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Localization of the drug content of digitalis Macdougall, Alice Pearce 1923

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%K^  Bk^ BA* J&2>^? ©1©  LOCALIZATION  OP  OP  THE  DRUG  CONTENT  DIGITALIS by  Alias  Pearoe Maodougall  A thesis submitted for the Degree of MASTER  OP  ARTS  in the Department of BOTANY  UNIVERSITY APRIL  OP  BRITISH  1923.  COLUMBIA  TABLE  OP  CONTENTS  INTRODUCTION BOTANICAL  1  PLANT PROCESSES  8  HISTORICAL MEDICAL  10 .  11  CLINICAL  13  PHSIOLOOICAL  16  CULTIVATION  17  COLLECTION  21  IDENTIFICATION TESTS  26  LOCALIZATION EXPERIMENTS  29  GLANDULAR HAIR EXPERIMENTS  52  GENERAL RESULTS  53  CONCLUSION  ILLUSTRATIONS  BIOGRAPHY  55  66  ILLUSTRATIONS 1.  FRONTISPIECE - PLANT BEGINNING 2nd. YEAR'S GROWTH  2.  LEAVES OF DIFFERENT SHAPES  3.  A LANCEOLATE AND AN OVATE LEAF  4.  PICTURE OF NON-GLANDULAR HAIR  5.  BIAGRAMS OF NON-GLANDULAR HAIRS  6.  PICTURE OF GLANDULAR HAIR  7.  DIAGRAM OF GLANDULAR HAIRS  8.  DIAGRAM OF VACUOLE OF GLANDULAR HAIRS  9.  PICTURE OF RETICULAR APPEARANCE OF LEAF  10.  DIAGRAM OF TYPICAL VENATION  11.  PICTURE OF VEIN GLAND  12.  ENLARGED PICTURE OF VEIN GLAND  13.  DIAGRAM OF VEIN GLAND  14.  PICTURE OF AREA BET .TEEN VEINS  15.  PICTURE OF MID RIB  16.  PICTURE OF BUNDLB  17.  PICTURE OF LONGTITUDINAL SECTION OF VEIN  18.  PICTURE OF ENLARGED LONGITITUDINAL SECTION OF VEIN  19.  PICTURE OF DIFFERENT SIZES OF LEAVES  20.  DIAGRAM OF SECTIONS OF LEAF  21.  APPROXIMATE LOCALIZATION OF DRUG CONTENT.  p i _, 7_  __DlG.rT-p.lii  j , I - Kl  CO'VNP^CACrN/  a  L_  Jdcon''  Yett  - 1 LOCALIZATION OF THE DRUG CONTENT OP DIGITALIS. The drug Digitalis, whioh is extracted from the leaves of the plant popularly known as Foxglove, has been used extensively for many years in the treatment of cardiao diseases and Is therefore of great pharmaoeutioal importance. Very little, howuver, is known of the aotual manufaoture of the drug in the plant itself.  The purpose of this investigation  is to disoover with what physiologioal prooesses its production is coraelated with a view to determining the most suitable oondition for its cultivation and collection. 1.  BOTANICAL  Digitalis purpurea is a member of the Scrophulariaoea family.  It is indigenous tc Central and Southern Europe,  and is naturalized in various other parts of Europe and in Canada and in the  United States as an ornamental plant.  In  British Columbia it is found on Tuxedo Island and in the Skeena and Fraser River Valleys and is rapidly assuming the character of a weed.  Its natutal habitat is in loamy and peaty soil but  it thrives well in ordinary well drained gardens, the soil of whioh is of open texture and of reasonable fertility.  Although  it is frequently found in fields that have been razed by fire, sowing in the open field so seldom gives good results that it oonnot be over recommended.  J.V. Lloyd ( A TREATISE ON  DIGITALIS, LLOYD BROS, page 3) states that it grows in  - 2 silioious lands but does not reproduoe itself in limestone soil, while aacording to B.M. Holmes ( PHARM. JOUR., JULY 1, 1905, 5) the soil should be loose and rich but not oaloareous and Bernbeok) A. J. P., 1879, 556) adds that it must be grown on a slope, supporting the theory that it is deleterious to the potency of the drug to have standing water in the ground. After the Parmer's Bulletin 663,  -  "the seeds are exceeding-  ly small and do not germinate well except under the most favorable conditions. They should be mixed with sand to ensure even distribution in seeding, and sown as early as February in seed pans or flats in greenhouses or in well protected frames. 7/hen danger of frost is past the plants should be hardened off and transplanted to the field where they may be set about a foot apart in rows spaced conveniently for cultivation." Fron a biennial or perennial fibrous root is sent forth the first year a dense rosette of leaves and in the following summer a single, erect, downy, and leafy stem attaining a height of 2-6 ft., terminating in an elegant spike of flowers which are attached with short peduncles in such a manner that they generally hang down on one side.  At the base  of each peduncle is a floral leaf which is sessile, ovate and pointed.  The blossoms are about 2 inches long, and are usually  lavender to dark purple in ooior but there is a variety that has white flowers known as Digitalis purpurea, var. alba. "The calyx is divided into five segments of which the uppermost is narrower than the others.  The corolla is monopetalous  bell-form, swelling on the lower side, irregularly divided at  - 3 the margin in short, obtuse lobes, and in shape and sixe not unlike the end of the finger of a glove, a olroumatanoe whloh has suggested moat of the names by whloh the plant is designated in different languages.  Its mouth la guarded by long, soft  hairs and the internal white ground is sprimcled with black: spots.  The filaments are white, curved and surmounted by large,  yellow anthers.  The Style supports a byfed stigma."- Villd  (Sp. Plant ill 383).  The seeds are numerous, very small,  grayish brown and oontained in a pyramidal, two-oelled oapsule. The leaves are simple in structure, being oomposed of a lamina and a petiole.  As they isaue in an approximately  horizontal poaition they are said to be dorsiverted and for this reason there la a stronger developement of chlorophyll tiaaue in the upper surface while in the lower the veina stand out more prominently and there la a greater number of stomata. The root leaves are from 7-10 inches in length and about 3 inches in width and stand on ahort, winged foot stalks but aa the leaves approach the top they are arranged alternately and become smaller and more sparse with shorter petioles* surfaoes have a velvet like appearance.  The  The upper is of a fine,  deep green oolor and is minutely hairy and somewhat wrinkled. The lower side is paler, grayish green and more downy.  The  general shape ranges from ovate to lanceolate and although the Med. Bot. (p. 216, t. 76) suggests that the lower leaves are oonsistently ovate and the uppers lanceolate this is not verified by B.O, plants where either lanceolate or ovate, or any stage between the two may be found in either poaition.  - 4Fig. 11. shows a number of leaves grown in a variety of places illustrating the faot that the location, in itself, does not determine the shape; Fig. 111. demonstrates by two root leaves, one lanceolate and the other ovate, taken from adjacent plants that this is not determined, either, by the position of the leaf on the plant.  These plants were grown so close together  that one could not be pulled out without the other and the leaves were taken from approximately the same positions on their respective plants.  The summit of the loaf is acute or  rounded and the base somewhat ouneate, tapering into the petiole which is about one third the length of the lamina in the rosette leaves, while in the upper ones it is almost wanting; the color Of this portion is grayish brown, it is laminated and of a fragile texture.  There are three distinctive features of this  leaf. A  Oalcium oxalate does not occur in this plant which dis-  tinguishes it from all other narcotic herbs. (£.11. Piatt, Thesis on Digitalis, U.of tf.). B  This plant, as has been intimated above, is definitely  villous and there are two distinct types of hairs. 1 Hon glandular- These are tubular outgrowths of the epidermal cells. Fig. iv v, and are characteristic of the Foxglove plant although they vary in form and in the number of cells.  Of these there are unicellular ones which  are merely outgrowths considerably longer than papillae and uniseriate hairs in which from 2-10 cells are connected on a chain.  The tip of these may be straight-pointed or  - 5 hooked-pointed or topped by a globular cell.  The cells  are of unequal length and are frequently placed at right angles to cells above or below (Mansfield, Histology of Medicinal Plants, Ghap. 11). Leaves covered with these hairs are characteristic of dry olimates and experiments show that shearing off the hair from the bearing leaf increases the loss of water by transpiration.  In fact,  the same species or individual becomes more hairy when subjected to a drier atmosphere. Glandular- These also are tubular outgrowths of the epidermal cells, but differ from those desoribed above i that they consist of 1-3 non glandular cells surmounted by a single or double glandular one, in which vacuoles or globules are observed (Fig. VI Vll). Kraemer, (Applied Eoonomic Botany, Wiley & Sons, Page 228)  de-  scribes the ocoasioner of this secretion as "the protoplasmic oontent of the cell" but points out that DeBary states that the secretion must originate in the wall itself.  To determine, if possible, the exact nature of  these cells in order to see what relation they hold if any, to the manufacture of the drug, sections were made somewhat similar to those suggested by Tummunn (Ber d d pharm. Gesellsch, 1908, Page 513) on Nov. 3rd to 4th, 1922.  