ABSTRACT
GLYCOALKALOID AND DEMETHYL STEROL CONTENT
IN SOME POTATO VARIE TIES AND CLONES
By

Samuel Lo -Tung Wang

A bisolvent mixture, MeOH -CHC13 (2:1), was developed for
the extraction of glycoalkaloid from potato tubers. Blending the
sample and the solvent mixture at a ratio of 1 to 20 completely
dehydrated the sample, denatured the soluble protein and eliminated
the adhesiveness of starch. A higher yield of glycoalkaloid was
obtained by the bisolvent extraction method than by the Soxhlet
extraction method. A complete recovery of added glycoalkaloid was
also obtained.

The glycoalkaloidisolated from the surface of potato tubers
was shown to contain both O(-solanine and (X-chaconine by thin-
layer chromatography. Both glycoalkaloids had identical absorption
spectra afterreacting with the Marquis reagent, the Clarke reagent

or 85% H3PO4.

Samuel Lo -Tung Wang

Demethyl sterols were obtained from the chloroform
fractionof the bisolvent mixture. After saponification, free sterols
and steryl glycosides were separated by thin -layer chromatography,
and both fractions were found to contain cholesterol, stigmasterol
and fl -sitosterol. The quantity of above sterols was determined by
thin ~1ayer and gas -liquid chromatography. ,8 -sitosterol was the
most abundant one.

The glycoalkaloid levels of Russet Burbank, Kennebec,

709 and 321 -65 potato tubers during control storage at 40 and 50F
generally-decreased. Most of the decrease occurred during the first
two months of storage and it was most pronounced in tubers with
initially high glycoalkaloid content. A rise in storage temperature
(45F .to 60F). resulted in an increase in glycoalkaloid content with

the greatest increase occurring in the RussetBurbank variety. The
glycoalkaloid content of tubers with an initially high glycoalkaloid
level was generally reduced by» sprouting, while in those withlower
glycoalkaloid levels, there was slight increase of glycoalkaloid.
None of the potato tubers had a glycoalkaloid level that could be con-
sidered undesirable.

Glycoalkaloidlevel was found to increase rapidly when
potatoes were treated with white light, but no increase was obtained

from the maleic hydrazide treated 709 after 2 months storage at 45F.

Samuel Lo -Tung Wang

Red and UV lights also induced synthesis of glycoalkaloid but to a
less extent, while far -red light caused less increase of glycoalkaloid
than did darkness.

Total sterol of potato tubers stored at 40F increased with
time, and an initial increase with a subsequent decrease of total
sterol was found in the MH ~30 treated potatoes stored at 50F. The
increase of total sterol was primarily due to the increase of
/8 ~sitosterol, although cholesterol decreased. The glycoalkaloid
level of the untreated potatoes stored at 50F did not have a consistent
pattern of change.

Potatoes with more than 15 mg/100 g w.b. of glycoalkaloid
were considered as unsafe for human consumption, but this level
would not be detected by the consumers according to the results of
this study. The glycoalkaloid level higher than 25 mg/100 g w.b.
was detected and rejected significantly. The taste of the glyco-

alkaloid appeared either as an astringent or burning sensation.

GLYCOALKALOID AND DEME THYL STEROL CONTENT

IN SOME POTATO VARIE TIES AND CLONES

By

Samuel Lo -Tung Wang

A THE SIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science

1970

ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude to his
major adviser, Dr. C. L. Bedford, for his inspiring advice and
constant interest in the course of this research.

Special appreciation is due to Dr. N. R. Thompson for his
assistance in collecting the samples and his valuable suggestions as
a member of the guidance committee.

The author also wishes to thank the other members of his
guidance committee, Dr. J. E. Varner, Dr. C. M. Stine and
Dr. P. Markakis, for their confidence and suggestions in preparing
this manuscript.

The generous help from Dr. C. C. Sweeley and Mr. J.
Harten of the Biochemistry Department, Dr. J. A. D. Zeevaart of
ABC] Plant Science Laboratory, and many of the faculties and the
fellow graduate students in the Department of Food Science are
greatly appreciated.

Finally, the author is indebted to his wife, Anne, for her
constant blessing during this study, and her invaluable assistance

in laboratory work.

ii

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES
INTRODUC TION
LITERATURE REVIEW .

The Occurrence and Distribution of
Glycoalkaloid in Potatoes

Factors Affecting Level of Glycoalkaloid
in Stored Potato Tubers . .

Chemical and Physical Properties of
Solanine and Chaconine

Quantitative Determination of
Glycoalkaloid of Potatoes

Mechanism of Glchalkaloid Synthesis
in Potatoes . . . .

Toxicity of Solanine

Relationship Between Glycoalkaloid and
Off- Flavor of Potatoes

MA TE RIA LS AND ME THODS

Materials . .
Treatment of Potatoes with Maleic
Hydrazide (MH -30) .
Storage of Potatoes .
Experimental .
1. Analytical procedure
A. Sample preparation
1. Surface sample
2. Boiled flesh sample
3. Diced fresh sample

iii

Page

vi

. viii

13

16
20

23

24

24

25
25
26
26
26
26
28
28

B. Extraction.
1. Reflux extraction method
2. Bisolvent extraction method .
3. Percent recovery by the bisolvent
extraction method .
4. Comparison between the bisolvent
and Soxhlet extraction method
C. Colorimetric determination of
glycoalkaloid

D. Chromatographic analysis of glycoalkaloid

and sterol of potatoes
1. Thin-layer chromatography of
glycoalkaloids
2. Thin -layer chromatography of
sterols . .
3. Thin -layer and gas -liquid
chromatographic analysis
of sterols .
II. Studies of the effect of storage . .
III. Studies of the effect of white light on
glycoalkaloid and sterol formation
IV. Studies of the effect of different lights on
glycoalkaloid and sterol formation
V. Studies of the effect of glycoalkaloid on
potato flavor
A. Flavor evaluation of the B5141 6
potatoes .
B. Determination of the threshold level
of glycoalkaloid affecting the
potato flavor . . . . .

RESULTS AND DISCUSSIONS

Sprouting . .

Methodology of Glycoalkaloid Determination ,

Thin- -Layer Chromatography of Glycoalkaloids

Thin -Layer Chromatography of Sterols .

Thin— —Layer and Gas -Liquid Chromatographic
Analysis of Sterols .

Change of Glycoalkaloid Level During Storage
and Light Treatment . . . . .

iv

Page
28
28
29
30
30
31
32
32
35
37
39
39
40

40

4O

41
43
43
43
51
55
60

63

Change of Sterols During Storage and
Light Treatment . .

Flavor of the B5141-6 Potatoes

Threshold Level of Glycoalkaloid Affecting
the Potato Flavor. . . . .

SUMMARY AND CONCLUSIONS
PROPOSALS FOR FURTHER RESEARCH .

REFERENCES CITED

Page
75
80
81
87
91

93

Table

LIST OF TABLE S

Summary of some physical properties of
solanine and Chaconine

Conditions of various lighting systems

Recovery of added solanine using the bisolvent
extraction method

Glycoalkaloid content of potato samples
extracted with the bisolvent extraction
and Soxhlet extraction methods. ..

Glycoalkaloid content of diced fresh sample
and/ or boiled flesh sample with the
bisolvent extraction and Soxhlet extraction
methods .

Sterol contents of potatoes (Russet Burbank,
Kennebec, 709 and 321 -65) at different
stages of storage at 40F and 50F .

Sterol contents of the MH —30 treated potatoes
(Russet Burbank, Kennebec, 709 and
' 321 -65) at different stages of storage at
40Fand 50F . . . . .

Sterol contents of potatoes (Russet Burbank,
Kennebec, 709 and 321 -65) with and
without light treatment for 6 days

Sterol contents of Russet Burbank potatoes

treated with white, far -red, red, UV
light and in darkness

vi

Page

11

40

47

48

49

76

77

78

79

Table

10.

ll.

12.

13.

Page

Glycoalkaloid content in surface and flesh of
some potato tubers (B5141 -6 stored at
45F and 65F, Russet Burbank stored
at40F)................... 81

Paired comparison tests on potato samples
using green potatoes as glycoalkaloid
source . . . . . . . . . . . . . . . . . . . 83

Paired comparison tests on potato samples
using albino sprouts as glycoalkaloid
source . . . . . . . . . . . . . . . . . . . 85

Singlesample procedure tests on potato

samples using albino sprouts as
glycoalkaloid source . . . . . . . . . . . . . 85

vii

LIST OF FIGURES

Figure Page
1. Structures of solanidine, CX-solanine
and CX-chaconine. . . . . . . . . . . . . . . 9
2. Biosynthetic routes of fl-sitosterol and
stigmasterol; campesterol; cholesterol
and solanidine . . . . . . . . . . . . . . . . . . 18

3. Storage and analysis scheme of the MH -30
treated and untreated potatoes . . . . . . . . . 27

4. Standard curves for glycoalkaloid
determination................ 50

5. Thin -layer chromatogram of glycoalkaloids
from the MH -30 treated potatoes
(Russet Burbank, Kennebec, 709 and
321-65) stored at 40F and 50F . . . . . . . . . 52

6. Thin -layer chromatogram of the glyco-
alkaloids extracted by the bisolvent
method and the Soxhlet method
(Russet Burbank tubers) . . . . . . . . . . . 54

7. Absorption spectra of solanine and chaconine
after reacting with (a) the Clarke
reagent, (b) the Marquis reagent,
(c)85%H3PO 56
4
8. Thin -layer chromatogram of the
unsaponifiable compounds in the
chloroform extract of the potato tubers,
sprouts and authentic sterols (solanidine,
cholesterol, stigmasterol and
-sitosterol)................ 58

viii

Figure Page

9. Thin -layer chromatogram of the unsaponi-
fiable compounds in the chloroform
extract of the potato tubers and Sprouts . . . . . 59

