errors have been silently corrected. All other
spelling and punctuation remains unchanged. In particular the author
uses Kavahin for what is now normally referred to as Kavain.

Italics are represented thus _italic_.




                                THESIS

                      presented for the degree of

                           MASTER OF SCIENCE

                                  at

                         THE COLLEGE OF HAWAII

                               JUNE 1915

                                  by

                            ALICE A. BALL.




The Thesis, herewith, on “The Chemical Constituents of the Active
Principle of the Ava Root” by Alice A. Ball, clearly demonstrates her
ability to do original work and to present her results in logical form.
Approved.

[Illustration: J. F. Illingworth]

  Chair of the Committee on
  Advanced Degrees.

  College of Hawaii,
  May 14, 1915.




                       THE CHEMICAL CONSTITUENTS

                                  OF

                           PIPER METHYSTICUM

                                  OR

                       THE CHEMICAL CONSTITUENTS

                        OF THE ACTIVE PRINCIPLE

                           OF THE AVA ROOT.




                               _INDEX._


                                                                    Page

  1. Historical                                                        1

  2. Method of Extraction                                              7

  3. Method of Separation of the Resins                                9

  4. Various Metallic Salts of the Resinous Acids                     12

  5. The Total Resins                                                 13

  6. The Barium Acid                                                  15

  7. Oxidation Products of the Barium Acids                           21

  8. The Iron Acids                                                   28

  9. Oxidation of the Iron Acids and
  the Free Acids                                                      33

  10. Alcohol Radicals                                                34

  11. Methysticin and Methysticinic Acid                              36

  12. Physiological Action                                            38

  13. Conclusion                                                      43




                             _HISTORICAL._


“Among the customs peculiar to the inhabitants of the South Pacific
Islands, perhaps the most noted is that of the preparation and drinking
of a narcotic beverage called _ava_, _kava_, or _yakona_. Much of
its notoriety arises from the repulsive way in which it is sometimes
made. Aside from this, it is characteristic of a certain oceanic
area, and seems to be as strikingly limited to this area as is the
stick-and-groove method of making fire. The custom, is not confined
to one ethnic stock, many notices in literature showing that both
Papuans and Polynesians practise it. In many of the islands the Liquor
is concocted by chewing the root of the Macropiper methysticum, or
long pepper, ejecting the comminuted mass into a bowl, adding water,
straining out the pulp, and drinking the fluid. In other localities it
is made by simply grating the root and adding water.

“The plant from which kava is made is a shrub of the natural order
Piperaceae. It is about six feet high with stems ranging from an inch
to an inch and a half in thickness; the leaves are cordate and from
four to eight inches long. This family is the source of the pepper
of commerce and contains several species that are of medicinal and
commercial importance.

In making kava, the root and base of the stem is used. The roots
usually weigh from two to four pounds, though sometimes as much as
22 pounds. Several varieties are distinguished by the natives; for
instance, in Tahiti there is a yellow variety called _Marea_; another,
which becomes pink on exposure to the air, is called _avini-ute_.

“Chewed when freshly gathered, the root first tastes sweet and
aromatic, then bitter, acrid and pungent. It provokes abundant
secretion of saliva and in a few seconds occasions a sensation of
burning on the tongue. The root contains about fifty percent of starch,
a little pale-yellow essential oil, two percent of an acrid resin, and
one percent of the neutral crystalline principle methysticin, called
kavahin. To the latter principle we must attribute the toxic qualities
of the kava preparation. The resin and the kavahin are insoluble in
water, but are soluble in saliva and the gastric juices.

“In Samoa, the ava root is grated or chewed, then soaked, the woody
pulp strained off, and the fluid drunk. The root is used either dry or
green. The flavor of the liquid is at first like that of soapsuds, but
immediately afterward a pleasant aromatic taste is imparted, faintly
bitter, as in quinine. In Samoa, ava drinking is the accompaniment of
all meetings of the men.

“Kava is at first stimulating, but the effect of an excess resembles
that of opium, producing a drowsy drunkenness, lasting for two hours.
The inebriate is usually peaceable, but sometimes is irritated by
noises, which is attributed by natives to the use of kava grown in
moist ground. The results of excess are skin disease, emaciation, and
general decrepitude. The peculiar whiteness of the skin caused by kava
drinking is said to be sought after in some islands as a sign that its
possessor is wealthy enough to devote his time to its acquirement.

“There is some misapprehension in regard to whether the liquid
undergoes fermentation before it is consumed, but it is positively
known that there can be no fermentation, for the liquor is drunk
immediately after the addition of water to the macerated root. Kava
that is prepared by chewing is said to be more palatable, which
is perhaps due to the conversion of the starch into a fermentable
substance by the ptyalin of the saliva.”[1]

 [1]—By Walter Hough—Reprinted from Smithsonian Miscellaneous
 Collections—No. 1472—August 1904. “Kava Drinking as Practised by the
 Papuans and Polynesians.”