The hairs were treated with a 50/50 solution of  zylene - absolute alohhol and the cuticle broken.  It  was discovered that the hairs resembled the second typo  - 6 described by this investigator - that is, the vaouolea consisted of a fine net work (Fig, 8 ) . Regarding their ooourrenoe Jelliffe (Drug Giro Sept. 1900, 176} states that no hairs appear on the upper epidermis but in the leaves examined both types of hairs were present on the upper and the lower surfaces (Table 111)* 0  The most distinguishing feature referred to by Mr, Piatt  is the characteristic venation of the leaf (Pig. 10). The vascular bundles known as veins, which enter a blade through the petiole and branoh in various directions have two functions - 1st.  Mechanical support;  materials to and from the leaves.  2nd.  To oarry nutritive  The venation of this leaf  is said to be pinnately reticulate.  A prominent, greyish  brown, mid rib runs from the base to apex and from it extends a number of prominent veins of the first order that diverge at angles of 30° to 60° and unite with one another near the margin.  Prom these arise many anastomosing veins which give  the leaf its extremely reticulate appearance (Pig. XI). The veins are exceptionally pubescent.  At the point of union of  the veins at eaoh tooth there is what Kramer describes as a single water pore, differing from true stomata in that they contain water and not air (A.J.P. 1912, 35). This is wider than the veins themselves and in some instances is found at the juncture of two or three veins and at other times it appears to be just an enlargement of a 3ingle terminus one (Pigs. 11, 12 & 13). The experiment, however, described below  - 7 attribute to this enlargement a totally different function. As Kramer himself states that true water pore  should give  off water in liquid form and no suoh exudation has been noticed on these leaves after careful examination of different ones under many conditions there is sufficient reason for further investigation of their structure. by this writer that the water pores  It is also stated  are distinctly a char-  acteristic feature of the upper epidermis but in this case the upper epidermis may be scraped away and this dilation remains intact (as in experiments below). The leaf of Digitalis purpurea oomplies with the typical green leaf structure in these respects*- A thin cuticle; a lower and upper epidermis oomposed of isodiometric cells larger on the upper than on the lower surface; wider lumen on the epidermal cells of the ventral side than on the dorsal; presence of hairs; stomata muoh more frequent on lower than on upper side; parenchyma differentiated into a palisade layer on the upper side; ohlorenohymat green ohlorophyll-bearing tissue; oollenohyma; and tracheae. (Figs. 14 18).  The leaf is, however, distinguished by the fact that  there is on the ventral side a layer of irregular cells outside the epidermis.  To this is attached the hairs.  referred to by many writers as the "Extra Epidermis".  This is At  times this layer is loose over the mid rib and on the petiole and the lower part of the lamina.  In the Fall this layer was  not observable but with leaves piolced Jan. 15th, after having  - a remained under aaow for •<>•• days this was remarked,  that  thia oonditlon la directly attributable to auperabundanoe of molature and not to frost, a laaf whioh did not ahow thlo oonditlon waa plaoad la a dlah of water for omo weak. the and of thia tlmo tha layer waa quit* looae.  At  These  •zparlaanti were made on a number of different leaves with Invariable result a.  2.  PLAHT PB00B3SSS  The manufacture of digitalis la neoeaaarily linked up with the physiological proooaaaa taking plaoa la tha laaf itself.  "The fundamental faot la the nutrition of all living  things to the oapaoity of green plante to make certain complex organio oompounda, namely carbohydrates, out of Carbon Dioxide and water by tha aid of light. la known as photosynthesis."  Thla unique proooaa  (Coulter, Barnes and Oowles,  fast Book of Botany, part 1, page 363, American Book Company). the organa roaponalble for thla oonveraion ara the oaloroplaata whioh ara found In the auporfioial parenchyma oella, tha ohlorenohyma. energy.  They poaseaa tha oapaoity of absorbing  thia process is, however, only tha flrat atago af  the ooaplete work of oonetruotloa.  from thoaa carbohydrates,  togathor with raw materials taken from tha eoil, tha plant builds up ita proteins and there la then stored up there aha energy to ha expended far tha work of growth,  tha proeess  - 9 of setting free the energy to perform this work, is known as respiration and it consists largely in the decomposition of protoplasm, either directly or as the result of the action of an enayme or of some internal force.  Ho two plants are  alike in the substances formed as a result of this aotion. These are, however, all included in the term "Bnd Products". These are divided into four general olasses. 1.  Reserve products - whioh are stored for food;  2.  Useful end products - the plant is benefitted directly by their presence;  3#  Intermediate products of metabolism;  4.  Waste products - in consequence of the destructive metabolism there arises a number of oompounds whioh cannot be used for the building of new parts and are therefore not again drawn into the metabolism. They are packed in some form where they will be out of the way.  Some of them are of considerable ser-  vice to the plant ass by a protective disagreeable taste and odour; covering wounds by gummy or resinous exudations; attracting by colour and odour insects to effect pollination.  It is among these  waste products that this drug must be placed.  On  account of its extremely nauseous taste and narcotl odour it protects the plant from predatory animals. An examination of the leaf (whioh will be detailed below) shows that it is stored in the enlargements  - 10 found at the ends of the veins.  3.  HISTORICAL  It is impossible to estimate with any degree of aocuraoy the number of years that this plant has been used for medicinal purposes. No doubt it was discovered by the peasants of the district of whioh it is a native, along with other herbs and compounded into a so-called "old wives* remedy". Following this purely impirioal use when polypharmacy oame into vogue it became a favorite for shot gun prescriptions.  From old records it is supposed to have been  used either as a poultice or in liquid form, the latter being obtained by crushing the leaves or boiling them in various solutions.  Dr. W. Withering, the physician to the General  Hospital in Berlin, was the first investigator of Digitalis (Pharm. Era, July 29, 1919).  In 1775 it was brought to his  attention that an old Shropshire woman was performing some remarkable cures for dropsy.  He investigated her methods and  disoovered that a "medicine" containing 20 substances was used.  Among them was Digitalis and of the twenty, this was  the only active principle.  He experimented on this drug in  his hospital work and in 1779 presented his results and observations to the Medical Society of Edinburgh.  Immediate-  ly he was greeted with a type of opposition approaching aotual derision.  He, however, was convinced of the efficiency  - 11 and of the ultimate establishment of the drug in medical aoienoe and wrote, "After all, in spite of opinion, prejudice, or error, time will fix the real value upon this and will determine whether I have imposed upon myself and others or contributed to the benefit of aoienoe and mankind".  Prom  that time forward the popularity of the drug, although receiving many rebuffs, has gradually increased until now its importanoe is suoh that there is a German adage which says "The experienced physician may be distinguished from the inexperienced one by the way in whioh he uses the Digitalis,n  4.  MBDIOAL  This drug is essentially a sedative as it allays irritation and assuages pain; the beating of the heart is slowed down and a feeling of faintneas or drouaineas results. It has been called the pendalum of the heart as it dimishes undue or abnormal action of that organ and aasists its driving power when that is deficient. Herbs)  (D. Ellis.Medicinal  The whole digitalis group of drugs increase the  tone of the heart but are all irritating to mucous membranes and are stimulating to the unstriped muscle fibre of the body.  "Small doees reduce the rapidity of the heart beat  with some increase in the size of the pulse wave but without any marked variation in the pressure.  If a large dose ia  - 12 given the pulse rate becomes extremely slow, the amplitude of the oardiac excusions and the blood pressure more or less elevated. summed upx  The changes In the activity of the heart may be slowing of Its rate, due to inhibitory stimulation;  increase of contractility and increase of tone, brought about by direct action upon the muscle; A debry of conduction probably through the action upon the vagus; and increase of irritability of the muscles.  