10. Thin -layerchromatogram of (a) sprout
glycoalkaloid, (b) spot 1, (c) authentic
solanine . . . . . . . . . . . . . . . . . . . 59

11. Two -dimensiona1 thin -layer chromatogram
of the unsaponifiable compounds in the
chloroform extract of the light treated

potatotubers................61
12. Two -dimensional thin -layer chromatogram

of the unsaponifiable compounds in the

chloroform extract of the potato sprouts . . . . 62

13. Gas -liquid chromatogram of (a) spot 4, the
free sterol fraction, (b) spot 1, the
steryl glycoside fraction after acid
hydrolysis 64

14. Mass spectra of (a) cholesterol, (b) peak 2,
(c)stigmasterol, and (d) [8-sitosterol . . . . . 65

15. Change of glycoalkaloid levels in the
surface samples of potatoes (Russet
Burbank, Kennebec, 709 and 321-65)
storedat40Fand50F . . . . . . . . . . . . 67

16. Change of glycoalkaloid. levels in the surface
samples of the MH -30 treated potatoes
(Russet Burbank, Kennebec, 709 and
321-65) stored at 40F and 50F . . . . . . . . . . 68

17. Average glycoalkaloid levels of the MH --30
treated and untreated potatoes (Russet
Burbank, Kennebec, 709 and 321-65) ,
6 months after harvest (4 months
controlled storage at 40F and 50F) . . . . . . . 70

Figure Page

18. Glycoalkaloid levels of untreated and MH -30
treated potatoes with and without light
treatment for 6 days . . . . . . . . . . . . . 72

19. Glycoalkaloid and anthocyanin synthesized
by potatoes (Russet Burbank) after
treatment with white light or white
light plus 100% CO2 for 6 days . . . . . . . . . 74

20. Glycoalkaloid and chlorophyll synthesized
by potatoes (Russet Burbank) after
treatment with different lights for
6Adays 74

INTRODUC TION

Due to degeneration. of the old potato varieties, breeding of
new clones is carried on continuously. New clones are evaluated
mainly by their total solids, yield per unit, disease resistance,
processing characteristics and flavor. At times, the presence of
glycoalkaloid is overlooked. The glycoalkaloid in potatoes is not
only a hazard to human health, but also causes off -flavor. With the
increasing number of clones and species hybrid seedlings being bred
each year, the possibility of introducing a potato variety with a high
level of glycoalkaloid is also increasing. Therefore, an efficient
and rapid method of glycoalkaloid determination is needed for
examination of the glycoalkaloid content of the new potatoes.

Studies were undertaken to determine the glycoalkaloid
content of two established commercial varieties, Russet Burbank
and Kennebec, which have excellent processing characteristics,
and are ranked number 1 and 3 respectively in total acreage in the
States (Talburt and Smith, 1967), and two clones, 709 and 321-65,
which are to be released by the Crop and Soil Science Department,

Michigan State University, during storage.

The effect of white, far —red, red and ultraviolet light on
the synthesis of glycoalkaloid was studied. It has been established
that potatoes exposed to sunlight prior to harvest generally contain
a high level of glycoalkaloid.

Representative plots of the potatoes were also sprayed with
maleic hydrazide, a preharvest sprout inhibitor, to evaluate its

effect on the synthesis of glycoalkaloid during storage.

LITERATURE REVIEW

The Occurrence and Distribution of
Glycoalkaloid in Potatoes

 

 

Solanum alkaloids, a group of steroidal glycoalkaloids
occurringlin the Solaneceae genus, has been a subject of interest to
scientists for many years. The study of steroidal glycoalkaloids in
potatoes was initiated when Desfosses (1820) reported the existence

' in berries of

of a new substance, which he designated as "Solanin,’
S. nigrum and S. dulcamara potatoes. Solanine was also found in
sprouts of S. tuberosum potatoes by Baup (1826), and in tubers
by Wackenroder (1843).

For many years, the steroidal glycoalkaloid isolated from
potato plant was thought to be a single compound, until Kuhn and
Law (1954), using paper chromatography, resolved the seemingly
single compound into six compounds, namely OC-, ,8 -, 7-solanine
and O(-, ,8-, 7-chaconine. ,8-, 7—solanine and [8-, 7-chaconine
are found only in potato plants, and can only be. isolated during the

active synthesis of CX—solanine and O(-chaconine. In potato tubers,

both CX-solanine and CX—chaconine are present (Allen and Kfic, 1968).

Therefore, solanine, defined as ”the steroidal alkaloidal
fraction of potatoes soluble in acidified alcohol and insoluble in
slightly alkaline aqueous solution" by Talburt and Smith (1967),
is a mixture of solanine and chaconine. However, it should be
mentioned that the word "solanine" appearing in literature published
since 1954 frequently refers only to OK -solanine. Many researchers
now use the term "glycoalkaloid" to avoid possible confusion.

Morgenstern (1907) investigated the solaninelevel in
potatoes of 39 varieties and reported that the average content of
solanine was higher in reddish varieties than inyellowish varieties,
and higher‘in food -potatoes than in fodder -potatoes. A number of
studies carried out in different countries (Lampitt fl. , 1943;
Wolf and Duggar, 1946; Arnold, 1950; Gull and Isenberg, 1960;
Zitnak , 1961; Simek, 1962; Gawecki and Ilecki, 1966; Vecher,
1967) confirmed that solanine level of different potato varieties
varied widely.

Solanine level of potato is a function of many factors in
addition to variety. Potatoes subjected to different climates, soils
and fertilizer treatments are likely to have different levels of
solanine (Simek, 1962). Potatoes from different crop years, if
not grown under identical environmental conditions, may be expected

to have different solanine content (Gawecki and Ilecki, 1966).

Solanine is present in all parts of the potato plant in different
concentration. Early analysis by Gmelin (1859) showed that different
parts of potato contained different amounts of solanine. Potato
sprouts had the highest content of solanine, and potato tubers had
the lowest, the ratio being 68 to 5 in the sample he used. Meyer
(1895) reported that the solanine content was higher in unpeeled
potatoes than in peeled ones, indicating that the solanine content was
higher in the potato cortex. He also found that the average level of
solanine decreased as the sprouts grew longer. Several studies on
the distribution of, solanine in different parts of potato plant (Lampitt
9131. , 1943; Street e21. , 1946; Wolf and Duggar, 1946;
Paseschnitschenko, 1957) agreed that sprouts, leaves, stolons,
roots, flowers and buds had very high concentration of solanine,
whereas the solanine concentration of the tubers was low, normally
ranging from 10 to 50 mg per 100 g (dry weight basis), and was
predominantly present in cortex of tuber (traditionally considered
as potato peel). Relatively higher concentration of solanine was
detected in the eyes of tuber (Lampitt _et_al. , 1943; Turapin, 1951).
Griebel (1924)_reported that unripe tubers had a higher solanine
content than mature ones and small tubers more than large ones.

Change of solanine level in various parts of potato plant

during the growing seasonlwas studied by Morgenstern (1907),

Street _e_t_a_l. (1946), and Wolf and Duggar (1946). The solanine level
in each part of the potato plant seemed to increase in the early
period of emerging and then gradually decreased until maturity.
Solanine content in tubers did not change in a consistent pattern.
Wolf and Duggar (1946) reported that a higher solanine content was
found between 50 and 70 days after emerging in tubers of White Rural
and Irish Cobbler, but solanine content in Russet Burbank tuber
decreased continuously with the growing period.

Factors Affecting Level of Glycoalkaloid
in Stored Potato Tubers

 

 

Potato tuber is metabolically active. Various biochemical
changes take place during storage. Without outside interference,
the tuber undergoes a resting period before change occurs. Upon
exposure to light, synthesis of chlorophyll (Larsen, 1949), antho-
cyanin (Stanko, 1962), chlorogenic acid (Zucker, 1963) as well as
glycoalkaloid in tuber is induced. Sunburned potato tubers have a
high level of glycoalkaloid (Morgenstern, 1907; B6mer and Mattis,
1924).

Conner (1937) reported that flourescent light with wavelength
of 300 nm stimulated the synthesis of solanine rapidly, and light
capable of promoting chlorophyll synthesis did not necessarily

increase solanine synthesis. Using freshly harvested potato tubers

as samples, Barug (1962) found that solanine content of potato
increased with the intensity and duration of light, and also pointed
out that solanine concentration in potato peels was highly correlated
with the intensity of light. Gull and Isenberg (1960) reported that
both solanine and chlorophyll increased with time of exposure up to
96 hours. Whether these two biochemical processes are closely
related was not certain.

Potato tubers exposed to different lights appeared to differ
in the amount of solanine and chlorophyll synthesized. Blue or
green cellophane papers were more effective than red ones in
reducing tuber greening undervlight (Larsen, 1949). Liljemark
(1960) reported that daylight induced more solanine and chlorophyll
than did red light, and greenlight induced little chlorophyll.

Solanine level of tubers in storage was not studied to any
extent. Arutyunyan (1940) reported that solanine level did not
change after 45 days of storage at -6C to -8C. Solanine was actively
synthesized when potato was sprouting, and most, if not all, of the
synthesized solanine was located in sprouts (Lampitt _e_t_al_. , 1943).
Moskaleva and Pogorelova (1969), using fluorescent microscopy and
histochemical analysis, found that distribution of both solanine and
chaconine in Sprouts was not uniform; maximum content was in
meristematic tissues of top and side buds. Synthesis of solanine

during sprouting was not light -dependent.