“In 1779 Captain King, R. N., who followed Captain Cook to the Islands,
describes the case of a priest as follows ‘a little old man of an
emaciated figure, his eyes exceedingly sore and red, and his body
covered with a white leprous scurf, the effects of an immoderate use
of ava.’ He also says, ‘The chiefs suffer dreadful effects from the
immoderate use of ava. Those who are most affected by it had their
bodies covered with a white scurf, their eyes red and inflamed, their
limbs emaciated, their whole frame trembling and accompanied with a
disability to raise the head.’”[2]

[2] “Leprosy Prize Essays,” 2nd series by Thompson and Cantile, 1897.

F. A. Griel, makes the following statement in a foot note. “The mixture
is a subnarcotic, and if drunk by European sailors produces highly
nauseous effects. If frequently taken a dry burning heat is produced
all over the body, the eyes become red, skin peels off in flakes
and then degenerates into leprous ulcers or the whole body becomes
emaciated and wastes away.”[3]

[3] Miquel, Systema Piperacearum.

Numerous attempts have been made to isolate the active chemical
constituent or constituents. As early as 1844 Morson discovered an
active principal _Kawine_. This is a greenish-yellow, strongly aromatic
and acrid resin. This was again studied by Cuzant in 1860.

Gobley isolated from kava root a crystalline principle (analogous to
piperin), _methysticin_, or _kavahin_, which is without odor and taste
and is probably inert.[4] In 1886 Lewin separated the resin into two
resins, of which the Beta resin is greasy and of a reddish-brown color,
appearing in mass almost black. This is less active than the alpha
resin which is yellowish brown, has the characteristic odor of the
drug, is freely soluble in alcohol, and placed upon the tongue produces
a burning sensation followed by local anaesthesia.[5]

 [4] J. P. C. Jan. 1860.

 [5] A. J. P. 1886, 450.

A volatile oil has been found in the root.[6]

 [6] J. P. C. March 1862.

Lavialle claimed to have obtained an alkaloid, _Kavaine_.[7]

 [7] L’U​nion Pharm. Jan. 1889.

The following statement was found in “Watts Chemical Dictionary”,
“Kawain—a crystalline resin occurring along with methysticin in
kawa-kawa, It is not a glucoside. On oxidation it yields benzoic
acid.[8]

 [8] Gobley, J. Ph. (3) 37, 19.

The following statement appears in the Encyclopedia Britannica.
“There appears to be little doubt that the active principle in this
beverage is a poison of an alkaloidal nature. It seems likely that
this substance is not present as such (i.e. as a free alkaloid) in the
plant, but that it exists in the form of a glucoside, and that by the
process of chewing, this glucoside is split up by one of the ferments
in the saliva and the free alkaloid and sugar is formed”.

Arthur Bossingham[9] communicates the results of a chemical examination
of Kava-kava. Besides the crystalline body, methysticin, which has
already been described by others, he was able to isolate and identify
three resins, one soluble in 5% solution of potassium carbonate, the
second insoluble in this, but soluble in 5% solution of caustic potash,
while the third was insoluble in both of these alkaline solvents, The
ash amounts to 2.495% of the air dried root, and contained besides mere
traces of Fe, Mn, mainly Calcium, Sodium and Potassium.[10]

 [9] Proc. A. Ph. A. 1898, 564.

 [10] Proc. Wisc. Pharm. Assos. 1898, 53, 55.




                        _METHOD OF EXTRACTION._


The fresh rhizome was chopped up and then ground up by means of a
meat chopper. In the preliminary work the material was dried in a
vacuum oven at a temperature not exceeding forty degrees Centigrade.
In the later work the material was rapidly dried in the sun. This
operation required about two days time. After being thoroughly dried,
the material was finely powdered, and then extracted with ether. The
following continuous extraction apparatus was used. Due to the extreme
rapidity with which the ether evaporated, it was necessary to surround
the coils with ice so as to keep the condensers cold.

[Illustration: Diagram of apparatus]




                 _METHOD OF SEPARATION OF THE RESINS._


After the removal of the crystalline methysticin from the extract, the
following method of separation of the resinous products was used.

The free acids were removed by shaking the total resins successively
with solutions of one percent ammonium carbonate, one percent
sodium carbonate and one percent sodium hydroxide. The acids thus
removed were recovered by treating the alkaline solution with dilute
hydrochloric acid and shaking out with ether. The three acids were not
markedly different in their physical properties. They possessed the
characteristic odor of the crude drug, were viscous, brown in color
and did not solidify at minus ten degrees Centigrade. These free acids
constitute about five percent of the total resins.

The remainder of the extract was treated with alcoholic potassium
hydroxide and saponified by heating the solution to about eighty
degrees for fifteen minutes. After the alcohol was removed by
distillation a small quantity of water was added. This resulting
solution was then extracted with ether to remove the alcohol radical or
radicals and the unsaponifiable material from the saponified product.
The ether extract was saved to be used in the work on the alcohol
radicals. The aqueous portion was evaporated to a semi-solid mass.
Carbon dioxide was passed over this mass for about fifteen minutes
to change any excess of potassium hydroxide to potassium carbonate.
Alcohol (95%) was then added to precipitate the carbonate. The
carbonate was then filtered off and water added to the filtrate.