Its greatest use is in chronic heart  conditions but it is often a valuable stimulant in the treatment of acute circulatory failure." and Therapeutics)  (H.O. Wood Jr. Pharooology  "By the first Influence It produces prolong-  ation of the diastole; by the second, an inorease putting forth of power in the systole.  After a toxic dose the systolio  irritation overbalances the diastolic stimulation, and the pulse becomes dicrotic because the diastole Is continually interrupted by systolio contractions and the aortic system remains empty, because the left ventricle never relaxes suffoperation iclently to receive blood. During the moderate/of digitalis there may be some sense of cerebral disturbance, brow tightness, and even a confusion of thought and giddiness.  After  toxic doses the symptoms are severe; a feeble, scarcely perceptible pulse, nausea and vomitting, stupor  or delirum,  hiocough, convulsions, and syncope have in several oases preceded the fatal issue".  (U.S. Dispensitory).  A secondary use of the drug is in the treatment of  - 13 dropsy as it is also a decided diuretic.  This disease is  frequently the result of general venous repletion which also interferes with the function of the kidneys.  Patients  suffering with dropsy are often in such a condition that the drug cannot be taken hypodermically or by tablet.  One of the  diffioult factors in the use of this drug is the danger of troublesome side effects and it is important that the drug should be prepared in such a way as to show a maximum of theropeutic action with a minimum of these disagreeable features.  It has proved useful in this case to apply it  externally, in the form of fresh leaves crushed, or dried leaves made into a poultice, or flannels soaked in the tincture.  Hoffner (J.P.O. Juillet, 1867, page 37) shows that it  is capable of being absorbed through the skin and thus can be obtained into the system by means of baths.  5.  CHEMICAL  As is the case with many other drug extracts, the chemistry is still incomplete.  It is known, however, to  depend for its activity on a number of gluooosides.  Our main  knowledge of this subject we owe to Sohmeideberg (P.J. *3', v 741) and to Kiliani (P.J. «3', XX11, 1061 and '4», 1, 29). "There are at least five gluooosides present in the leaves and seeds of foxglove and these are the aotive principles in the medicine."  Thatcher - Chemistry of Plant Life.  - 14 1«  Digitoxin C 3 4 Hg. 0 1 1 '- This is the most active in its physiologioal effects and from a quantitative p  p  point of view is the most important. However, its effects are not the same as those of the whole leaf*  The work of Frankel which demon-  strates its tendency to culminative action has been confirmed by other experimenters and it is generally conceded that it acts more slowly than digitalis.  The experiments of J. P. Arnold,  H, 0, Wood Jr., Zeltner and Cushy prove that it affects the circulation of lower animals as does digitalis and it has been recommended as better than the older preparations by Potain, Stark, Cowan, and other clinicians.  However, it is  locally very irritant and as prone as digitalis to derange digestion so that there is no apparent advantage.  Attempts have been made to find a  relationship between the digitoxin content and the activity of the preparations but all results are failures. When it is hydrolyzed it yields digitoxigenin Q C-- H  H_„ 0. and a sugar digitoxose  0 , which is supposed to be a dimethyl  tetrose.  Digitoxin is a glucoside, insoluble in  water but soluble in alohhol and in chloroform.  - 15 E.  Digitalin 0 _ H g 6 014r - This is found mainly in the seed. It breaks up into inactive digitalegin C <)3 and two sugar*.  H1Q  This is soluble in alcohol  but insoluble in chloroform and water. 3.  Digitophyllin.  4.  Digitalein - Resembles digitalin closely but it is insoluble in water.  It is claimed by Kraft  (Aroh. d. Pharm. 250, 1912, 118) to have been obtained in as pure a condition as Gitalin °49 H 80  <5  23#  Jt  is n o t  Probable that digitalein  represents digitalis. 5.  Digitonin - 0 g 4 H 9 g 02e - This is a physiologically inactive saponin-like body present in the leaves. It has no characteristic effects but has been apparently demonstrated to be the physiological antagonist of digitalis. These constituents of the leaves are divided by Kol-  vert (Munich Med. Wooh. Aug. 20/l2, p. 1865) into gluoosides of the digitalis group and gluoosides of the saponin group. The action of all these upon the heart is similar.  These  terms are not used consistently and there is a tendency in this country to popularize them and this probably accounts in part, for the evident difference in our digitalis terminology and in that of Kuropean pharmacists.  The group known as  saponins form oolloidal solutions in water, whioh produces a soapy foam when agitated.  (Hoas & Hill. Chemistry of Plant  - 16 Products).  They have oharaoteristio reaction which play an  important part in the investigation of the character of the specific drug.  6.  PHTSIOIOOIOAX,  As the aotivity of the drug depends upon no single, active principle it is impossible to obtain a chemically standardized preparation and attention has naturally been directed towards the development of physiological methods of assay.  These are extremely numerous and are all more or less  approximate owing to the faot that standardization is made on the basis of a lethal dose and many other factors, other than the actual poisoning by digitalis, may enter into the death of the animal.  Oats, guinea pigs, frogs, goldfish and mice  have all been used but as the first three are most important a short resume of a common method of using each will be given. Guinea pigs of about the same weight are injected beneath the skin of the abdomen with the drugs to be tested. The dose is reckoned aooording to the weight of the animal and is increased until the lethal dose is arrived at. In the case of cats the toxic dose is determined by a first injection of Ice of a 10$ solution and • further injections of ,5cc each, administered at intervals of not less than five minutes until the death of the animal.  The toxic doses  were compared on the basis of the number of milligrams of the  - 17 drug used per kilogram of the body weight of the oat. (Bdmunds & Hale, The Physiologioal Standardisation of Digitalisi Page 36). The tozio dose for frogs is found aooording to Houghton*s method (J. Am, M. Ass. Chioago 1898, xzzl, 959) which is based on the theory that the killing power of oardiao drugs for frogs of definite size and speoies, kept under proper oonditiona, is constant per gram of body weight.  Frogs may  be of any one speoies or weight providing allowanoe is made for variation. handled.  They should be freshly caught and carefully  The dose of the drug to be tested is calculated  aooording to the weight of the frogs to be employed and made up with salt solution so that it will measure about l/2 oo and is then introduced into the abdominal lymph sac by means of a pipette.  The frogs are placed in jars for twelve hours,  then examined and deaths recorded.  A second series, to lessen  the doses, is made which will narrow it down so that in the third series, this time of five injections, three frogs should be killed.  The standard preparation can then be obtained by  calculating the relative strengths.  7.  CULTIVATION  The recognition of the exceedingly great amount of variation in different preparations has forced Investigators to examine carefully not only conditions of growth but the  - 18 collection of leaves as well.  No doubt there are many causes  partially responsible for these differences and the futility of cultivating correctly and then lowering the toxicity by improper collecting is readily recognized.  The matter of cul-  tivation has been an occasion for many opinionated statements and as many heated arguments and discussions.  There are the  problems as to the amount of light and moisture required and there is much controversy too as to the relative potency of wild and of cultivated plants and when cultivated, what fertilisers, if any, should be used.  A short recital of these  problems will be given with a view to determining the oonaensus of opinions. Regarding the amount of light required by the leaf there is comparative agreement.  The majority of those who have  stated their views on the subject agree with Martingale (TitleDigitalis Assay, H. K. Lewis, London) when he says, "The most potent leaves I have examined were seoond year leaves grown in this oountry In a sunny exposed condition."  Duncan recom-  mends that the plant should be grown in elevated plaoes exposed to the sun.  There are some, however, who are unremitting  in their efforts to oonvinoe pharmooologists and growers that a wooded locality is the natural habitat and therefore that leaves grown with a much reduced amount of light must be more potent than those whioh are grown in the open.  