Varietal differences of potatoes in the response to light
irradiation was also found. Genetic difference in the greening of
potato tubers was reported by Gull and Isenberg (1958), and Akeley
M. (1962). Barug (1962) compared the potential of solanine
synthesis in 9 varieties exposed to daylight for 72 hours, and sug-
gested that varietal difference with respect to solanine increase in
potatoes upon exposure to light also existed.

Chemical and Physical Properties of
Solanine and Chaconine

 

 

Both solanine and chaconine are steroidal glycoalkaloids;
this group of compounds is characterized by consisting of three
moieties: sugar, steroidal structure and alkaloid. Their aglycone
is solanidine. The only difference between solanine and chaconine
is in the sugar moiety.

The structure of solanine was studied soon after its dis-
covery (Blanchet, 1838; Zwenger, 1861). The correct empirical
formula was not worked out until Sh6rpf and Hermann (1933)
proposed a formula C H ON for solanidine; andlater, Clemo (1936)

27 43

proposed a formula C47H73015N for solanine with one secondary

alcohol group and one tertiary amino group. Structural formula of

solanidine, A5 -solaniden -3,8 -ol (Figure 1) was proposed by Prelog

and Szpilfozel (1942) and confirmed by Uhle and Jacob (1945) by

 

 

 

R = HO - SOLANIDINE

OC -SOLANINE

O< -CHACONINE

 

 

Figure 1. --Structures of solanidine, CX-solanine and CX—chaconine

10

converting a known compound, sarsasapogenin, to solanidine
derivative.

The sugar moiety of solanine, solanose, consists of one
mole each of glucose, galactose and rhamnose. Brigg and Vining
(1953) studied the mode of linkage of sugar moiety of solanine by
means of periodic oxidation, and reportedathat it was O(-L-
rhamnopyranosyl-(1+2 galact) - [8 —D —glucopyranosyl -(1—.3 galact) -
B -D-galactopyranosyl- A5 -solaniden 6,8 -ol (Figure 1). ,8 - and
7 4'solanine are compounds formed by depleting one and two sugars
from CX-solanine respectively (Kuhn and L6w, 1954).

Chaconine was first separated from a solanine preparation
using paper chromatography and the solvent system, pyridine -HOAc -
H20 (3:1:3) (Kuhn and L6w, 1954). Its sugar moiety, chaconose,
was found to be CX-L-rhamnopyranosyl -(1—-2 g1uc)-O(-L-
rhamnopyranosyl (1—-- 4 gluc) 78 -D -glucopyranosyl - A5 -solaniden—
33-01. ,8- and 7—chaconine are isomers lacking either one or two
rhamnose molecules.

A summary of the physical properties of these compounds
is givenin Table 1. It should be noticed that the different melting
point of CX-solanine and CX-chaconine reported by investigators is
probably due to low purity of sample. The highest value is regarded

as authentic.

11

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Solanine and chaconine are identical in the following aspects.
Both are very soluble in dilute acid or acidified alcohol, and insoluble
in alkaline aqueous solution (Reuling, 1839; Swenger, 1859).

According to Autenrieth (1928), solanine is slightly soluble
in water, about 1 part in 8000 parts of water, soluble in 500 parts
of cold and 125 parts of boiling ethanol and 4000 parts of ether.
Precipitation of bothcomponents from solution is completed as the
pH value is raised to 9. 4, where, according to Wolf and Duggar (1946),
the solubility of solanine is in the order of 0. 1 mg per 100 ml.

Both compounds give identical colors with the following

color tests: SbCl reagent (Wierzchowski and Wierzchowska, 1961),

3
red; Marquis reagent (Alberti, 1932), purple; Clarke reagent

(Clarke, 1958), blue; H PO , orange.

3 4

Solanine and chaconine can be recrystallized from aqueous
alcohol. Solanine precipitates after repeated recrystallization in
needle form. Chaconine does not crystallize readily, and crystalliza-
tion is induced by lowering the temperature slowly to avoid gel
formation (Allen and Kfic, 1968).

The major difference rests on the chromatographic
behavior of these two compounds. They have been separated on

paper chromatography, column chromatography, and thin -layer

chromatography. With paper chromatography using ethyl

13

acetate -HOAc -H20 (3:1:3) as solvent system, relative Rf value for

(X-solanine and CX-chaconine was found to be 1. 00 and 1. 61
respectively (Kuhn and L6w, 1954). Paquin and Lepage (1963)

obtained Rf values 0. 22 and 0. 54 for (X-solanine and CX-chaconine

respectively with 95% EtOH —HOAc (3:1) and silica gel plate. Using

H20 saturated n-butanol as solvent system, Allen and Kfic (1968)

reported that R for O(-solanine and a-chaconine on Al O plate

1' 2 3

was 0. 16 and 0. 28 respectively, and R on silica gel plate, 0. 36 and

f

0. 52 respectively. Column chromatography was first employed by
Paseschnichenko and Guseva (1956) for separating chaconine from

solanine. They eluted the mixture with H O saturated n-butanol on

2

dry neutral A1203 column and obtained pure chaconine and solanine
successfully. - Patt and Winkler (1960) purified solanine with an
ion -exchange column.

Infrared absorption. spectra of solanine and chaconine only
differ slightly at 8 and 12.2 [1. according to Allen and Kfic (1968).

Quantitative Determination of
Glycoalkaloid of Potatoes

 

 

The gravimetric method of solanine determination involved
isolation and purification of solanine from potato tissue and direct
weighing after it was dried in an oven to a constant weight. Morgen-

stern (1907) heated the ground potato samples with dilute HOAc for

14

one hour, filtered and rinsed the residues with water. The residues
were then re -extracted with 96% alcohol for 12 hours, filtered and
the filtrate condensed. Solanine was precipitated by NH 4OH from
the combined filtrates. B6mer and Mattis (1924) proposed a method
which called for extraction of ground potato samples with HOAc

4 times, and subsequent extraction of the dried crude precipitates
with 95% ethanol in Soxhlet extractors for 5 hours. Gmelin (1859)
estimated the solanine content by measuring the reducing power of
sugar after hydrolysis of solanine with H2 SO4. Conner (1937) in his
study of light effect on solanine synthesis hydrolysed solanine with
HCl and measured the reducing power. These methods, however,
were laborious and their precision depended entirely upon the
technique in isolating and purifying the compound.

Alberti (1932) reported the first colorimetric method using

conc. H SO

2 4 and 1% HCHO, known as Marquis reagent. Solanine

was allowed to react with conc.. H SO for a short period, and a

2 4
purple color‘developed upon addition of 1% HCHO. This method was
modified by Pfankuch (1937), Rooke et__a_l_. (1943), Wolf and Duggar
(1946), Dabbs and Hilton (1953), Baker _e_t_a_l_. (1955). The last
method called for Soxhlet extraction with 3% HOAc in EtOH for

18 hours, evaporation of ethanolic solution to near dryness, coagula -

tion of soluble protein with 5% H2 SO 4, and filtration. The filtrate

15

was diluted with 1% H2804 to volume. Three m1 of sample solution

was added dropwise to 3 ml of conc.. H2804 in a 3 -minute period,
and kept cool with constant swirling in an ice bath. , Three ml of 1%
HCHO was then added and purple color was measured at 570 nm
after 90 minutes.

Clarke (1958) discovered that a mixture of cone. H3PO4
and dilute para -HCHO could produce a color reaction for determina-
tion of solanine; he also noted that chaconine underwent the same
reaction. This reaction was adopted by Patt and Winkler (1960) for
solanine determination, and was modified by Schwarz (1962).

Solanine was extracted with 5% HOAc, precipitated by NH OH,

4

centrifuged, and the precipitate dissolved in 3 ml of conc.. H3PO4
plus 3 drops of para —HCHO saturated aqueous solution. The blue
color was measured at 595 nm after 60 minutes. Vecher (1967)
employed the above method to determine the solanine content of the
important and new potato varieties in Russia.

Sachse and Bachmann (1969) made a comprehensive com-
parison of the two methods described above and concluded that the
Clarke reagent was superior to the Marquis reagent. They found
that refluxing of the potato sample in 80% ethanol or 90% methanol

for 30 minutes yielded more solanine than did the Soxhlet extraction

with 3% HOAc in 95% EtOH and proposed the following procedure for

16

solanine determination: Reflux the potato sample in 80% EtOH for

30 minutes, filter, precipitate the glycoalkaloid from filtrate with
NH4 OH, filter again, and dissolve the residue into. 1% H3PO4. Mix
1 m1 aliquot of sample solutionwith 10 ml of reagent s’olution made
of 0. 03 g of para ~HCHO and 100 ml of 85% H3PO4. Measure the blue
color after 30 minutes at 600 nm.

Wierzchowski and Wierzchowska (1961) determined the
solanine concentration with a mixture of 30 g of SnCl3 and 20 ml of
cone. HCl successfully. This method did not gain popularity due to
the toxic nature of the reagent. A polarographic method of glyco-
alkaloid determination was reported by Pierchalski and Mrozowska
(1968).

lMechanism of Glycoalkaloid Synthesis
in Potatoes

 

 

Guseva and Paseschnichenko (1957) found that an enzyme
preparation from potato sprouts was capable of cleaving the sugars
from solanine. Studying with C14-acetate, Guseva and Paseschni-
chenko (1958) found that, if the sprout was grown in light, most C14
was incorporated into aglycone, and if in darkness, C14 was
incorporated -into both sugars and aglycone. If radioactive mevalonic
acid was used, the rate of incorporation was higher. However,

more radioactivity was incorporated into the non -saponifiable

fraction than the glycoalkaloid.