To the clear filtrate a solution of barium chloride was added.
Immediately a dense thick cream yellow precipitate formed. Barium
chloride was added in excess. The barium salt was filtered off by
means of a suction pump and the precipitate washed with water. The
filtrate was treated with a few drops more of barium chloride so as to
be sure that an excess had been added, which was proven if no further
precipitate formed. To this clear filtrate an excess of a solution of
ferric chloride was added. Immediately a thick heavy precipitate of the
iron salt came down, which was filtered off by means of a suction pump
and the precipitate washed with water.

The barium precipitate was treated with dilute hydrochloric acid and
heated to boiling to decompose the barium resinate, and to liberate
the free resin acid. The liberated acid was a brown resin of a thin
consistency and had a characteristic odor. This acid was removed by
shaking out with ether. The ethereal solution was dried with anhydrous
sodium sulphate, filtered, and the ether removed by distillation. The
last traces of ether were removed by heating the resin acids in a
vacuum oven at sixty five degrees Centigrade. This resinous acid is the
acid spoken hereafter in this paper as the BARIUM ACID.

The iron precipitate was treated with dilute sulphuric acid (ferric
chloride is soluble in ether and ferric sulphate is not) and heated to
boiling to decompose the iron resinate. The acid when liberated had a
strong characteristic aromatic odor. It was extracted with ether in the
same manner as the barium acid. This resinous acid is the acid spoken
of in this paper as the IRON ACID.

The resinous extract left after the removal of the crystalline
methysticin is spoken of as the TOTAL RESINS.

The resin acids removed by the preliminary shaking out with the various
aqueous alkalies are spoken of as the TOTAL FREE ACIDS.




          _THE VARIOUS METALLIC SALTS OF THE RESINOUS ACIDS._


After obtaining the potassium salts of the acids and having freed
the same from the excess of the potassium hydroxide and potassium
carbonate, the possibilities of the formation of different metallic
salts were tried.

The soluble salts of the following metals yielded precipitates:—

  Manganese
  Barium
  Cobalt
  Silver
  Mercury
  Iron
  Zinc
  Copper
  Calcium
  Lead

The following metallic salts gave a complete precipitation:—

  Silver
  Iron (ferric)
  Manganese

Since the barium and iron (ferric) salts gave the best means of
separation, they were used to separate the resins.




                          _THE TOTAL RESINS._


The total resins were brown in color, thick syrupy consistency and
possessed the characteristic odor of the drug.

A molecular weight determination was made in the same manner as with
the Barium acids, and the following data obtained.


  Wt. of pipette before              18.7660
  Wt. of pipette after               14.7160
  Wt. of resin used                   4.0500
  Temperature before                  3.52
  Temperature after                   3.71
  Change in temperature                .18
  Volume                              37 cc.
  Constant for solvent              3280
  Approximate molecular weight      2000

Combustions were made and the following data obtained.

  Wt. of boat                  3.4722     3.4720
  Wt. of boat and resin        3.6355     3.6220
  Wt. of resin                  .1633      .1500

  Sulphuric acid tube         79.1051    79.3865
  Tube plus water             79.2073    79.4770
  Water                         .1022      .0905
  Hydrogen equivalent           .01136     .01006

  KOH bulb                    49.4316    50.8720
  Bulb plus carbon dioxide    49.8350    51.2430
  Carbon dioxide                .4034      .3710
  Carbon equivalent             .12102     .1010

  Percent Carbon              67.31%     67.3
  Percent Hydrogen             6.9 %      6.7
  Percent Oxygen              26.8 %     27.0

The total resins when placed on the end of the tongue produced a marked
stinging sensation followed by a local anaesthesia. After the first
stinging was produced the sensation was rather pleasant. The local
anaesthesia persisted a long time, giving a sensation much the same as
that produced by cocaine. The barium and iron acids also produced this
local anaesthesia, but the initial stinging sensation was much more
pronounced, which was probably due to the acid nature of the substance.




                          _THE BARIUM ACID._


The resinous material used and spoken of as the “Barium Acid” is
the material prepared and so named as given under the “Method of
Separation”.

PHYSICAL PROPERTIES:—Dark reddish brown in color, syrupy in consistency
and has a characteristic odor; heavier than water; soluble in benzol,
ether, alcohol and acetone, but insoluble in petroleum ether and water.

This resin constitutes about sixty percent of the total ester resins;
i.e. the resins left after the free acids have been removed with
aqueous potassium hydroxide.

An analysis of the barium salt obtained by precipitation from the
potassium soap was made and the following data obtained:—

  Wt. of substance used      1.1715     .5860
  Wt. of barium sulphate      .4430     .2205
  Barium equivalent           .2606     .1298
  Percent barium            22.2%     22.1%

The following method was used in making the above analysis. The weighed
material was ignited in a platinum crucible by gently heating until the
combustible gases formed were given off. The crucible was then more
strongly heated to completely burn off the carbonaceous material left.
The residue was extracted with nitric acid and the barium precipitated
as the sulphate with dilute sulphuric acid, and the weight of the
barium sulphate determined.