Again, with  regard to the question of moisture, the writer above is  - 19 generally agreed with hut as before, there is a popular idea that the plants thrive best in a low lying district of whioh the earth ia soggy, drainage oonditions being entirely disregarded*  The question of the excessive moisture does not  refer so much to the water whioh falls on the leaves (although this has a very definite bearing on the subject) but to the healthy oondition of the roots, whioh are an important organ in the plant process of which the drug is a by-product. .7ith the exoeption of the comparative value of first and aeoond year leaves, by far the most vexing question ia that oonoernlng the relative potency of the drugs extracted from wild and from cultivated plants.  The opinions  offered are given below (Table 1) and to facilitate clarity the full references can be found in the bibliography and are indicated by numbers.  BOU I lili 1.  Berateok  3.  Brown National Herb Ass.  41.  Sohmeideberg  19.  Hirohashi  40.  Sharp & Brown  43.  Thatcher  It is stated in Oregon that wild leaves have the highest toxicity, while the reverse is the oase in Washington. This leads to the obvious oonolusion, that when such  - 20 differences of opinion exist between reliable investigators and that these two conditions are in direot antithesis to eaoh other, that were one or the other absolutely correct, this would not exist*  It follows then that, although in some  instances wild plants may be best and in others cultivated ones are more toxic, it is not because of this character in itself*  Lastly there arises the question that if these plants  are cultivated what fertiliser should be used*  At the Botan-  ical Gardens at Point Grey, B.C., there are four beds of digitalis, all grown under different conditions.  The plants  that are fertilized with oow manure are by far the finest and are followed by those treated with superphosphate*  Those in  the ammonium sulphate bed are small and many of them have not survived the winter.  Those plants of whioh the growth was  checked and are unfertilised are the poorest of all*  Mr.  Daughinee (U.B.O. *22) in his experiments on the relative toxicity of the leaves of these plants discovered that tincture from those fertilized by oow manure was the best, followed by those whose growth was checked.  The other two fertili-  sers produced a plant of whioh the tincture is poor.  In other  words as far as the drug oontent is ooncerned the plants grown under the most natural condition prove most satisfactory* Digitalis then should be grown in rich, well drained soil, in suoh a way that it will be exposed to as much sunlight as possible..  21 8.  COLLECTION  The question of oolleotion is slightly more involved owing to the faot that there a greater number of faotors whioh must be taken into oonsideration.  Since a great deal  has been written on this subject it will be possible to give only a digest of the material at hand.  As has been intimated  above, the most argumentative problem is whether leaves of the first or the second year plants are preferable.  It has  been discussed by pharmacologists since 1870 when the results of investigators (Bull. Soo. Pharm. 1870, 164; T.B.P. 1870, P. 75; Y.B.P. 1871, P.97) indicated that the leaves should be collected in the late summer and that the radical leaves could be included.  Immediately the controversy arose as to  the advisability of plucking leaves the first year of growth. Bernbeok (Ph. Zeitung, 1879, P. 506; T.P.B., 1880, P. 173) was in favor of leaves from plants of the second summer. It might be supposed that physiological assays oould settle this question finally but it is discovered that two experimenters Parr and Hynes (P.J. l/l907, P. 198; Jl. Am. Ph. July 1908, Pg. 330) supports the original view while Hale asserts that first year leaves are ZQ% - 40$ stronger than those obtained in the second year.  Pooice states that seoond  year leaves have their highest value at the time of flowering and the first year ones in the late summer.  The British  Pharmooopia agrees with that of the T7.&. whioh says "the  - 22 dried leaves of Digitalis purpurea Lumme (Fam. Seroph.) collected from plants of the second year's growth, at the oommenoement of flowering."  Geiger holds that they should be  gathered then but F. Sohnleder, on the other hand, asserts that they are better in the early Pall of that year (A.J,P. 1870, Pg. 221). Dr. Robert A. Hatcher in a paper entitled "Digitalis and its preparations" points out that actual experiments have shown that leaves of cultivated, first year plants are most active.  This given in tabulated form indi-  cates the extreme disparity of opinion, Table 2 First Year  Second Year  42.  Sohnieder  47.  Withering  13.  Promme  33.  Natibelle  12.  Parr  1.  Burnbeck  16.  Hart  3.  Brown  Any time from June to November of either the first or second year, has its supporters and some suggest that the season makes no difference at all.  It is oonparable to the  problem of the wild and cultivated plant and there is no doubt that, as in that case, this in itself is not the controlling factor.  If the leaves are grown in proper conditions otherwise,  the faot that they are from first or second year plants makes no difference.  The only danger is that leaves of the firs*  year plant may be too small to be potent or that plucking these leaves may kill or seriously retard the growth of the plant.  - 23 Owing to the rather distinctive appearanoe of the glandular hairs in this leaf the possibility of them being concerned in the aotual manufacture of the drug has been repeatedly suggested.  There is no doubt that their numbers are  proportionately greater in some plants than in others and it follows that, should they be for this purpose, it would be advantageous to cultivate these varieties* has a two fold nature:-  The question then  Whether there is any direot relation-  ship between the number of hairs and the conditions of cultivation; and whether the hairs as such are directly connected with the manufacture of the drug. Probably on aooount of the superior numbers of the purple flowers there has been little controversy regarding the relative value of these and of the white flowers. Generally speaking, experimenters agree that there is little, if any, difference in the leaves of plants which produce these two kinds of flowers.  This can be readily understood  when it is remembered that the drug is a waste product of a life process going on in the leaf itself. There was a time when pharmacists used the entire plant for the manufacture of the drug but as its chemistry advanced and it was discovered that some active substances were found only in certain parts of the plant and that other portions had a very small drug content in proportion to them, it was decided that the potency of the preparation would be raised if suoh were discarded.  "The seeds contain more of the  - 24 aotlve principle than the leaves, are leas likely to suffer in drying, and keep better.  They are, however, little used*  As far as the relative strength of these two parts can be determined from their alcoholic extracts it would appear from the experiments of Hirtz (A.J.P. xxxlll) that the seeds are ten times stronger than the leaves'*. U.S. Dispensatory, 19th Edition, Wood, Remington & Sadtler.  Hirohashi gives the same  proportion and it is verified by others who suggest that the seeds are anywhere from 8 to 15 times as strong as the leaves. As far the leaf itself, any writers who have referred directly to this, state that the mid rib and petiole should be removed before the powder is prepared.  The only aotual results ob-  tained are from the work of Fromme who used Keller's process and deoided that the average leaf yields ,3% by weight and the petiole, "frequently".1$ to *3%.  No record oan be found of  anyone having experimented with the roots but Broeker states that the parenchyma yields six times as much drug as the stem. It is noteworthy too that these last parts have deoidedly, if gradually, fallen into disuse. In searching for explanations of these problems a far fetched suggestion has been made that the size and shape of the leaf governs its value as a drug producer.  Coupled with  this is the theory that different conditions account for different shapes of leaves.  In Pig. 3 are two leaves - one  lanceolate and one ovate - from two plants grown under identioal conditions.  An allusion has been made also, to the position of  - 25 the leaves on the stem and Hirohashi contributes that leaves increase in toxicity from the top of the plant to the bottom. As to the size, however, it is known definitely that leaves are only toxic from 8 to 30 cm.  In Fig, 19 a number of leaves  of different sizes are photographed.  A. is 32 om. and is  therefore too large, while S. and H. are both less than 3 cm. so they too would have to be discarded.  (An experimental  verification of these lengths is given below). After the leaves have been chosen and plucked there is some technique to be followed regarding their drying and preparing for the market.  Withering recommends that the leaves  should be dried, either in the sunshine or by a gentle heat before the fire and oare should be taken to keep them separated when drying.  Others, among whom is Pereira, state that  a preferable mode to dry them is in a basket in a drying stove. The Farmers' Bulletin 663 P. 30 says that leaves should be dried carefully in the shade and should be stored in such a manner that they will not be exposed to light or moisture. Generally speaking, then, the plant should be dried under moderate conditions and should be stored in air tight, darkened vessels. Sxporiments indioate that yields of 450 to 600 lbs. of dried leaves per acre may be obtained under certain conditions.  As the plant is biennial the crop occupies the  soil for the greater part of two seasons and requires closer attention than most truck garden crops.  Although it is of  - 26 great medioal importanoe its potency is suoh that it is administered in small quantities and the annual market requirements are only a few 1,000 lbs.  It would follow then  that from an economic standpoint it ia somewhat doubtful whether it would be profitable to grow digitalis commercially in B.C., unless it oan be shown that olimatio and soil conditions are suoh that a superior drug is produced. From this discussion it follows that the real cause of variation aeems to have an intrinsic connection with the nature of the soil and the character of the season.  The  problems left open, whioh will be dealt with in the experiments desorlbed below, ares- The influence of light and moisture on the potency of the leaf; The importance of different parts, leaf (hairs), seeds, roots and stems; and the size, shape and position on the plant of the leaf to be used. 9.  IDBNTIFIOAIION TB3TS  The problem of ohemioal tests is not only one of obtaining a definite identifying test for digitalis but it is desirable to procure a means of readily determining the different strengths of various preparations.  As has been said  before, no satisfactory means of doing this has yet been devised but for the purpose, merely of discovering in what portions of the leaf the drug is found, only a test whioh will differentiate clearly with the oolour of the leaf itself is quite satisfactory.  A list of tests was arranged which were  successful with a Parlce Davis tincture.  O&ZI - 27 la 2#  Treat with tanic aoid - dense pero. "  »  ferrioyanid* of pot. 12 to 15 mln., strong turbidity.  3  4#  •  «  *  "  sulphurio aoid - red oolor (a)  Intensify with addition of bromine water.  (b)  Add 5$ ferrio sulphate solution - violet,  (o) • ferrio ohloride solution - blue green. aoetic aoid and dll. sol. ferlo ohloride blue green. (a)  Add ferrio sulphate aoetic-blue t  (b)  Add sulphurio aoid - greenish yellow.  5.  »  ••  Ammonium ohloride - green.  6.  •»  "  ferrio ohloride - mossy. Add hydroohlorio aoid - olear.  7.  Reduoes ammonioal silver to metallic silver.  8.  Merourio iodide in potassium iodide - odourless amorphous preoipate.  9.  Phosphotungstio aoid - colourless amorphous preoipate.  10.  Sol. pot. ferrioyanide in ferrio ohloride - Turnbull's blue.  11.  Dilute sulphurio aoid - warm with Pehling's solution.  12.  Prohde'8 test - Ammonium molybdate 1% in H 2 SQ^ - Red.  13.  lofer's test - Aloohol and H 2 S Q 4 - Violet brown.  14.  Keller - Kiliainl's test - This is the most notable one whioh has yet appeared.  10 grms. of the tinoture  with 10 grms. of water are evaporated to 10 gfcms.  - 28 in a water bath.  After precipitating with  liquor Plumbi Subaoetatis, the filtrate i3 shaken out with 10 oc ohloroform.  The chloroform  is evaporated and. the residue is dissolved in 3 oc aoetic acid to which a trace of ferric chloride is added and layered beneath with concentrated H  S  .  Oolouration is produced.  If  the tincture contains no gluooside there is no blue or green colour but a brown zone and the addition of commercial digitoxine in certain proportion e.g. 0.001 grms. and upwards to 10 cc is clearly discernible.  (W. H. Martingale,  Digitalis Assay H. K. Lewis, Page 15,} Of this number only those could be used for the purpose of microscopical examination which were simple enough to give a single colour in this reaction.  Of the 14 then, numbers  1, 2, 5, 6, 7, 8, 9, 14 were eliminated. A portion of the leaf, after the under layer had been removed, was placed on a watch glass and treated with eaoh test and the results noted.  Owing to the difficulty of distinguishing the green  of the leaf from blue, green or yellow, only 3, 11, 12 and 13 remain.  The leaf when treated aa direoted in 11 turned  a consistent withered brown but with 13 and 14 a good test was obtained.  The a,b,o, portions of 3 were either failures  or added nothing to the test but the reaultwith concentrated H 0 S~. was satisfactory.  A number of comparative experiments  - 29 were run through and No. 3 was the only one to giTe consistent results, so this test was adopted for the experimental work* 10.  LOOALIZATIOH BXPKRIMBNTS  The experimental section of this speoies may be separated into two divisions - 1st. The treatment of various parts of the leaf and plant to Identify the drug in it. 2nd.  Comparative and histological work on the glandular  hairs. The plants used in these experiments were A.  Wild plants 1st.  Prom Burnett* Greek, Burquitlam, B.C., grown in peaty moist soil hut in a sunny looation.  2nd.  Prom Poreat Glen, Burnaby Lake, B.C. Grown in a loamy well drained soil in an exposed situation.  3rd.  Prom the woods behind the Botannioal Gardens at Point Grey, B.C.  Grown on decay-  ing matter in a poorly drained, shaded area. B.  Cultivated plants - Taken originally from the Burnaby Lake Distriot where they were grown in a shaded position. 1st.  Fertilised with oow manure.  2nd.  Pertilised with superphosphate.  3rd.  Pertilised with ammonium sulphate*  - 10 4th.  Growth oheoked. flMao four wora pleoed ae aeedllnga la 8 a 10 bode oa the •••torn alope of the gardens la tho Fall of l»t«, hawing baaa grown to this otage la eold fraaea.  5th.  toodllafrs grown la oold fraaoo froa aeede plant** la tao lata oaoaoor of 1911*  fhooa  are being foroed In order to hawe thoai bloom In 1923, thus eliminating oa* itaion. At, la all that* experlaeate, tha laaf waa oat la Mitloni for trtatmant, thaaa portlona will ba lndloated by auaber and aro ahowa dlagramatloally la Fig. 19*  ••rlti I A.  these experlaeats aero parforaad oa Jan. 8th, a bright  sunny day.  fh* oztro layar  Bxperlaent 1 -  tti«T9ntril alda la aot looao.  Toaag loaf Of plant froa eeed aown 8opt. I til. Sorapa lower opldoralo froat oootloa * with aola oa rtital alda Taint laaadlately beooao oloar aad tha portloa of loaf between thoa a bright green toea oado if welae appear a browa red  taperlaeat t - taaw laaf 3 a « a p a upper  apldarmlt  - 31 -  (Treat seotion 17 on dorsal side Bo glandular hairs observed One Juncture of veins appears reddish Edges red as above Ex. 3 -  Same leaf Sorape and treat seotion 10 on venlml side Edges and tips very red  Ex. 4 -  Petiole of leaf of plant whioh has bloomed Treat and sorape seotion 1 ventral side No effeot exoept consistent yellowing  Ex. 5 -  Same leaf, petiole Sorape and treat section 2 on dorsal side Same result as four  Ex. 6 -  Same leaf Treat and sorape seotion 18 on ven1»l side Diffusion, edge very red  Ex. 7 -  Same leaf Sorape and treat seotion 14 venijal side Not so vividly coloured but veins showed direct effect  Results -  Prom this part of the series it would appear that the drug is found in increasing quantities from the centre of the leaf to the edge and that it is not found in the petiole at all.  - 32 23rd, dark: foggy morning. 1 - Old leaf - after flowering The leaf Is pricked by a pin in a number of places treat and scrape section 7 on dorsal side No immediate change Extreme edge becomes red, then outermost veins Pin prioks show no change Edge becomes extremely red and diffusion all about it Only non glandular hairs show and these are clear 2 - Same leaf, no pin prioks Scrape both sides, treat venfal side Veins and edge red Non glandular hairs unaffected Veins nearest edge the reddest 3 - Leaf of unflowered plant Scrape and treat ventral side section 6 Results as in 2 4 - Same leaf as above In these tests ventoal side soraped and treated Sect. 7 - 10$ H 2 S Q 4 , no effect •  8 — 50jS "  "  »'  "  M  18 - 75$ "  •  turns brown all over  •  19 - Gone."  "  same effect as in Ex.3  - 33 Bx. 5 - Same leaf Scrape and treat ventaal aide section 17 Veins nearest edge are reddest No glandular hairs show Bx. 6 - Fresh leaf of plant in Ex. 3 Left in sunlight 6 hours Scrape and treat vental side seotion 6 Veins and edges beoome brilliant red Near margin red patches at vein junctures Sx. 7 - Gross section of petiole of Ex. 6 Treat without scraping Extreme edges appear browner than the rest Bx. 8 - Petiole of Bx. 1 Treat and scrape vental side All yellowish Bx. 9 - Petiole of Bx. 1 Scrape and treat vental side of Sec. 5 Edges slightly reddened Bx.10 - Leaf as in Bx. 1 - leave 3 hours Scrape and treat vental 3ide Sec. 18 Veins slightly red Colour of edge not as intense as in Ex. 3 Results- That there is not found in the petiole as much of the drug as in the lamina and that leaves of flowered plants are not more toxio than those of unflowered ones.  It was also discovered  - 34 that concentrated acid is moat adviaable and that the patches of colour oooasionally seen in the interior of the leaf are not due to wounds. C.  January 31st, 3 P.M.  Bright day but no direct sunlight.  Leptome of mid rib of second year plant Treat either end with acid Both ends become brown red Disintegrates but reddened fibres remain The aoid is not carried down as the centre is unaffected. Series 2 On January 31st, 1923, four leaves of about equal size, 20 cm*, were taken from a plant of 1922.  On this day  there was no direct sunlight. A.  This leaf had been oovered roughly on the plant for 48  hours. Ex. 1 - Scrape and treat venlral side section 10 Whole section turns brown Veins appear slightly lighter than the rest Bx. 2 - Sorape and treat dorsal side seotion 19 Same result as in Sx. 1 Sx. 3 - Sorape and treat ventral side section 5 Bdge nearest the petiole slightly red but the rest is a dull brown Results - It appears that light is essential to the production of the drug and it is noteworthy  - 35 that in Ex. 3 the portion nearest the edge was loosely covered and there was some appearance of digitalis. B.  This leaf was in water on lab. table 18 hours.  Extra  layer ia noticeable over mid rib. Ex. 1- Both aide8 acraped veittal side of section 7 treated Veins become clear and remain bright green Enlargement of tooth becomes intensified as well as veins leading to it One or two patches at junctures Ex. 2 -Scrape and treat ventral side section 1 All turned yellow Slight brown streak up the centre edge Ex. 3 - Teat water in which it was placed No indication of digitalis Ex. 4 - Section 3 treated Many non- glandular hairs appear oily and break: off easily No glandular hairs observed In this section many more non-glandular hairs comparatively than glandular onea Gradually a alight brown red edge diffusion Ex. 5 - Examine section of 4 with high power microscope Diffusion is not due to colour of hairs for they are all transparent and apparently  - 36 unaffooted. Bx. 6 - Sxtra layer from section 2 is treated Cells irregular Turns green yellow Some parts are yellow brown Bx. 7 - Extra layer from section 6 is treated Same result Bx. 8 - Bxtra layer from section 18 is treated Same result Vhen first examined all these sections appear wrinkled as a re.lief map but straighten out when treated. Ex.10 - Scrape and treat ventjal side section 14 Veins clear and remainder bright green Anastomosing veins become definitely brown Bx.ll - Scrape and treat venial side section 8 Leaf bright green - veins yellow transparent Edges red Hairs are transparent, appear to have oil globules around them In this specimen the glandular hair is observed but it unaffected Ex.12 - Leptome of mid rib Both ends treated Appears very dark Place oover on section and separate into  - 37 fibrelilce strips. Prom these there is a red diffusion Results-That the extra layer, to whioh the hairs are attached shows no indication of the drug. layer is loosened by water.  This  The vein enlarge-  ment and the reins leading to them show the presence of the drug.  Neither type of hairs show  any indication of it. 0.  Placed in water in a microsoope oase in lab. 36 hours.  The extra layer is muoh looser than in B and it is almost detached over mid rib and lower part of leaf. Ex. 1 - Test for digitalis in the water in whioh specimen was placed. Turns slightly brown but shows no definite digitalis colouration Ex. 2 - Treat extra layer from sec. 1 No indication of dig, Ex. 3 - Treat extra layer from sec. 3 Same as above Ex. 4 - Treat extra layer from sec. 10 Same as above Ex. 5 - Treat extra layer from s e c 18 Same as above Ex. 6 - Treat extra layer from sec. 15 In this a faint brown line is seen in part above mid rib  - 38 Ex. 7 - Treat extra layer from seo. 12 Same result as experiment 6 Ex. 8 - Treat extra layer from seo. 8 Before treatment one or two dark spots were observed.  When treated these turned  bright pink Remainder yellow Ex. 9 - Treat extra layer of seo. 7 Before treatment outline of venation oan be seen, also two dark spots When treated these spots appear very pink and with a miorosoope it is seen that they are at the juncture of veins Ex. 10 - Sec. above from which extra layer removed Extreme tip dull red Veins do not show good colour Ex. 11 - Scrape and treat vental side seo. 13 Veins slightly brown No indication of digitalis colouration Sx. 12 - Scrape and treat vent»l side seo. 19 Veins not definite Edge merely dry brown not intense red Results - Digitalis is not manufactured in the hairs but the close connection of the extra layer and the vascular system seems to account fo the slight colouration.  The drug is  - 59 •Tidently not as great in this leaf, whioh has been kept moist in the dark. D.  This leaf was examined in fresh condition.  The extra  layer is not loose or baggy. Bx. 1 - Sorape and treat rental side seo. 3 Edge red veins definitely coloured Sx. 2 - Sorape and treat dorsal side seo. 6 More intense oolour than Bx. 1 Bx. 3 - Treat venfral side seo. 7 Same result as Bx. 2 Bx. 4 - Bxtra layer from seo. 5 soraped off and treated At vein junctures turns pink Bx. 5 - Sorape and treat ventral side seo. 17 Tips of this are slightly withered When treated these tips turn red and there is a diffusion from them Results- The drug is beat in this leaf.  It is definite-  ly looated in the enlargement at the tip of the vein.  Upper and outside parts of leaf  most potent. Series 3 On Feb. 1st three 1922 plants, in a healthy growing oondition, were taken.  A. was partially covered in lab.  by throwing over it a dark oloth.  B. was put in a box in  the lab. so as to insure complete darkness.  C. was kept in  the lab. in a box with a saturated atmosphere.  - 40 A.  Young leaf, about 12 am. long. In rather a wilted oondltion  and most of tne tips ara brown.  In this leaf the extra layer  It not notloeably loose. lz. 1 - Treat venttal side seo. 7 Teins nearest aid rib unaffected Those nearest edge are brownest Kdgea slightly red Kx. 2 - Treat rental side seo. 5 ••ins olear but unoolored Xdge has only a slight diffusion Kz. 3 - Treat vent*«.l side seo. 1 Teins olear and the rest is bright green So diffusion or oolouration Kx. i - Sorape and treat rental side seo. 17 Sdge is not wilted Same result as Sx. 1 Kz. 5 - Seo. 8, tips very wilted Diffioult to remove extra layer from these Treat Some red diffusion from wilted tips Xx. 6 - Treat extra layer from seo. 15 Turns yellow but no brown over aid rib Iota - Remainder of leaf was put in water covered, when removed the extra layer was looser. Xz. 7 - Sorape and treat venwal side seo. 6 Xo diffusion, veins and edges brown.  <  - 41 Results - Show that neither wilted condition nor lack of light layer*  cause loosening  of extra  Partial darkening, however, seems  to have arrested the manufacture  B.  drug and this condition  is  by plaoing  oovering,  This plant was completely  in water and oovered  removed it was in a alightly withered layer adhered  of the  intensified  for one week, when condition but  extra  closely.  E x . 1 - Scrape and treat veitfral side sec, 7 Chocolate brown  diffusion  Hairs are unaffected B x . 2 - Treat oross section of petiole Disintegrates  rapidly  No definite digitalis  oolouration  Bx. 3 - Treat venial side sec. 10 Slight  reddish brown diffusion from extreme tip  Hesults - Complete darkening  serves to arrest  manufacture even more effectively conditions for A,  the  than those  This lessening of light  does not affect the adherenoe  of the extra  1 ay e r . C.  This plant was kept  for one week.  in a saturated atmosphere  in a box  It was noticeably withered when removed.  it was taken a healthy  dark green leaf about 20 cm. long.  Prom  - 42 The specimen used was in much better condition than that of A. or B. Bx. 1 - Scrape and treat ventral side sec. 1 Turns brown Bx. 2 - Scrape and treat venlral side sec. 3 Turns brown In this case some brown diffusion Sx. 3 - Treat sec. 5 Definite brown diffusion at edge Yeins dear and remainder light coloured Bx. 4 - Sorape and treat sec. 18 dorsal side Brown diffusion as above Bx. 5 - Water which was in evaporating dish in box tested for dig. In small quantities gives a distinot brown color. Results w That a moist darkened condition is most detrimental to the manufaoture of the drug in the leaf although the plant itself may show a good healthy oondition.  