17

Not much progress was made in regard to the elucidation
of biosynthetic mechanism of glycoalkaloid until newer chromato-
graphic techniques were introduced in the 1960' 8. Successful
isolation of many intermediate triterpenes and sterols from plants
such as soybeans and potatoes made it possible to postulate the
metabolic pathways toward glycoalkaloid synthesis.

Phytosterols of potatoes were first isolated by Schwarz
SHELL (1955). Using conventional methods, they identified these

phytosterols as IB-Sitosterol and stigmasterol. Schreiber et al.

 

(1961) reported that the major sterol in both potato and potato beetle
was B-sitosterol. Johnson et_a_l. (1963) discovered cholesterol
from extracts of a growing potato sprout with gas -liquid chroma -
tography. This was the first time that cholesterol was found to be
present in plant tissue.

Subsequent isolation of triterpenes and phytosterols from
leaves by a group of workers led by Dr. K. Schreiber in Germany
confirmed the presence of cycloartenol (Schreiber and Osske, 1962),
24-ethylidene lophenol (Schreiber and Osske, 1963), 24-methylene
cycloartanol (Osske and Schreiber, 1964), 24-methylenealophenol
(Osske and Schreiber, 1965) in potatoes. Based on these findings,
Schreiber and co ~workers (Ardenne 121' , 1965) proposed a scheme
of the possible routes leading to the syntheses of cholesterol,

campesterol, B-sitosterol, stigmasterol and solanidine (Figure 2).

18

 

Detailed pathway CH
not known

HO

 

Figure 2. "Biosynthetic routes 01,8 -:1touterol and stigmasterol; camputm‘ol; cholutorol and
solanidine

Note: I. Cycloatenol; II. 24-methylene cycloartenol; Ill. Lophenol; IV. 24 thylene lophenol:
V. “ethylldene lophenol; VI. Cholesterol; V11. Camp sterol; VIII. «hostel-01;
IX. Stigmuterol; X. Yamogenln: XI. Tomatld —5-en~3 -ol; XII. Sohnldlne.

19

According to Schreiber and Osske (1962), the raw fat
extract of potato. leaves was first saponified, and the unsaponifiable
fraction was precipitated with digitonin. The digitonide of tri-
terpenoids and sterols could be fractionated into 3 fractions on a
column of silica gel or florisil. Fraction A, comprising 60-70% of
the digitonin -precipitable material, was composed principally of
cycloartenol and 24-methylene cycloartanol; fraction B, 7 -13%,
was composed of lophenol, 24-methylene lophenol and 24-ethylidene
lophenol; fraction C, 18-33%, was composed of cholesterol,
campesterol, ,B-sitosterol. An analysis of fraction C (Ardenne
et a1., 1965) showed that it contained 49% fl-aitosterol, 36%
stigmasterol, 12% cholesterol and 3% campesterol.

The biosynthetic route from cholesterol to yamogenin,
tomatid -5 -en -3/8 -ol and solanidine was based. on the finding that
both yamogenin and tomatid —5 -en -3,8 -ol could be obtained from the
hydrolysis product of solanine (Schreiber, 1957), that tomatid -5-
en -3,8 --01 could be synthesized from yamogenin -like compound
(Schreiber and Rénsch, 1965a), and that solanidine could be synthe -
sized from tomatid -5 -en -3/8 -ol (Schreiber and R6nsch, 1965b).

Rees 3111. (1968) reported the isolation of a phytosterol,
40¢, 14O(.dtmethy1oho1esta—8, 24 —dien-3B -ol, from potatoes. They

hypothesized that cycloartenol, likelanosterol, an intermediate for

20

animal sterol biosynthesis, might be the most important intermediate
for phytosterol biosynthesis. The scheme of biosynthetic pathway
for cholesterol formation was suggested to be cycloartenol -—- 31 -
norcycloartenol ——-- 4a, 14a-dimethylcholesta -8, 24 -dien ~3/B -ol
—-lophenol———-- cholesterol, although 31 -norcycloartenol has
not beenisolated from any plant source yet. This scheme can be
added to that of solanidine biosynthesis shown in Figure 2. Further
details in the solanidine biosynthesis are unavailable.

Since relatively significant amount of cholesterol exists in
potato leaves along with [3 -sitosterol, stigmasterol and campesterol,
it is very likely that cholesterol is an important intermediate for

solanidine biosynthesis (Bennett and Heftmann, 1965).

 

Toxicity of Solanine

A number of outbreaks of potato poisoning were reported.
Harris and Cockburn (1918a, 1918b) cited that 56 Berlin soldiers
were ill after a meal of greened potatoes in 1899, and at the end of
1917, quite a few poisoning cases were reported in Glasgow, England,
where 61 individuals were involved with one death. The symptoms
included gastro -enteritis, diarrhea, colicky pain, headache and
depression. The suspected potatoes were examined and it was found

that the uncooked potatoes contained 38 mg solanine per 100 g fresh

21

potato and the cooked potatoes contained 24 mg solanine per 100 g.
Poisoning by potatoes was attributed to the unusual high content of
solanine. The hazard of eating sprouted or sunburned potatoes was
emphasized.

Potato varieties with a high level of solanine were reported
frequently during the 1920' 8. One variety examined by Alfa and
Heyl (1923) contained 68. 7 mg, 40. 0 mg and 29. 8 mg of solanine
per 100 g of potatoes respectively for three different lots. Similar
results were also reported by Griebel (1923), B6mer and Mattis
(1923, 1924).

According to a USDA report cited by Hansen (1925), 64 cows
were poisoned after being fed with some potato leaves; 6 pigs died
after eating sprouted raw potatoes. The solanine concentration in
stomach content of the poisoned animals was found to be 60 to 85 mg
per 100 g of fluid, (Lowe, 1929).

Riihl (1951) reported that ingestion of berries of a potato
plant caused death of a 2 —year -old girl after 13 days, and found that
solanine was the primary factor causing physiological lesions. He
noted that large doses of solanine irritated the gastro -intestinal
mucosa, and caused vomiting and diarrhea. Kingsbury (1964)
recorded the symptoms of solanine poisoning as being apathy,

drowsiness, salivation, dyspnea, trembling, progressive weakness

22

or paralysis, unconsciousness, gastro-enteritis, anorexia, abdominal
pain, constipation, diarrhea, and headache.

Sprouted potatoes fed to rats caused marked weight loss and
death, but the similar potatoes had no toxic effect on rats when peeled
(Smerha and Ceskoslow, 1948). K6nig and Staffe (1953) reported that
the injection of 2 g of solanine into 40 kg sheep caused a change in
hemoglobin, erythrocyte and leucocyte, and shock in 30 minutes.
Abbott fl. (1960) found that solanine had a high anti -cholinesterase
activity 111m. Similar results were also obtained by Orgell and
Hibbs (1963a, 1963b). Kline _et_a_l. (1961) studied the toxic effects
of potato sprouts or-crystalline solanine on pregnant rats, and he
found that rats fed with either one had a lactation deficiency, which
was probably due to anti -hormonal action of solanine.

Toxicity level of solanine on human beings has not been
established. However, it will be regarded as a dangerous level if
the potato contains higher than 100 mg per 100 g (dry basis) (Talburt
and Smith, 1967).

The resistance of certain potato plants to potato beetles
was attributed to the presence of glycoalkaloids in potato leaves.
Sturkow and L6w (1961) found that crystallized solanine and chaco-
nine when infiltrated into potato leaves in concentration of 0. 02 to

0. 8% had repellent effect against both normal and DDT-resistant

23

potato beetles. Allen and Kfic (1968), using a paper bioassay
technique, found that solanine and chaconine were the principal
fungitoxic compounds in addition to caffeic acid in extracts of Irish

potato tubers.

Relationship Between Glycoalkaloid and
Off-Flavor of Potatoes

 

 

Glycoalkaloid is a bitter substance, which in small amount
is not noticeable, and is probably regarded as a flavor constituent
of potato tubers. If the level of glycoalkaloid in potato tubers
increases, a bitter taste arises (Zitnak, 1961). Higher concentration
of glycoalkaloid. is found in cortex of potato tubers, particularly
those exposed tolight. Thus off ~flavor of potato is likely to be
enhanced by consuming the unpeeled tubers.

From ,the rather scarce information available, that off-
flavor of potatoes is entirely due to the high level of glycoalkaloid has
not been confirmed. An organoleptic test on flavor of light -treated
or sunburned potatoes, and controls did not show any significant
difference in score (Gull and Isenberg, 1958). Barug (1962) also
failed to correlate the flavor change with change of glycoalkaloid

concentration. Instead, he found that the flavor score was highly

correlated with surface discoloration of potato tubers.

MATERIALS AND ME THODS

Materials

 

The potatoes used in this study were grown at the
Montcalm County Experiment Station Farm, Michigan, by the
Department of Crop and Soil Science, Michigan State University.
The crops were harvested at the end of September and in early
October, 1969.

Two commercial varieties, Russet Burbank and Kennebec,
and two clones, designated as 709 and 321 -65, were chosen.
Russet Burbank and Kennebec were used mainly as controls. 709,
a clone of S. tuberosum, was chosen because it was one of the new
clones found to have marketing potential. 321 -65, a S. tuberosum
and S. stoloniferum species hybrid, was selected on the basis of
its high solids content (28. 3%) and commercial value as a yellowish
potato product (Pope, 1969). In addition, B5141 -6, a potato
reported to have off ~flavor (Thompson, 1969), was used in the

flavor study.

24

25

Treatment of Potatoes

with Maleic'Hydrazide (MH -30)1

 

 

Potatoes (all except B5141-6) were sprayed with a 4000 ppm
water solution of MH -30, 1 a sprout inhibitor, four weeks before
harvest, and the vines were killed by sodium arsenate solution 10 days

before harvest.