Expressed as the ACID NUMBER, or the number of milligrams of potassium
hydroxide required to neutralize the free acids in one gram of the
substance, the following data was obtained:—

  Barium equivalent                    .2606      .1298
  Barium expressed as KOH equiv.       .2131      .1062
  Wt. of material used                1.1715      .5860
  Milligrams of KOH per gram        181.9      181.3
  ACID NUMBER                       181.9      181.3

The following gives the ACID NUMBER obtained by direct titration of
the barium acid; in “A”, barium hydroxide was used and in “B”, sodium
hydroxide was used.

A small quantity of the material was dissolved in a sufficient quantity
of neutralized alcohol to give a liquid of a light yellow color,
Phenolphthalein was used as the indicator, and the alkali was added
until a red color was produced.

                              “A”        “B”

  Wt. of substance            .6160      .2345
  Cc. of alkalie             4.54       1.75
  KOH equiv. per gram       41.40      41.90
  Acid number               41.40      41.90

On evaporating a portion of the alcohol from the material left after
titrating with the sodium hydroxide, and adding water to obtain an
aqueous solution of the sodium salt, an emulsion was formed, and on
standing globules of the free resinous acid separated. From the data
thus obtained, it can readily be seen that the acid or acids which
constitute the BARIUM ACID must have a number of carboxyl groups and
form a different series of salts by precipitation than by direct
titration. The salt or salts formed by direct titration, although
neutral to phenolthalein may be acid in structure. This is further
shown by the fact that the potassium salts produced by direct titration
are readily hydrolyzed. The acid number obtained by the precipitation
of the barium salt may be called the COMBINING VALUE, and the acid
number obtained by titration the TITRATION VALUE.

A number of molecular weight determinations were made on the free
barium acid. McCoy’s Boiling Point Apparatus was used and Merk’s benzol
(free from thiophene) was used as the solvent. A weighing pipette with
a bulb was used to introduce the material, the bulb being weighed
before the material was introduced into the apparatus, and afterwards,
the difference being the weight of the material used.

  Wt. of pipette (before)      16.3670      14.8895
  Wt. of pipette (after)       14.8895      12.4610
  Wt. of material used          1.5225       2.4285
  Original temperature          3.53         3.53
  Final temperature             3.60         3.62-3
  Change in temperature          .07          .09
  Volume of solution           37 cc        43.5 cc
  Constant for solvent       3280         3280
  Approx. Molecular Wt.      2000         2100 or 1800

Combustions were made using the barium acids. By qualitative tests it
was found that the acids contained only carbon, hydrogen and oxygen.
The following gives the results of the combustions.

  Wt. of boat                 2.8402      2.8402
  Wt. of boat and resin       3.0500      3.0250
  Wt. of resin                 .2098       .1848
  Wt. of H2SO4 tube          78.0250     78.1415
  Wt. of tube plus water     78.1520     78.2550
  Wt. of water                 .1270       .1135
  Hydrogen equiv.              .0143       .0126

  Wt. of KOH bulb            48.8140     51.7095
  Wt. of bulb plus CO2       49.3800     52.2085
  Wt. of CO2                   .5660       .4990
  Carbon equiv.                .1543       .1361

  Percent Hydrogen           78.5%       73.6%
  Percent Carbon              6.8%        6.8%
  Percent Oxygen             19.7%       19.6%

An attempt was made to make the potassium salts of the barium acids by
saponification with alcoholic potash. A small quantity of the acid was
treated with an excess of ten percent alcoholic potash and heated to
eighty degrees Centigrade to complete the saponification. Instead of
the formation of the potassium salts, a thick dark brown solid, gummy
mass separated. On cooling it solidified to a brittle solid which had
all the physical properties of a true resin. This solid is soluble
in ether, chloroform and benzol, slightly soluble in alcohol and
insoluble in petroleum ether and water. It burns without the formation
of an ash. Evidently, this brittle material is a condensation product
of the original barium acid.




               _OXIDATION PRODUCTS OF THE BARIUM ACIDS._


A small amount of the Barium acids was sealed with concentrated nitric
acid in a hard glass tube and heated in a bomb furnace for an hour
and a half at 115 to 120 degrees Centigrade. On cooling a yellow
solid separated. Qualitative tests showed that this oxidation product
contained no nitrogen, combined with sodium hydroxide readily, is
soluble in hot water, slightly soluble in cold water, easily soluble in
ether, alcohol and benzol and slightly soluble in carbon tetrachloride.
It decolorizes alkaline permanganate but does not decolorize bromine
water.