It follows  therefore that because the natural habitat for foxglove is in a peaty soil this oondition is not valuable from an eoonomio standpoint. was loose.  It was observed in this leaf that the extra layer This can therefore be directly attributed to the  abundance of moisture in the air.  -43 Series 4 The plant for this series was picked and experimented upon at Forest Grlen, Burnaby Lake.  It was found  growing in an exposed plaoe on a southern slope in rich soil. A.  Leaf taken from this plant 22 cm. long in healthy  condition, dark green colour beginning second year's growth. In this specimen the extra layer on the ventral side was not notioeably loose* Bx. 1 - Scrape and treat ventral side, sec. 1 Ho result except brown discolouration near mid rib Bx. 2 - Scrape and treat ventral side, sec. 3 Some slight red colour at edge but generally speaking unaffected. Bx. 3 » Scrape and treat ventral side, sec. 15 Brown discoloration over mid rib Ex. 4 - Scrape and treat ventral side of sec. 14 Same result Bx. 5 - Scrape and treat ventral side, sec. 12 In this some red discoloration at vein junctures Ex. 6 - Sorape and treat ventral side, sec. 11 This condition is more pronounced and rib itself appears to be definitely ooloured. Bx. 7 - Sorape and treat sec. 10, ventral side  - 44 Enlargement at end of mid rib becomes brilliant red and diffusion is formed all along edge.  This is so great that  non-glandular hairs appear to be affected.  Under higher magnifioation,  however, it la seen they remain quite olear. Ex, 8 - Scrape and treat see. 8, ventral side Enlargements in teeth stand out prominently and are colored as tip a brilliant red. All the veins of this section too show the presence of digitalis Ex, 9 - Scrape and treat ventral side of sec, 6 As in 8, dense diffusion and definite colouration of enlargement.  However, only  the veins directly leading into this appear red.  In this section near the  edge there i3 a red blotch formed at the juncture of two veins. Results - Prom this it appears that the drug content is limited to a very definite area, that is the edges and upper part of the lamina.  Pig,  21 gives diagramatically the approximate localization of the drug showing (a) the area in which the drug is most found (b) the area in whioh there is no indication of it.  - 46 B.  This is a young leaf of one year.'s growth.  It is the  same length as the leaf used in A., and is similarly in a healthy condition, although its colour is lighter green.As might be expected the extra layer on this leaf is not loose. Kx. 1 - Scrape and treat ventral side, sec. 1 This turns brown, being slightly darlceft nearer the edge. Sx. Z - Sorape and treat ventral side, sec. 3 No red oolour is seen, although there is a brownish red diffusion from the upper edge. Bx, 3 - Sorape and treat ventral side, sec. 5 There is now a definite red oolour in the vein enlargement and a diffusion at the edge. Ex. 4 - Sorape and treat ventral side, sec. 7 A very large vein enlargement is situated in this section, into which empties the contents of three veins, all of these are red almost to the mid rib and from it a dense diffusion is formed along the edge. In this section a glandular hair is plainly seen.  The glandular material has re-  mained intact and gives no evidence of having been affected by the acid.  - *• -  Ex. 6 - Sorapo and troat too. 11 ^antral aid* • aaabar of rod M I M I  aro •••n at vols  jaaoturoa and *Ton th* aid rib lttolf whan a 00T*r bai boan plaoad on it braaka Into flbraa froa whioh tharo la • rod diffusion. Bx. d - Sorapo and trtat vontral a Ida, too. IS Im thla aaotloa nono of that* rod aaaaaa art aoon and thooffh all tho Taint ara olaar only an oooaalonal ono ffiraa any oTldonoa of tho drag.  Thla appoara by  a thla rad lino, lrraffularly traaod throoffh tho oontro and lnoroaalnff and atralffhtonlnff ont aa It approaohoa th* a Iff a. Bx. T - Sorapo and traat aaotlon IB, vantral aldo Show* no ovldonoa of tho druff, only a allffht brown oolourlnff aronnd aid rib Bx. 8 - Troat tha axtra layar froa oao. 6 T a m o yoilow loaodlataly and ono or two plak apota appear, whloh woro ••ldontly anparlapoaod apon w*ta jnnotarea. ax. » - Traat th* *xtra layar froa aoa. 4 T a m a yollow aa abowa bat ao *olor*d apata appear, with th* *xooptloa of •aa* irraffalarly plaood brawalah araaa.  - 47 Results - It is evident then that the same looalization of drug exists in this first year leaf as in the second year leaf described above.  Prom these  experiments too there is every reason to believe that this leaf is just as potent as the other. Se,rj,ea 5, From the plant used in series 4 the stems and roots were talcen and the examination of these, together with the examination of similar seeds to that from whioh this plant grew form this series. A.  ax. 1 - ft oross seotion is made of the stem at the point where it arises from the ground. No indication of digitalis Ex. 2 - A similar seotion was made higher up with like results. Ex. 3 - From a longtitudinal seotion of the cell no indication of digitalis was received. Results -  It appears then that the drug does not exist as 9uoh in the stem of foxglove plants and there is ample just ifioation for discarding them in the preparation.  B.  Ex. 1 -  The root hairs were examined and although  there appeared to be a very definite brown diffusion no red oolour was observed.  - 48 fix. 2 - A cross section was made of the root from a number of plaoes and all results were similar to above. Results - Pharmacists are justified too in discarding this part of the plant, C.  Seeds were crushed and when treated with ashes yielded  a very brilliant red similar to that formed in the vein enlargement of the leaf.  There is some reason then for ex-  cepting the statement that these seeds have a real toxio value. Series 6. This is a purely comparative series made from representative leaves of plants of different fertilisers and leaves of different sizes.  In eaoh oase section 8 is scraped  and treated on the underside. A.  Leaf 3 cm. long.  There is a slight amount of drug  in this leaf but noticeably small when compared to B.  A leaf 10 om. long whioh showed a very definite  digitalis colouration as did 0.  15 om.  D.  20 om.  B.  25 cm.  P.  30 om.  G.  This leaf is 35 om. long and shows very little  oharaoteristio reaction. H.  Leaf from plant fertilised by oow manure,good reaction.  - 49 I.  Fertilized by ammonium sulphate, no diffusion and only slight colouration,  J,  Fertilized by superphosphate.  Similar results.  K.  Plant from bed whioh was oheoked. Results as in H» Results - These results serve to verify the statements of  other investigators and substantiate the general theory of conditions of growth and oolleotion.  - 50 11.  GLANDULAR HAIR EXPERIMENTS  The second part of the experimental work was done with the glandular hairs found in the leaf.  Series of hand  sections and' of freezing microtome sections were made of leaves of the various specimens used for the purpose of discovering (a)  whether there were glandular hairs on both  sides of the leaf; (b)  if there is any indication of  segregation; (o) if their numbers and position varied with different plants and if this variation follows a general rule. 1.  The list of specimens is as follows:  Dig. purp. seeding, growth retarded by drought 8/9/22 tl  tt  »t  it  tt  2.  11  si  3.  11  II  it  n  n  tl  /20  •  , retarded from blooming  5.  n  11  tt  it  , bloomed, leaves piolced, shows vitality to produce new growth  6.  n  II  /21  •  , grown in boggy land, healthy  7.  11  tl  8.  it  't  /22  "  very young leaf 2 cm.  9.  11  11  /21  •  very large leaf 35 cm.  10.  >i  11  tt  11  grown in shade on decaying vegetable matter  11.  11  It  12.  11  11  13.  11  11  14.  tt  11  15. 16.  11  11  'i.  H  "  11  alba  "  11  11  8/9/22  •  naturally in well drained land  sseeds e e d s sown summer of 21, fert. with sulph. of am. alba  •  N  »  n  tt  M  i»  it  t«  tt  11  n  11  11  tt  "  n  it  M o o w manure  •I  «  11  n  it  it  8uper.phos. 11  n  it  - 51 The table below gives the results of these experiments. The numbers 1 to 10 are used for comparison of the relative number of glandular hairs in eaoh speoies. Table 111. No.  No. of gland.hairs  upper side , pn, r.ibs bet f jibs  x  lower side on ribs bet.ribs  1  7  X  2  6  X  3  5  x  4  8  x  5  7  x  X  X  6  8  X  X  X  7  7  X  X  X  8  7  X  X  X  X  X  X  X  x  y  X X  X  * 9  7  X  X  X  10  9  x  X  X  11  6  X  X  12  7  X  X  13  6  X  14  6  X  15  5  16  7  X  X X  x  X  X  X  X  r.  I  x  X  X  X  X  Prom the above results it will be seen 1.  That all these have glandular hairs on both sides.  2.  That there is no relation between number of hairs and the  - 52 tonicity of the leaves, as Ho. 10 has a low potency but relatively the greatest number of hairs, while the potenoy of 15 to 16 is the same and there is a difference of two in the numbers of hairs. 3.  