Storage of Potatoes

 

Potatoes were stored at 40F and 50F to provide for-long-
term storage and alsoto permit different physiological activities.

The MH ~30 treated potatoes were harvested on September 25,
1969, stored immediately at 45F in the Experiment Station Farm,
and brought to the Food Science Department on October 14, 1969.
Theywere divided into two lots, and stored at 40F and 50F respectively.
During the first 10 days, the temperatures rose to 44F and 58F due
to the respiratory heat generatedin the close chamber. Small lots
were stored at 45F for-light treatment studies.

Untreated potatoes were harvested on October 10, 1969,
stored at 60F, which gradually dropped to 45F in 35 days, and brought
to the Food Science Department on December 1, 1969. They were

similarly divided and stored at 40F and 50F respectively. Small

 

1A product of Uni -Royal Chemical Co. containing 58%
diethanol amine salt of 6-hydroxy -3 -(2H) pyridazinone as active
ingredient.

26

batches of untreated potatoes were brought in earlier (November 1,
1969) and stored at 45F for light treatment.

B5141 -6 potatoes were harvested in September, 1969, and
stored at 45F and 65F respectively. They were brought to the Food
Science Department on January 13, 1970, for flavor evaluation and

glycoalkaloid determination.

Experimental

 

The chemical analyses were initiated about 1 month after
harvesting and were continued at monthly intervals until vigorous
sprouting of the potatoes occurred. The storage and analysis scheme

is presented in Figure 3.

 

I. Analytical procedure

A. Sample preparation

 

Three types of sample were employed:

1 . Surface sample

 

Three tubers from each sample lot are blanched
in live steam for 20 minutes, 10 minutes on each side,
cooled with cold waterspray immediately and hand -
peeled. The 2 to 3 mm thick surface of the entire tuber
is scraped into a small plastic cup with a stainless

steel spatula and mixed thoroughly.

27

 

 

 

 

 

 

 

 

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28

2 . Boiled flesh sample

 

. Three tubers from each sample lot are boiled in a
stainless steel kettle for about 30 minutes, drained,
cooled with cold water and hand -peeled immediately.
The peeled tubers are then mashed and mixed with

mortar and pestle.

3. Diced fresh sample

 

The tuber chosen for glycoalkaloid determination

is peeled, trimmed and diced into small pieces.

All samples used for glycoalkaloid determination were
dried at 60C in a vacuum oven for 18 hours, weighed and

percent dry material computed.

Extraction

 

1. Reflux extraction method

 

To evaluate the reflux extraction method, 200 g of
potato sprouts were refluxed with 1000 m1 of acidified
95% ethanol (3 ml HOAc per 100 ml) for 24 hours. The
ethanolic solution was cooled, filtered and sufficient
saturated lead acetate solution was added to precipitate

colloidal materials. The mixture was centrifuged, the

29

supernatant removed and evaporated to a small volume,
and diluted with ca. 200 ml of 2% HOAc. The pH was
.3. 8.

Differential precipitation was induced by continuous
addition of dilute NH 4OH, and the. precipitates were
collected by, centrifugation at pH 4. 5, 7. 0, 8. 0 and above
9.0 as recorded by a glass electrode pH meter. The

nature of the precipitation was evaluated in terms of

purity of the precipitates.

Bisolvent extraction method

 

Five gram samples in triplicate were blended with
100 ml of a mixture of MeOH-CHCI3 (2:1). in a semi-
micro stainless steel blender assembly for 4 to 5 minutes.
The suspension‘was filtered through a sintered glass
Biichner funnel (medium porosity), and the residue
rinsed twice with a 25 ml portion of the same solvent
mixture. The filtrate was transferred into a 500 ml
separatory funnel, 60 m1 of O. 8% Na2 804 added, and
the mixture vigorously shaken. The layers were allowed

to separate and the lowerlayer (CHC13) was discharged

into a 150 m1 beaker. The methanolic layer was

30

2804, and the

bottom layer (CHC13) again collected in the 150 ml

re -extracted with. 10 m1 more of 0. 8% Na

beaker. The combined CHCl3 fractions, which con-
tained various pigments and steroids, were air -dried,
andkept for chromatographic analysis of sterols.

The methanolic fraction was collectedinto a 250 ml
beaker, and separatory funnel rinsed with 1% HOAc.
This solution was concentrated on a steam bath to less
than 10 ml and made to volume with 1% HOAc (10 to
50 ml depending on the concentration of glycoalkaloid).

It was used as the sample solution for glycoalkaloid

determination.

 

Percent“ recovery. by the bisolvent extraction method

T05 g surface samples with either low or high
level of glycoalkaloid, 1 mg of pure O(-solanine (K & K
Laboratory, Plainview, N. Y.) was added, and glyco-
alkaloid determinedwith or without addition of the pure
solanine.

Comparison between the bisolvent and Soxhlet extrac -
tion method

 

 

Diced ,flesh samples of raw potatoes as well as

boiledflesh samples were extracted with 150 ml of

31

3% HOAc in EtOH for 18 hours with Soxhlet extractors.
The ethanolic solution was collected into a 250 m1
beaker, and evaporated to near dryness on a steam

bath. Five ml of 5% H280 was added .to coagulate the

4
colloidal substance. The solution was filtered, and

rinsed with 5 ml of 5% H280 The glycoalkaloid was

4.
precipitated from the filtrate by raising the pH to 9. 4
with NH 4OH, centrifuged and redissolved in 10 ml of

1% HOAc. Identical samples were also extracted with

the bisolvent extraction method. The yields of glyco-

alkaloid from both methods were compared.

Colorimetric determination of gl_ycoalkaloid

A modification of the colorimetric method described by
Schwarze (1962) was used. Five or 10 ml of the sample
solution were pipetted into a 40 ml centrifuge tube (heavy
duty, bottom tapered). The pH value was adjusted to 9. 4
with 5M NH4OH, the tube heated at 80C for 1 minute and
left to stand for-10 minutes. It was centrifuged at 2, 000 rpm
for 15 minutes, the supernatant siphoned off, the precipitate

washed with 0. 5 to 1 ml of water, recentrifuged and the

water decanted off.

32

To the precipitate, 5 ml of 85% H3PO was added,

4
followed immediately by 5 drops of saturated para -HCHO
solution. It was stirred well and left to stand for 60 minutes
at room temperature. The absorbance was read at 600 nm
on a Beckman DB Spectrophotometer. The concentration of
glycoalkaloid was determined from a standard curve
obtained for a series of (X-solanine concentrations from
0.06 mg to 0. 6 mg prepared in the same manner as the
sample. Higher concentration of glycoalkaloid was deter-
mined with a standard curve ranging from 0. 1 mg to 1.2 mg

constructed by replacing 85% H3PO with 70% H3PO and

4 4

developing for 120 minutes at room temperature.

ChrOmatggraphic analysis of glycoalkaloid and sterol of
potatoes

 

1. Thin -1ayer chromatography of glycoalkaloids

 

The glycoalkaloid precipitate obtained in the same
manner as described for its colorimetric determination
was dissolved in a 1% HOAc solution in MeOH. It was
spotted on a thin -layer plate (E -merck Silica Gel F -2 54,
abrasion resistant), and developed in one of the following
solvent systems until the solvent front travelled 12 to

15 cm:

1)
2)
3)
4)

5)

33

MeOH -EtOAc ~HOAc-H20 (20:30:10:1)
95% MeOH -EtOAc -HOAc (10: 15:2)
BuOH -HOAc-H20 (40:8:20)

3 -Heptanone -HOAc -H20 (8:5 :1)

EtOAc -Pyridine -H20 (3: 1:3)

The plate was then air -dried, and sprayed with one of

the following visualizing reagents:

1)

2)

3)

Lowry reagent: A mixture of 50 mg of FeCla. 6H20,

90 ml of H O, 5 ml of glacial acetic acid and

2

5 ml of conc. H2504.

SbCL3 reagent: A mixture of 30 g of SbCl3 and
40 m1 of conc. HCl.

Dragendorf reagent: Made by mixing 5 ml of
solution A (0. 8 g of Bismuth subnitrate, 10 ml
of glacial acetic acid and 40 ml of H20), 5 m1
of solution B (40% aqueous KI), with 20 m1 of

glacial acetic acid and diluted to 100 ml with

H20.

After spraying, the plate was heated at 90C in an oven

for5 to 10 minutes for'visualization. A long -wave UV

lamp (Ultra -Violet Products, Inc. , San Gabriel, Cali-

fornia) was used to aid the visualization of faint spots.

34

Preparative thin —layer chromatography was also
employed for the preparation of a large amount of pure
(X -solanine and (X-chaconine. The glycoalkaloid
isolate was redissolved in 1% HOAc, treated with active
carbon, reprecipitated, and then dissolved in 1% HOAc
(in MeOH) after waterwashing. It was applied to a
0. 5 mm thick preparative plate (E -merck Silica Gel
PF-Z 54), and developed in the no. 1 solvent system for
90 minutes. Small quantities of the isolates were
spotted on both sides of the plate as markers, and were
sprayed with the no. 1 visualizing reagent after develop-
ment. The bands corresponding to CX-solanine and
(X -chaconine were collected and eluted with warm
methanol. The methanolic solution was condensed and

made aqueous prior'to addition of 5M NH OH for pre-

4
cipitating solanine and chaconine. The respective
precipitates were washed with water, and the “-solanine
and CX—chaconine obtained were tested with the following
reagents:
1) Marquis reagent: Six ml of cone. H2804 were
added to 0. 2 mg of each glycoalkaloid, with

cooling in an ice bath, and followed by 3 ml of

1% HCHO.