That there are a number of intermediate products formed and that
nitration also takes place during the formation of these intermediate
products is shown by the following. A small quantity of the acids were
placed in a test tube and covered with concentrated nitric acid. This
was suspended in an H2SO4 bath and a thermometer inserted so as to
observe the temperature. On being gently warmed the nitric acid and
the resin began to react with a rapid evolution of carbon dioxide and
oxides of nitrogen. As soon as the reaction had modified and before the
temperature rose above one hundred degrees Centigrade, a small amount
was removed and added to water. Some of the solid acid was formed and
also a number of globules of oil and there was a strong persistent odor
of nitrobenzene. The original test tube was heated to about one hundred
and twenty degrees centigrade and a small portion again removed. There
was more of the solid material formed and the odor was similar to
vanillin or coumarin or cinnamic aldehyde. The test tube was again
tested when the temperature had reached one hundred and thirty five
degrees Centigrade. There was no aromatic odor and a large amount of
the solid formed. When viewed under the microscope the substance had
the appearance of curled threads.

When the barium acids were treated with the standard nitrating mixture,
a solid was obtained which showed the presence of nitrogen when the
standard test was applied.

After the preliminary tests were made, the following method of
preparation was used. Two or three grams of the barium acids were
introduced into a hard glass tube of about thirty centimeters in length
and fifteen or twenty cubic centimeters of concentrated nitric acid
added. The reaction which is very vigorous at first was regulated by
keeping the tube under running water. After this vigorous action was
over the tube was placed in a sulphuric acid bath, and the temperature
gradually increased until it had reached one hundred and twenty five
degrees Centigrade, at which temperature it was kept for about five
hours. It was necessary to add a small quantities of nitric acid from
time to time to make up the loss by evaporation. When the oxidation
was completed the product was poured into water and then heated to
boiling. The resulting solution was filtered and the filtrate allowed
to cool. On standing a quantity of a pale yellow substance separated.
The following data gives the percent yield of this oxidation product.

  Wt. of container                    6.7220
  Wt. of container and substance      7.5920
  Wt. of substance                     .8700
  Wt. of oxidation product             .1240
  Percent yield                      14%

The oxidation product was dried by placing it in a vacuum over
sulphuric acid for several days. The neutralization equivalent of this
crude oxidation product was 157.

  Wt. of substance used               .0502
  Number Cc. of NaOH N/10            3.2 cc.
  Neutralization equivalent        157

The oxidation product was heated on a watch crystal and the sublimate
allowed to collect on a funnel. The first sublimate gave a melting
point of 109 degrees Centigrade.

Combustions were made on this sublimate with the following results.

  (1) C        66%
      H         4.7%

  (2) C        65.7%
      H         4.8%

  (3) C        65.15%
      H         4.8%

  (4) C        65.8%
      H         4.85%

The neutralization equivalent was obtained by titrating an alcoholic
solution of the sublimate with standard sodium hydroxide. The following
results were obtained on two different lots of the sublimed oxidation
product.

  Wt. of substance              .09      .1006
  Cc. of alkali N/10           7 cc     7.9cc
  Neutralization equiv.      128.6    127.3

Using a third sample the neutralization equivalent was obtained from
the analysis of the silver salt. The silver salt was formed by adding
silver nitrate solution to a carefully neutralized solution of the
sublimate. The insoluble silver salt was filtered off, washed with
water to remove the excess of silver nitrate, and dried in a vacuum
over sulphuric acid for several days. A weighed quantity of the silver
salt was ignited in a platinum crucible and the residue of metallic
silver was weighed. The following data were obtained using material
from the same sample for each analysis.

  Wt. of dish                    12.8825     12.8826
  Wt. of dish and substance      13.0060     13.1310
  Wt. of substance                 .1235       .2484
  Wt. of dish and silver         12.9400     12.9980
  Wt. of silver                    .0575       .1154
  Neutralization equiv.         125         125.3

Using the same sample, a neutralization equivalent was obtained by
titration with standard NaOH.

  Wt. of substance                 .0912
  N/10 NaOH                       7.25cc
  Neutralization equiv.         125.8

The above data shows that the sublimate is a mixture. No empirical
formula can be calculated from the combustions, and different samples
give different neutralization equivalents although the duplicate
determinations on the same sample showed good agreement thus
demonstrating the reliability of the methods.

By fractional sublimation it was possible to obtain fractions with
different melting points. The first sublimate melted sharply at 109
degrees. From the last fraction it was possible to separate some
crystals that melt at 200 degrees Centigrade. These might possibly be
p-acetyl-benzoic acid, as its properties of solubility, crystalline
form, its melting point and power of sublimation agree with those of
p-acetyl-benzoic acid.

Those crystals that appeared identical with benzoic acid were placed
in a melting point tube, and some known benzoic acid (from toluol)
was placed in another tube. These two tubes were placed in the same
sulphuric acid container and their melting points taken at the same
time. They melted at the same temperature.

The sublimate had a very pleasant aromatic odor resembling benzoin.
It gave no coloration with ferric chloride, thus eliminating a large
group of aromatic compounds. Some of the crystals were found to be
identical with benzoic acid when examined under the microscope.
The characteristic odor of methyl benzoate was produced when a
small quantity of the crystals were heated with methyl alcohol and
concentrated sulphuric acid. On treating some of the carefully
neutralized product with ferric chloride solution, a flesh colored
precipitate was formed. It agreed closely in its analysis with the
precipitate formed with known benzoic acid.