There is no difference in the two varieties of flowers,  •s In one and two the hairs are less with the alba, in 13 and 14 they are equal, and in 15 and 16 there are more found in the white variety. 4*  There is no general rule regarding their position on  the epedermis.  The results are so varied that one concludes  that should a sufficient number of specimens be examined these hairs would be found in all positions. 5*  The relative numbers are not governed by the plant, as  there is no very great difference between the minimum and maximum. Therefore the glandular hairs as such, in numbers or position, show no direct relation to the potency of the leaf and are not responsible for the manufacture of the drug.  It  follows then that an advantage oould only be gained by cultivating for a much-haired variety in this way; hairs are a safeguard against dry conditions which are advantageous to the drug manufacture but are not natural to the plant itself. In an endeavour to discover the exact nature of these hairs their content was examined both from the sections mentioned above and by other experiments.  Prom a superficial  examination there was observed what appeared to be one or two  - 53 oil globules in the glandular top oell of the hair.  They were  not oil as they could not be dissolved out with zylene.  ,7hen  treated with zylene and alcohol a few specimens were obtained with a broken cuticle and the inside structure could be readily made out.  The vacuoles, or globules, appeared as a fine  net work of glandular material.  12.  GENERAL RESULTS  The results may be divided into two main divisions (a) proper cultivition of the plant (b) selection of the leaf to be used. A.  As has been demonstrated by experiments, a great deal  of sunshine but only a moderate amount of moisture are the two main conditions necessary for the manufacture of the drug in the foxglove leaf.  Although cultivation, per se, is not necess-  arily advantageous to the plant, the soil used is often richer, looser and better drained, all being factors which tend to increase its potency.  It follows then that of the plants  treated experimentally those grown at Forest Glen, Burnaby Lake, and those in the beds fertilized by oow manure at the Botanical Gardens, Point Grey, are most toxic.  Thosetaken from Brunette  Greek, Burquitlam, B.O., and from the woods behind the garden are lacking respectively in good drainage and in sunlight and are therefore distinctly poorer in drug content. B.  The leaf chosen should be as nearly as possible 10 to  25 cm. in length and should be in a healthy condition.  As the  - 54 extra layer is loosened by moisture and excess of moisture lessens the potency of the leaf oare should be taken to see that this condition does not exist in leaves from which a tincture is to be made.  To insure this, as well as to pluck  the leaf at a time when it is receiving an abundance of sunlight it is inadvisable to collect them between September and May of any year and as the removal of a leaf necessarily retards the growth of the plant the orop may well be left till the second year to be gathered when the leaves will be more mature, numerous and uniform.  In conclusion, the drug is manufactured in the leaves as a waste product, justifying its retention by a bitter taste and disagreeable odour; it is stored in a form, which is oolored red, by concentrated sulphuric acid, in an enlargement of a vein or of veins in the tooth of the leaf; in the veins leading up to this; and in the seed.  In order  to obtain the greatest yield these parts alone should be used and oare should be taken to provide for the plant proper climatic and soil conditions.  I L L U S T R A T I O N S  .  af'iii'Wr  • i  -  55  -  i  it  - 66 -  ? P^  * * ! » BMA l M f  ft*. 4 -  •  -  •*t4«r»l«  •f wm gkmmtoX** **!»••  '*  -  57  St".  Fl«. * - «l«alilar kai»  * %  tpitenlii  » l # . r - »ia*ra* mt Altfrmn*  «lan*al*r aalr«.  - 58 -  fl*. 6 - Biagraa at glandular Malarial mt brak«n hair*  w>^  \  Vew  Fig, t - Pietara •bowing large  aa« »!«• af train*,  -  tt«« ! • -  59  -  ty»l«*l  Jk  60 -  FlC* H - » ! • • • * • t a. t — f  a m l a i g — » t aft ftlp «f * • ! » .  acftr* lay«*t  - 61 -  F i g . 13 - Biagraa of •»largo*oat a t t i p of * » l n .  fefe.  »*** 1* * *!•%«•» of *»•» of !»»* b«%»««a • * ! » • * , ?«ll»a*« la? or* o, SatAorais;  t , i n *la»*»lar h a l r o ( b r a w n )  - 62 -  '»  -  W «PS®~  ;V.->>,  Mm&w i9T-r  *!«• **  -  .-» -  -  63  -  Fl«. i f - *i»%«r« *r iftagitaiftMl M o t i o n of *•*» ft* tWNHFiift*  ttftll*  »f  ft»i««rs»l«t  » , M i l s »f v e i n .  »!«• * • * **l**t»« ( » * • • • ) showing t r a o h o a .  -  7^ I I t_*Jt  T  64  -  - 65 -  /$ •  <—  r^T  X9  n* i t \  l-wfe  f-Q  \%  —I 5  i i t —  - 1 *r * —W—  5. 8  1  -  r*  F ^ .  = S  P T J  i  VlC* tO • Dlagraa of ••otloas Into whioh loaf out.  »1#.ftl- Biagraa Of aooroxlmto localisation of tno Arc*. a, b,  Positive oontent Negative oontent  ^J  B I B L I O G R A P H Y  6* 1.  Bernbeok  A.J.P. 1879, 556 Ph. Zietung. 1879, 506 Y.B.P. 1880, 173  2.  Borneman  A.J,P. Deo. 1912, 546-554 Jour A. Ph. A 1912, 114 Suooeasful oultiyation of medioinal plants in Pennsylvania. A.J.P. 1907, 547  3.  Brown  Proo. Mun. Phar. Assoc. 1909, 74-80 Jour A. Ph. A. 1910, 176 Eistorioal, ohemioal and olinloal review of the digitalis. Pharm. Era July 29, 1909, 111.  4.  Bulletin  of Soo. Pharm. 1870, 164 Parmer's 663, page 30, No. 5  5.  Casar & Laret* A.J.P. 1899, 145 Value of digitalis leaves Jour A. Ph. A. 1900, 587 Percentage of Digitoxin before and during the flowering period Jour A. Ph. A. 1912, 154 Precautions against the immature collection of digitalis leaves  6.  Coulter, Barnes & Kowles - Text Book Of Botany, Part 1. American Book Company.  7.  Dispensatory  19th Edition U.S. Puhlishers-Wood, Remington & Sadtler  8.  Duquesnel  Jour A. Ph. A. 701, Title - Digitalis Leaves  9.  Sdmounds & Hale The Physiologioal Standardisation of digitalis.  - 67 Hyg. hab. Bull. No. 48. Public Health & Marine Hospital Service of U.S. 10.  Ellis  Medicinal herbs. London. Sons Ltd. 1918.  11.  Porr & Hynea  P.J.I. / 0 7 , 198.  Blackie &  New reference J.L.A.M.P.H. July 19 08.  12.  Porr  Pharm. Jour, Feb. 13th 1907, 198.  13.  Promma  Pharm. Jour, Sept. 25th 1897, 283  14.  Hass & Hill  Chemistry of Plant Products 180-184 Longmans Green & Go. New York:.  15.  Hale  Pharm. Bra, Oct. 7th 1919, 372  16.  Hart  Pharm. J. 60, 440, A., A.O.S.,/08,1859 Pharm. Jour 1908, 440 Jour A. Ph. A. 1908, 192  17.  Hartwioh & Bohny  Jour A. Ph. A. 1906, 735, Charaotera of Digitalis Leaves. J. Am. Pharm. Assoc. 1913, Quantitative  18 #  Hathoer  Estimation of Digitalis 19.  Hirohashi  J. Pharm. Soo. Japan, July 1911 A.J.P. 1911, 452  20.  Hirta  A.J.P. XXX111  21.  Holmes  Pharm. Jour July 1st 1905, 5. Cultivation of Digitalis Journal A Ph. A 1906, 73  22.  Hoffne»  J.P.O. Juillet 1867, 37  23.  Houghton  24.  Drug  Jour Am. Med. Assoo. 1898, XXXI, 959 Am. Jour Pharm. 1909 volume 81, p. 461 The Pharmaoeutioal Assay of Heart Tonios Ciro. Sept. 1900, 176  - 68 Jour. A. Ph. A. 1901 672 Histology of Digitalis  25.  Kilianl  P. J. (3) XX11, 1061 and (4) i,29.  26.  Kobert  Munioh Had. <7ook, Aug. 20th 1912, page 1865  27.  Kraamer  A.J.P. 1912, 35. Applied and Soonomio Botany, page 228, Wiley & Sons Am. Jour. Pharm. Aug. 1911, Variations in forms of Digitalis Hairs. Soientifio and Applied Pharmooognosy, p. 718, Wiley A Sons  28.  Kraft  Aroh. Pharm. 250, 118 (19121  29.  Lloyd  A Treatise on Digitalis, Lloyd p. 3.  30.  Mansfield  Histology of Medioinal Plants,Chap. 2 Wiley 4 Sons 1916, New York.  31*  Martlndale Digitalis Assay H.K. Lewis London, 1913, p.218t78  32.  Med. Bot.  P. 218 t78  33.  Kativelle  Ph. central* 47, 1874, 38.  34. 35.  Newoombe Patoh  A.J.P. May 1912, 201-214 J.A.P.A. 1912, 153 Jour. A. Pha A. 1891, p. 151. Is there a reliable prooess for the assay of Digitalis?  36.  Pharmooopea  37.  •  U.S. 3¥Great Britain  38.  Piatt  Thesis on Digitalis D. of W.  39.  Pitenger  J.Am. Pharm. Assoo. April 1915. Bioohemio Drug Assay Methods 1914, P. Blaokiston & Son Oompany.  40.  Sharp & Brown Trans. Brit. Pharm. Issoo. 1912, 442-447  41.  Sohmiedeberg  P.J. (3) B. 741.  - 69 Bull. Soc. Roy. de Pharm. Bruxelles 31-340 Determination of the Pharmooologioal Value of the Dtied Leaves of Dig. Purp. 42.  Sohnleder  Pharm. oentralb 1867 No. 49. A.J.P. 1870, p. 221  43.  Thatcher  Chemistry of Plant Life  44.  Pharm. Era Deo. 23, 1909, 649. The Future of Pharmooognosy. Drug Giro. Oot. 1912, 616 Caution regarding microscopical examination of drugs.  45.  Tunmann  Ber. dd Pharm. Gesellsoh 1908, p. 513  46.  fender  A.J.P. 1892, 375 1893, 381 Alkaloids oolour reaction  47.  Withering  A.J.P. July 29, 1901. Aooount of Foxglove and some of its uses, 1785.  48.  Wolff  Jour. A. Ph. A. 1904, 659, Method of collecting and preserving and disposing of Digitalis.  49.  H.C.Wood Jr. Pharmooology and Therapeutics  50.  Ypunlcer  Jour. Am. Pharm. Assoc. 8, 1903-8 (1913)  

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