35

2) Clarke reagent: Five ml of conc. H3PO4 were
added to about 0.2 mg of each glycoalkaloid
with shaking, followed by 5 drops of saturate
para -HCHO solution.

3) Cone. H3PO4: Five ml of conc. H3PO4 were
added to about 0. 2 mg of each glycoalkaloid.

Their absorption spectra were determined with a Beckman

DB Spectrophotometer.

Thin -layer chromatography of sterols

 

The air -dried CHCl3 fraction from the bisolvent
extraction procedure was redissolved in 20 ml of dry
ethyl ether and saponified with an equal amount of KOH
saturated MeOH. The solution was shaken vigorously
for 2 minutes and allowed to stand for 20 minutes in a
250 m1 separatory funnel. The mixture was separated
into 2 layers with the addition of 80 ml of distilled water
and moderate swirling. The lower layer was discarded,
and the ethereal layer was washed with additional water
.until the washing showed neutral reaction to litmus paper.

The washed ethereal fraction was filtered through a

SO into a small beaker, and the

layer of anhydrous Na2 4

36

separatory funnel rinsed with dry ethyl ether. This
ethereal solution was spotted on a thin -layer plate
(E -merck Silica Gel F-2 54, abrasion resistant), and
developed in one of the following solvent systems for
45 to 60 minutes:
1) Benzene -EtOAc (1:1)
2) Hexane -EtOEt-HOAc (80:20zl)
. 3) 3-Heptanone -HOAc -H20 (8:521)
The plate was dried andsprayed with one of the following
visualizing reagents:
1) Lowry reagent
2) Liebermann -Burchard reagent: A mixture of
chilled glacial acetic aCid and cone. HZSO4
(19:1).
3) Iodine vapour
4) Benzidine -NaIO4 reagent: A mixture of solution
A (0. 1% NaIO4) and solution B (2. 8 g of benzidine

in 80 ml of ethanol, 70 ml of H O, 30 m1 of

2

acetone and 1. 5 ml of 1N HCl).
The sprayed plate was heated at 90C in an'oven for 5 to
10 minutes, and the spots examined with the aid of a

short- or long -wave UV lamp (Ultra -Violet Products,

Inc. , San Gabriel, California).

37

Two -dimensional thin -layer chromatography of
sterols was also carried out using a combination of
solvent system no. 1 and 2, other conditions being the
same.

Thin -layer and gas -liquid chromatogaphic analysis
of sterols

 

 

The entire ethereal sample solutions obtained from
three 5 g potato samples were combined and streaked
onto a O. 5 mm thick plate (E-merck Silica Gel PF -254)
with small quantities as markers on both sides, and
developed in the no. 1 solvent system for 45 minutes.
The plate was air -dried, and the markers sprayed with
the no. 1 visualizing reagent and heated at 90C in an
oven for 5 to 10 minutes. The bands of steryl glycosides
and free sterols were collected and eluted with CHC13 -
MeOH (2:1). To the eluent, 40ug of authentic 5 0(-
cholestane was added as internal standard (The Anspec
Co. , Inc. , Ann Arbor, Michigan), and the mixture
dried in a warm (about 60C) water bath under a flow of
nitrogen gas.

Quantitative determination of sterols with GLC has

been carried out by the use of trimethyl silyl ethers of

38

cholesterol (Wells and Makita, 1962), campesterol,
stigmasterol and fl-sitosterol (Rozanski, 1966).
Therefore, trimethyl silyl derivatives of the sterol
fraction were made in this study for quantitative
analysis. The silylating reagent was made of 3 parts
of hexamethyl disilazane, 1 part of trimethyl chlorosi-
lane and 9 parts of water -free pyridine. The steryl
glycoside fraction (spot 1) was first hydrolysed with
1N HC1 in MeOH at 75C over night, and the sterols were
extracted with chloroform, dried and silylated with 40 ul
of the silylating reagent. The free sterols from spot 4
were silylated directly; After 10 minutes, the mixture
was injected directly into a F & M 5075 Gas Chromato-
graph (Hewlett Packard Co. , Southfield, Michigan).

The gas —liquid chromatography was performed on

1

an 8-foot long glass column (0. D. 3- in) packed with 1%
0V —1011 on 100 -120 mesh Gas -Chrom Q (The Anspec
Co. , Inc. , Ann Arbor, Michigan) under the following
conditions: Column temperature, 240C; Injection port
temperature, 290C; Detector block temperature, 295C;

Flow rate of Helium, 30 ml/min; Hydrogen, 20 mI/min;

Air, 350 ml/min. Each injection was allowed to run

 

1OV -101 is a methyl derivatove of silicone oil.

II.

III.

39

for 40 minutes, and duplicate injections were made for
each sample. The relative retention time for each
component was computed with reference to 5 (X-

cholestane, the internal standard.

Studies of the effect of storage

 

Monthly analyses were made on the untreated and MH -30
treated potatoes (Russet Burbank, Kennebec, 709 and 321 -65)
stored at 40F and 50F for the determination of the glycoalkaloid
and sterol levels using surface samples. Six months after
harvest (4 months controlled storage) at 40F and 50F, average
levels of glycoalkaloid in these potato tubers were determined
using boiled flesh samples.

Studies of the effect of white light on glycoalkaloid and sterol
formation

 

 

The MH -30 treated and untreated potatoes (Russet Burbank,
Kennebec,. 709 and 321 -65) after storage at 45F for about 60 days
were randomly sampled and exposed to white light (both fluorescent
and incandescent) for 6 days, 3 days on each side, at 52F. To
retain the moisture, the tubers were sacked in punched poly-
ethylene bags. Glycoalkaloid and sterol were determined using

surface samples .

40

IV. Studies of the effect of different ligfhts on glycoalkaloid and
sterol formation

 

 

Untreated Russet Burbank potatoes after storage at 45F for
60 days were exposed to white, far -red, red, ultra -violet and
no light for 6 days, 3 days on each side, under the conditions
listed in Table 2. The levels of glycoalkaloid and sterol were

determined using surface samples.

 

 

 

Table 2. - - Conditions of various lighting systems
Li ht Range of Energy Temperature

g Wavelength nm erg/sec/cm ° C
Dark - - - - — - 1 9
UV <400 - - - 25
Red 600-700 3770 24
Far-Red 615-775 11426 28
White 400 -7 00 - - - 2 5

 

 

 

 

V. Studies of the effect of glycoalkaloid on the potato flavor

A.

Flavor evaluation of the B5141 -6 potatoes

 

The mashed potatoes were prepared for flavor evaluation

control, Russet Burbank (40F storage).

of the B5141 -6 potatoes (45F and 65F storage) against the

41

Potatoes were peeled, boiled for 30 minutes in a stainless
steel kettle, and blended with 0. 1% salt, 2 -3% margarine
and 15 -17% milk on wet weight basis. Using a flavor dif—
ference procedure, the samples and the control were
presented to a 24-member taste panel for comparison.
Panel members were instructed to judge whether the samples
were similar, better or worse than the control. Glycoalkaloid
level of these potatoes was determined without addition of
ingredients.

Determination of the threshold level of glycoalkaloid
affecting the potato flavor

 

A dilution procedure was employed to determine at what
level glycoalkaloid would affect the potato flavor. Surface
material of the light —treated potatoes, or juice of the albino
sprouts, were used as sources of glycoalkaloid. The surface
material was collected in the same manner as was the sur-
face sample, and was blended to homogeneity in a Waring
blendor. The juice was expressed from the sprouts, which
were steam -blanched for 10 minutes, cooled, chopped and
blended. The glycoalkaloid and moisture content of both
materials were determined before use. Increasing amounts

of glycoalkaloid were added to several aliquots of the plain

42

mashed potatoes which contain very little glycoalkaloid.
The samples were presented to the taste panel using either
the paired comparison procedure, or the single sample
procedure.

In the paired comparison procedure, the simulated
samples were paired with the control samples, and were
presented to the taste panel in an increasing order. In the
single sample procedure, the simulated samples were pre-
sented alone. The panelists were asked to detect the off -
flavor of the samples and to judge the acceptability of the

samples .

RESULTS AND DISCUSSIONS

Sprouting

 

Potatoes stored at 40F up to nine months were still free of
sprouts. At 50F storage, sprouts began to appear on the MH -30
treated 709 and 321 -65, untreated 709 and 321 -65 three months after
harvesting. Vigorous sprouting took place 1 month later. Russet
Burbank and Kennebec seemed to respond to the sprout inhibitor
more favorably and were not vigorously sprouting until the fifth
month of storage. Therefore, monthly chemical analyses were
carried on until the fifth month for the MH -30 treated potatoes, and

until the fourth month for the untreated potatoes.

Methodology of Glycoalkaloid Determination

 

Since it is well -known that the peelings of raw potatoes
contain a highlevel of glycoalkaloid (Lampitt £131. , 1943), a change
in glycoalkaloid level should be pronounced in the surface area of the
tubers. Therefore, in this study, a surface sample composed
primarily of the cortical tissue rather than the composite sample of

the whole tuber was usedto determine the change in glycoalkaloid

43

44

during storage. Steam -blanching provided the most satisfactory
method for removing the skin and separating the cortical tissue, and
it also inhibited the enzymatic browning reaction, which takes place
rapidly in the fresh tissue of potato.

Boiled flesh samples were used for evaluating the average
level of glycoalkaloid in potato tubers, and diced fresh samples were
used for comparison purpose.

Since the glycoalkaloids, solanine and chaconine, have high
melting point and low solubility in water, it is expected that there
would be little or no loss of the glycoalkaloids during steam -blanching.
Theloss of glycoalkaloid from the whole tuber after boiling for
30 minutes was not determined, but it was offset by the identical
experimental condition of each sample.