The filtrate left after the removal of the iron precipitate was
acidified and extracted with ether, and the ether removed by
evaporation. The resulting substance decolorized alkaline permanganate
solution, but did not decolorize bromine water. When the leaflet
needles that melt at 200 degrees were mechanically removed from the
original sublimate, the substance left after precipitating with ferric
chloride melted at 109 degrees. When these crystals were not removed,
the melting point of this material was not definite, but was over a
range of five degrees, from 110 to 115 degrees Centigrade.

The oxidation product contains at least three distinct substances,
benzoic acid, a substance melting at 200 degrees and—which is probably
p-acetyl benzoic acid and a third substance melting at 110 degrees.




                           _THE IRON ACIDS._


The resinous material used and spoken of as the IRON ACIDS is the
material prepared and so named under the “Method of Separation”.

PHYSICAL PROPERTIES:—Transparent and reddish brown in color, oily in
consistency and has a characteristic tea like odor, heavier than water,
freely soluble in benzol, ether, alcohol and acetone, but insoluble in
petroleum ether and water.

This resin constitutes about eighteen percent of the total ester resins.

By qualitative tests it was shown that the acids contained only carbon,
hydrogen and oxygen. Combustions made on the iron acids gave the
following results.

  Wt. of boat                    2.6950      2.6950
  Wt. of boat and substance      2.8470      2.8495
  Wt. of substance                .1520       .1545

  KOH bulb                      50.9620     51.0805
  Bulb and CO2                  51.3370     51.4610
  Wt. of CO2                      .3750       .3805
  Carbon equivalent               .1023       .10376
  Sulphuric acid tube           76.2448     76.3450
  Tube and water                76.3400     76.4453
  Wt. of water                    .0952       .1003
  Hydrogen equiv.                 .0106       .01114

  Percent Carbon                67.3%       67.2%
  Percent hydrogen               7.0%        7.2%
  Percent Oxygen                25.7%       25.6%

An analysis of the iron salt obtained by precipitation from the
potassium soap gave the following data.

  Wt. of substance used           .2955       .3387
  Wt. of FeSO4                    .0445       .0509
  Ferric equiv.                   .03208      .03676
  Percent Iron                  10.85%      10.85%

The following method was used in making the above analysis. The weighed
material was ignited in a platinum crucible by gently heating until the
combustible gases formed were given off. The crucible was then strongly
heated until the carbonaceous material was completely burned off. The
residue was weighed and the percentage of iron determined.

Expressed as the ACID NUMBER, or the number of milligrams of KOH
required to neutralize the free acids in one gram of the substance,
the following data was obtained.

  Ferric equivalent                .03208      .03676
  Fe expressed as KOH equiv.       .0965       .1107
  Wt. of material used             .2955       .3387
  Mg. of KOH per gram           326.6       326.7
  Acid number                   326.6       326.7

The following gives the ACID NUMBER obtained by direct titration of the
Iron acid. The method is the same as that used in getting the titration
value of the Barium acid.

  Wt. of substance                 .2450       .2472
  Cc of alkalie                   2.2         2.3
  KOH equiv.                       .01232      .01288
  KOH equiv. per gram            53.87       52.

An attempt was made to saponify some of the iron acid, but it was
impossible. The alcohol was partially distilled off, and the acid
freed by making the mass acid with sulphuric acid, and shaking out
with ether. The ether was distilled off, but the remaining acid had
different physical properties from the acid with which the experiment
was started. It was lighter in color, and solidified at zero degrees.
At room temperature it was almost solid. On ignition it left no ash.
This probably is a polymerization product of the original acid.

Since many organic acids whose salts cannot be prepared by the ordinary
methods can be prepared by passing dry ammonia gas through a solution
of the acid in anhydrous ether, this method was tried with the iron
acid. The iron acid was dissolved in anhydrous ether, and the dry
ammonia gas was bubbled through this ether solution. At first no change
was noted, but after several minutes there was a flocculent thready
precipitate formed which was light brown in color. The experiment was
repeated. At first the precipitate was a very light brown, but after
forming it quickly darkened. After standing a few hours the flocculent
precipitate changed to a sticky brown mass. This same change was
produced immediately if the precipitate was exposed to the air. The
resulting mass had no odor of ammonia.

The flocculent precipitate formed at first was probably the ammonium
salt of the iron acid, which like most ammonium salts, it was
precipitated due to its insolubility in ether. Due to the ease of
hydrolysis this salt immediately decomposed to the acid and ammonia
when traces of moisture were present.




           _OXIDATION OF THE IRON ACIDS AND THE FREE ACIDS._


When the iron acids were oxidized in the same manner as the barium
acids the amount of the oxidation product formed was about one fourth
of that produced with an equal amount of the barium acids. The time
necessary to completely oxidize the iron acids was much less then
required for the barium acids. On sublimation the iron acid gave a
product melting at 110 degrees Centigrade and is probably identical
with the one formed from the barium acids. The iron acid also yielded a
sublimate melting at 208 degrees C.