The total solids content of the potato samples, Russet Bur-
bank, Kennebec, 709 and 321 -65, at the beginning of storage were
24.1, 19.5, 19.0 and 28. 3% respectively, and 21. 3, 22.1, 19.9 and
26. 5% respectively at the end of 4 months of storage (Pope, 1969).
The total solids content of the surface samples after steam -blanching
varied from 15 to 23. 5%, and hence the levels ofglycoalkaloid in dif-
ferent lots of potatoes were compared on dry weight basis.

In the study of the reflux extraction method, it was observed

that, as the time of refluxing increased, the solution became more

45

brown in color. .This was probably due to polymerization of the
phenolic compounds. It was impossible to remove the brown
polymerized pigments to any significant extent by saturated lead
acetate precipitation in the purification procedure. The extract
after being diluted with 2% acetic acid had a tea -brown color.

The precipitate collected at pH 4. 5 was a dark brown, clay-
like substance, which had a high affinity for glass and could not be
redissolved by 2% acetic acid. The bulk of the glycoalkaloid started
precipitating at pH 6. 3, and completed precipitation at pH 9. 3. The
mother liquor, highly buffered, was light brown in color. Precipi-
tates collected at pH 7.0, 8.0, and 9.0 were suspended in 95% ethanol,
and heated in 70C water bath. Recrystallization failed to remove the
polymerized pigments. It was thought that the reflux method with
aqueous ethanol was infeasible for quantitative determination of
glycoalkaloid because of the polymerization of phenolic compounds,
and also, the presence of a large quantity of starch and protein in
the extractfurther lowered the purity of the precipitates.

The bisolvent system, MeOH -CHCl3 (2:1), modified from
MeOH -CHC13 (1:2) employed by Folch (1957) for lipid extraction,
was found to be anexcellent mixture for glycoalkaloid extraction,
which is polar in nature. Blending this solvent mixture with the

sample at a ratio of 20 to 1 (100 ml of solvent to 5 g of sample)

46

completely dehydrated the sample, denatured the protein and
eliminated the adhesiveness of the starch. This permitted rapid
filtration of the solution through a sintered glass Biichner funnel
(medium porosity).

The non -polar chloroform fraction was. readily separated
from the polar methanolic fraction in the filtrate by the use of

0. 8% Na SO . After the two —step separation, less than 1 m1 of

2 4

chloroform. remained in the methanolic fraction. It was noted that
in the presence of either excessive water or excessive salt,
separation could not be achieved because hydration of the chloroform
fraction took place.

The methanolic fraction which contained glycoalkaloid was
normally colorless, or occasionally pinkish if the content of
anthocyanin was high. It is important that the methanol be evapo-
rated completely from the methanolic fraction before the glyco-
alkaloid is redissolved in 1% acetic acid. The glycoalkaloid sample
solution turned brownish uponaddition of 5M NH 4OH due to the
destruction of anthocyanin by alkali. However, the precipitated
glycoalkaloid had alight gray appearance and could be redissolved

in 1% acetic acid readily. There was no evidence of contamination

with polymerized compounds.

47

Two sets of surface sample with different levels of
glycoalkaloid were used in the recovery study, one with 1. 37 mg/
5 g w.b. l and another with 4. 20 mg/5 g w.b. 1 The added solanine,
which is equivalent to 73% of the glycoalkaloid in the first sample

and 24% in the second sample, was completely recovered in both

cases (Table 3).

Table 3. --Recovery of added solanine using the bisolvent extraction

 

 

 

 

method
Glycoalkaloid
Glycoalkaloid) After Addltlon Recovered
Sample m l5 w b of 1 mg of 0/,
g g ' ' solanine °
mg/5 g w. b.
Lla 1. 37 2. 45
L2 1.37 2.40 Ave. 105
L3 1. 37 2. 40
H18L 4. 25 5. 10
H2 4. 10 5. 30 Ave. 105
H3 4. 20 5. 30

 

 

 

 

aL and H designate potato samples with low and high level

of glycoalkaloid respectively.

bmg/5 g w.b. is mg per 5. 0 g of potato sample on wet basis.

 

1mg/5 g w.b. is mg per 5. 0 g of potato sample on wet basis.

The glycoalkaloid yield obtained from the surface samples

and from the diced fresh samples using the bisolvent extraction

method was higher than that obtained using the Soxhlet extraction

method (Tables 4 and 5).

Table 4. --Glycoalkaloid content of potato samples extracted with
the bisolvent extraction and the Soxhlet extraction methods

 

 

 

 

Sam le Bisolvent Met od Soxhlet Method Yield
p mg/5gw.b. mg/5gw.b. %
L1a 1. 37 1. 05
L2 1.37 1.00 Ave. 79
L3 1. 37 1. 20
H1a 4. 25 3.10
H2 4.10 3. 05 Ave. 74
H3 4.20 3.15

 

 

 

 

aL and H designate potato samples with low and high level

of glycoalkaloid respectively.

bmg/5 g w.b. is mg per 5.0 g of potato sample on wet basis.

With the surface samples the yield with the Soxhlet method was about

75% of that obtained by the bisolvent method. With the diced samples,

the yield varied. Analysis of diced fresh samples gave poor repro-

ducibility of glycoalkaloid content for replicate samples, whereas the

results obtained using peeled, cooked and mashed potatoes as samples

were reproducible (Table 5).

49

Since different areas of the tuber have

different levels of glycoalkaloid (Lampitt et a1. , 1943), it is obvious

that the diced fresh samples will not be as homogeneous as the boiled

flesh samples.

Table 5. —-Glycoalkaloid content of diced fresh sample and/or boiled
flesh sample with the bisolvent extraction and Soxhlet

extraction methods

 

 

Bisolvent Method

Soxhlet Method

 

 

 

 

Sample Diced Fresh Boiled Flesh Diced Fresh
Sample b Sample Sample b
mg/5gw.b. mg/5gw.b.b mg/5gw.b.
a
L-45 0. 34 0. 41 0.31
L—45 0. 39 0. 41 0. 30
L-45 0. 46 -—-- 0.31
RB -403 Nil 0. 04 o. 10
RB -40 Nil 0. 04 0.07
RB—40 0.03 ---- 0.12
a
L-65 0.81 0.41 0.37
L-65 1.04 0.41 0.58
L-65 1.24 ---- 0.39

 

 

 

 

aL-45 and L-65 are B5141-6 potatoes stored at 45F and 65F
respectively, and RB -40 designates Russet Burbank potatoes stored

at 40F.

bmg/5 g w.b. is mg per 5. 0 g of potato sample on wet basis.

50

The standard curves for the determination of glycoalkaloid
using 70% and 85% H3PO4 are given in Figure 4. With 85% H3PO4,
the color reaction was more rapid and sensitive than with 70% H3PO4.

The use of 70% H3PO for color development gave a less viscous solu-

4

tion and was more suitable for determining higher levels of glyco-

alkaloid such as that in potato sprouts.

/ 85% H3PO4

Ii / /°

/

0.0 4 1 1 1 1
0.2 0.4 0.6 0.8 1.0

Glycoalkaloid mg

70% H3PO4

Absorbance (600 nm)
o
A
l

 

 

Figure 4. —-Standard curves for glycoalkaloid determination

51

Thin-Layer Chromatography of Glycoalkaloids

 

The solvent systems which had been reported in the
literature, such as‘BuOH eHOAc -HZO (40:8:20) (Szendey, 1957),
3-heptanone -HOAc-HZO (8:5:1) (Lepage, 1964), and EtOAc -pyridine-
H20 (3:1:3) (Kuhn and Low, 1954) were first employed. The results
obtained with all three solvent systems were not satisfactory. It
took 390 and 200 minutes respectively for the solvent front of

BuOH -HOAc -H O and 3 -heptanone -HOAc -H

2 O to travel the necessary

2
distance, and yet the separation was very poor. Spots were not
clearly detected after the glycoalkaloid precipitate was developed in
EtOAc -pyridine -HZO for 330 minutes. The low mobility and poor
separation of the glycoalkaloids in these solvent systems necessitated
the search of a new solvent system, and hence 95% MeOH -EtOAc-
HOAc (10:15:2) was used. Early TLC results with this solvent system
showed that small quantities of solanine from 0. 5 to 4 [lg could be
chromatographed and visualized as distinct spots after being sprayed

with the SbCl reagent. This solvent system was then modified to

3
MeOH -EtOAc -HOAc-HZO (20:30:10:1). This mixture was very
efficient as to the time consumed and the separation achieved.
Roughly 70 minutes were required for the solvent to travel 12 cm.

A typical result (Figure 5) showed that all potato tubers

contained both solanine and chaconine with Rf values 0. 18 and 0. 35

Rf Value

52

RBT -50
KennT -50
709T -50

32 1 T -50
RBT -40
KennT -40
709T -40

32 1 T -40
CX-solanine

:OOOOOOOO
300000000.

 

Figure 5. -- Thin -layer chromatogram of glycoalkaloids from the
MH -30 treated potatoes (Russet Burbank, Kennebec,
709 and 321 -65) stored at 40F and 50F

Solvent system: MeOH -EtOAc—HOAc-H20 (20:30:10:1)
Front and time: 10. 9 cm and 70 min
Spray reagent: The Lowry reagent

53

respectively. The density of color seemed to correspond to the
concentration of the glycoalkaloid, and hence a densitometer might
be used to quantify the two compounds separately. However, the
ratio of solanine to chaconinewas estimated as 1 to 1 without using
any instrument.