The free acids when oxidized in a like manner gave as one of the
products a low melting crystalline compound that contained nitrogen.

The iron acid although related to the barium acid as shown by the
formation of a common oxidation product is different in structure as
shown by the difference in the amount of the oxidation product formed
and the time to complete the oxidation.




          _THE ALCOHOL RADICALS AND UNSAPONIFIABLE MATERIAL._


The material that was shaken out with ether after the saponification
consists of the unsaponifiable material and the alcohol radicals of the
acids produced by saponification.

On allowing the ether to evaporate from this material, feathery
needles separated. These were removed by means of a suction pump and
recrystallized from hot acetone. The melting point of this product was
122-125 degrees Centigrade. The precipitate was dissolved in benzol and
allowed to slowly crystallize. It formed long prismatic needles with
melting point of 130 degrees Centigrade. When heated with concentrated
sulphuric acid, a brown green fluorescent solution was produced.

The remaining material was steam distilled, and the distillate
extracted with ether to remove the oil. The ether was dried with
anhydrous sodium carbonate, the solution filtered and the ether removed
by distillation. The oil that remained was light yellow, specific
gravity less than that of water, and possessed a very characteristic
odor resembling that of musk. The material left after the steam
distillation was cooled and shaken out with ether. The ethereal
solution was dried with anhydrous sodium sulphate and the ether
removed by distillation. The resulting mass was a dark brown resin
without any characteristic odor, solidifying at zero degrees.

The crystalline product with a melting point of 130 degrees and the
essential oil are evidently the alcohols formed through saponification
of the resin esters. The crystalline product could be readily separated
from the resinous material because of its slight solubility in
acetone. Since this product was not precipitated when the unsaponified
resin esters were treated with acetone and because it did not make
its appearance until after the process of saponification, it is
quite probable it is a product of saponification. The essential oil
can be detected in extremely small amounts due to its penetrating
characteristic odor. Before the process of saponification it could not
be detected, but as soon as saponification took place the odor was very
marked. From this it is quite evident that this oil is a product of
saponification.

The dark brown resinous mass that remained may be either an alcoholic
resin formed through the hydrolysis of the ester resins or it may
be a resin belonging to the class known as resenes[11] which resist
saponification.

[11] Com. Organic Analysis. Allen Vol. II, 146.




                 _METHYSTICIN AND METHYSTICINIC ACID._


The crystalline product obtained from the ether extract was
recrystallized several times from absolute alcohol to remove all
traces of any resinous material. It had a melting point of 122-123.
It therefore was not pure methysticin, which has a melting point of
138-139 degrees. On examining this product under the microscope it
was found to contain two distinct forms of crystals, long needles and
prismatic plates. Some free methysticinic acid, melting point 180
was examined under the microscope and found to consist entirely of
prismatic plates. Therefore the original crystalline body contains some
methysticinic acid.

Some of the original precipitate was crystallized once from absolute
alcohol and thoroughly dried. This was used for the following data to
determine the percent of free methysticinic acid in the crystalline
product. A weighed amount of the crystalline product was dissolved in
carefully neutralized alcohol, and titrated with tenth normal NaOH,
using phenolthalein as the indicator.

  Wt. of substance used      .2590
  NaOH N/10                  .5 cc
  % methysticinic acid.

The potassium salts of the various resin acids formed by the
saponification of the ester resins should yield a resinous mass syrupy
or oily in consistency when acidified, if it consists entirely of the
two groups of acids spoken of as the iron acids and the barium acids.
Also the weight of the barium acids plus the weight of the iron acids
should nearly equal the weight of the ester resins, if these are the
only two acids, because the alcohol radicals constitute not more than
two percent of the ester resins. But when the total potassium salts
were acidified the product formed contained a crystalline substance
in addition to the resinous acids. This crystalline substance was
separated from the resinous acids by means of their difference in
solubility in ether. On recrystallization from absolute alcohol,
it had a melting point of 179 degrees Centigrade. It is therefore
methysticinic acid. This acid constitutes about twenty percent of the
total acids from the ester resins because the barium and iron acids
constitute only about seventy five percent.

This methysticinic acid cannot be entirely formed by hydrolysis of
methysticin which was incompletely removed from the resinous material.
The methysticinic acid existed either combined with some other alcohol
than methyl alcohol or was combined with one of the resin alcohol
radicals.




                        _PHYSIOLOGICAL ACTION._


Due to the impossibility of preparing the alkali salts of the barium
and iron acids so as to be positive that the resulting preparations
completely represented the barium and iron acids, and due to the
insolubility of the acids and the total resins in solvents from
which they would not again be precipitated when introduced into
the circulation, it was found necessary to make an emulsion. The
emulsifying agent used was acacia (gum arabic). It was possible by
careful preparation to make a permanent emulsion that was miscible
with water in all proportions to form a homogeneous mixture. The
animals used were rabbits of about six pounds weight. The injections
were made by Dr. Geo. McCoy, director of the Leprosy Investigation
Station at Kalihi. All the physiological experiments were made at the
bacteriological laboratory at the Leprosy Investigation Station.

One cubic centimeter of the emulsion of the total resinous extract
(strength 1 in 5) was injected into the ear of a rabbit; the animal
died immediately. Another rabbit was injected in the same manner
with one half cubic centimeter of the same preparation. The animal
immediately stretched out and became rigid, or appeared to be
paralyzed, but after several minutes these symptoms lessened and after
about five minutes the rabbit appeared normal as far as activity was
concerned. As soon as the rabbit had recovered a second injection of
one quarter of a cubic centimeter of the same preparation was injected
in the same manner into the same animal. The same symptoms were
produced and with equal intensity, but ten minutes passed before the
animal again became conscious. For several minutes after recovery the
animal appeared somewhat drowsy and stupid but it soon regained its
former activity.

One half cubic centimeter of the iron acid emulsion (strength 1 in 7)
was injected into the ear of a rabbit. Immediate paralysis and apparent
anaesthesia set in lasting very pronouncedly for eight minutes. The
rabbit’s head was drawn backwards and its legs stiffened giving
symptoms similar to strychnine poisoning but they did not persist.
When a second injection of one quarter cubic centimeter was given to
the same animal, the same symptoms were produced, the animal remaining
under the influence of the injection for about fifteen minutes.

The iron acid injection was repeated on another rabbit with the same
pronounced symptoms of strychnine poisoning but the effect lasted only
about ten minutes.

One half cubic centimeter of the barium acid emulsion (strength 1 in
7) was injected into the ear vein of a rabbit. The animal uttered
several loud cries and after moving several feet it became spastic and
went into a sort of a stupor, beginning to come out of it after ten
minutes. As soon as the animal recovered, a second injection of one
quarter cubic centimeter was made. The animal again uttered loud cries
and then it went into a stupor, but it was not spastic. This lasted
approximately ten minutes. The animal did not completely recover until
about twenty minutes.

Thinking that the action might be largely mechanical and that the
symptoms produced were from the emulsion itself and not from the effect
of the material that was emulsified, an emulsion of olive oil was used
in a like manner. This olive oil emulsion was made in approximately the
same consistency as the resinous emulsions, and one cubic centimeter
was injected into the ear vein of a rabbit. No visible effects followed
this injection during the one hour’s time the rabbit was under
observation.

One cubic centimeter intraperitoneal injections of the total resinous
extract emulsion and the same amount of the iron acid emulsion were
made into rabbits. During the three hours the animals were under
observation no symptoms were produced. This may have been due to the
extreme slowness of absorption as compared with the rate of elimination
from the circulation.

Dr. J. F. Illingworth states as the result of his observations while
in the Fiji Islands that the kava beverage even when taken in large
amounts, does not apparently affect the brain to an extent as to cause
the drinker to appear as if under the influence of alcohol, but he
appears as if the muscles from the hips downward are paralyzed. These
symptoms last for about half an hour, at the end of which time the
person is perfectly able to walk home.

In general the active constituent of any drug produces a more
pronounced but more fugitive effect than the crude drug itself, and one
might therefore expect more violent reactions from the isolated active
constituents of the Ava then from the crude infusion. Judging from the
negative results obtained from the injection of the olive oil emulsion,
it is probable that the physiological effects described above
following the intravenous injections of the various preparations were
not mechanical but must be ascribed to the action of the constituents
of the Ava.




                             _CONCLUSION._


The crystalline product consists, largely of methysticin, the methyl
ester of methysticinic acid. In addition to this there is about five
percent of the free methysticinic acid present.

The resinous product consists of about five percent of free resinous
acids, which acids can be separated into three different acids or
groups of acids according to their solubility in the different
alkalies. The remaining resinous product is composed of ester resins.
On hydrolysis these esters yield three distinct acids, two resinous
acids and methysticinic acid. The two resinous acids are distinctly
different, both in physical properties and in chemical properties. They
can be sharply separated by means of the difference in solubility of
their barium and iron salts. These acids may be separate individual
substances, or a group of related substances. The barium and iron acids
although different chemically, have some groups in common as shown by
the formation of a common oxidation product. There are at least three
alcohol radicals formed through hydrolysis, a resinous alcohol radical,
a crystalline substance with a melting point of 130 degrees, and a
volatile oil.

The physiological action of the Ava is due to the ester resins. The two
resinous acids formed through the hydrolysis of these esters seem to be
the two active constituents of the resin ester.

From the above outlined work it appears that the ava root does not
contain any alkaloidal substance, as none of the constituents were
found to contain nitrogen.

That the aqueous infusion used by the ava drinkers contains the same
constituents as are extracted by the ether is shown by the following.
An infusion of the ava was made by allowing some of the powdered drug
to remain in contact with water for about twelve hours. The infusion
was then filtered and the filtrate extracted with ether. A resinous
mass remained which from all appearances was identical with the resins
obtained from the ether extract. It also gave the same action when
placed on the tongue as was produced by the resins from the ether
extract.