Using the same solvent system, the glycoalkaloid precipitate
obtained from the Soxhlet extraction method was compared with that
obtained from the bisolvent extraction method, and identical compo-
nents were resolved (Figure 6). These results therefore showed the
differences in glycoalkaloid content obtained by the bisolvent and
Soxhlet method were only due to the difference in extraction efficiency.

Among the three reagents, the Lowry reagent, first used by
Lowry (1967) for detection of cholesterol and its esters, was most
satisfactory. A purple red color which fluoresces under long UV
light developed after heating for 5 to 10 minutes at 90C. Although
the SnCl3 reagent was very sensitive in detecting the glycoalkaloid,
it was not desirable because of its toxic nature and short shelf life.
Dragendorf reagent was not satisfactory for detection of solanine and
chaconine.

Successful separation of solanine and chaconine with TLC
permitted preparative TLC of the two glycoalkaloids. In the prepara—

tion of a large amount of glycoalkaloid, preparative TLC is preferred

R 1' Value

54

Bisolvent extraction
CX-solanine

Soxhlet extraction
Soxhlet extraction
Bisolvent extraction

33

0.

O O
CO

00
GO

 

Figure 6. -— Thin -layer chromatogram of the glyco-
alkaloids extracted by the bisolvent
method and the Soxhlet method (Russet
Burbank tubers)

Solvent system: MeOH -EtOH -HOAc -H 0 (20:30:10:1)
Front and time: 12 cm and 70 minutes
Spray reagent: The Lowry reagent

55

to column chromatography because TLC requires less time and
achieves better separation. After elution of the glycoalkaloids with
MeOH, it is necessary to displace MeOH with 1% HOAc before the
glycoalkaloids are precipitated since the glycoalkalids can only be
precipitated by alkali from aqueous solution.

Absorption spectra of solanine were identical to those of
chaconine with all three reagents tested (Figure 7). With the Clarke
reagent, both glycoalkaloids showed the maximum absorption at 660,
600 and 512 nm. Use of 600 nm for quantitative determination of the
glycoalkaloids was quite appropriate. This also confirmed that
earlier data of the colorimetric determination of total solanine,

which was the sum of solanine and chaconine, were acceptable.

Thin-Layer Chromatography of Sterols

 

In the chloroform fraction of potato sample, three components
including sterols, carotenoids and chlorophylls were shown by thin-
layer chromatography. After saponification, the majority of
chlorophyll was lost to the aqueous fraction in the form of chloro-
phyllide, and the esterified sterols were freed from fatty acid.

Benzene -EtOAc (1:1) was the most satisfactory solvent
system for the resolution of sterols in this study. Six spots (exclud-

ing carotenoids) were resolved from each of the potato tubers or

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57

sprout extracts. Spot 3 was found only in potato sprouts, and spot 2
was found only in the potato tuber (Figure 8). Spot 6 absorbed short
UV light without spraying. After spraying with the Lowry reagent,
spots 1 to 5, and 7 fluoresced under-long UV light, and spots 1 and 4
showed specific purple -red color of sterols. The Rf values of these
spots were 0. 00, 0.21, 0. 36, 0.60, 0.67 and 0. 72 respectively.
Co-chromatography with the standard sterols indicated that spot 4

had similar R value as cholesterol, stigmasterol and fl -sitosterol.

f

With hexane -EtOEt-HOAc (80:20:1), the R values changed

f
considerably. Spots 2, 3 and 5 had similar Rf values, and spot 4
was next to spot 1. Spot 7 was resolved into 3 spots. The Rf values
of these spots in numerical order are 0. 00, 0. 10, 0.10, 0. 05, 0. 10,
0. 13, 0. 47, 0.62 and 0. 66 (Figure 9). With the more polar solvent
system, 3 -heptanone -HOAc-HZO (8:5:1), spot 1 was moved with a Rf
value 0. 68, while the others were poorly resolved. The Rf value

of spot 1 indicated that it was a steryl glycoside (Lepage, 1964). It
was once suspected that spot 1 might contain solanine due to poor
partitioning procedure between the chloroform fraction and the
methanolic fraction. However, results with TLC showed that spot 1
did not contain any substance with a Rf value corresponding to
solanine (Figure 10).

Two dimensional TLC of non -saponifiable from potato

tubers or sprouts was carried out using a combination of

58

Stigmasterol
[B -s itosterol

Cholesterol
Solanidme
Tubers
Sprouts

 

@063
®O®D

3 0.36

2 0.21 O

0.16 0

1 0.00 - ‘ - Q Q

V}

Figure 8. -- Thin -layer chromatogram of the unsaponifiable com -
pounds in the chloroform extract of the potato tubers,
sprouts and authentic sterols (solanidine, cholesterol,
stigmasterol and -sitosterol)

Solvent system: Benzene -EtOAc (1:1)
Front and time: 12.8 cm and 45 minutes

Spray reagent: The Lowry reagent

@ -- Purple -red color

59

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60

benzene —EtOAc (1:1) and hexane -EtOEt-HOAc (80:20:1), In tuber
sample, all spots were clearly separated, but spot 1 and 7 were split
into 2 spots in each case (Figure 11). Chromatogram for the sprout
sample (Figure 12) was obtained by developing the plate in the same

solvent systems, and spraying with the benzidine -NaIO reagent and

4
the Lowry reagent successively. After spraying with the benzidine -
NaIO4 reagent which reacts with sugars and glycosides, 2 spots, G1
and G2, were detected, and after spraying with the Lowry reagent
on top of the first reagent, spot 1 showed same color as G1 and G2.
This proved that spot 1 was a glycoside. Spot 3, the unique sub-
stance in sprouts, appeared to be a major substance accumulated
during sprouting (Figure 12), but it was not identified.

Among the reagents for-sterol visualization, the Lowry
reagent was preferred although the Liebermann -Burchard reagent
had similar visualizing function. Iodine vapor, on the other hand,
was used only for temporary location of the spots, and plate was
re -sprayed with other reagents after iodine sublimes.

Thin -Layer and Gas -Liquid
Chromatographic Analysis of Sterols

 

 

The 8-foot long glass column packed with 1% CV -101 was
very satisfactory in separating the standard sterols. Cholesterol,
campesterol, sigmasterol and /8 -sitosterol had relative retention

times of 2.072, 2. 665, 2. 880 and 3. 263 respectively.

61

Sample: White light treated tuber

.3

e

g 4

e

E @151

 

Figure 11. -- Two-dimensional thin -layer chromatogram of the 7
unsaponifiable compounds in the chloroform extract
of the light treated potato tubers

1. Benzene -EtOAc (1:1)

2. Hexane -EtOEt-HOAc (80:20:1)
1. 13 cm and 45 minutes

2. 13. 9 cm and 45 minutes
Spray reagent: The Lowry reagent

Solvent system:

Front and time:

62

Sample: Sprout _______________
r. __________________________

1""

\ O

\M

\-
'7‘
\
0.
HI
‘_I

 

@4—

Figure 12. -- Two-dimensional thin -layer chromatogram of the
unsaponifiable compounds in the chloroform extract
of the potato sprouts

Benzene -EtOAc (1 :1)
Hexane ~EtOEt-HOAc (80:20:1)
15.5 cm and 52 minutes

Solvent system: 1
2
1.
2. 14.6 cm and 44. 5 minutes
1
2

Front and time:

Spray reagent: Benzidine -NaIO4 reagent

The Low ry reagent

63

Eight peaks were resolved from Spot 1 (Figure 13). Peak 4,
6 and 7 were cholesterol, stigmasterol and IG-Sitosterol respectively.
Proof of the presence of the above sterols was obtained by the mass
spectrometry (Figure 14). Peak 3, which has a mass number 397,
was present in larger amount than was cholesterol. Campesterol
was not present.

Five peaks were resolved from spot 4 (Figure 13). Peak 1
was cholesterol; peak 2, unknown; peak 3, unknown; peak 4, stig-
masterol; and peak 5, ,8 —sitosterol. The chromatogram indicated
that campesterol was present in a very small quantity in the potato
tuber’while 3% of campesterol was found in the potato leaves
(Ardenne i111; , 1965). Mass spectra of peak 1, 4 and 5 confirmed
the presence of the above sterols. The mass spectrum of peak 2
(Figure 14) is given because of its possible significant role in the
biosynthesis of glycoalkaloid.

Change of Glycoalkaloid Level During
Storage and Light Treatment

 

 

Glycoalkaloidlevel in the surface of potato tubers generally
tended to decline with the storage time. If the glycoalkaloid level
was high at the beginning of storage, an obvious decrease would be
seen, but if the tuber started at a low level of glycoalkaloid, change

during storage was normally insignificant (Figure 15, 16).

64

 

 

 

 

 

 

 

 

 

 

 

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Figure 13. --Gas -liquid chromatogram of (a) spot 4, the free sterol
fraction, (b) spot 1, the steryl glycoside fraction after
acid hydrolysis

65

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Month

Figure 15. --Change of glycoalkaloid levels in the surface samples of

potatoes (Russet Burbank, Kennebec, 709 and 321 -65)
stored at 40F and 50F

 

\

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Glycoalkaloid mg/100 g d.b.

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\\ . e o RBT-40
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180
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Month

Figure 16. --Change of glycoalkaloid levels in the surface samples of the
MH -30 treated potatoes (Russet Burbank, Kennebec, 709
and 321 -65) stored at 40F and 50F

69

It was noticed that the greatest change took place in the
untreated potatoes between the first and second month of storage,
and in the MH -30 treated potatoes between the second and third
month of storage. This seemed to correspond to the sprouting
activities, which started in this period. Decrease of glycoalkaloid
was probably due to the vigorous sprouting activities. However,
glycoalkaloids of tubers in both 40F and 50F storage tended to reach
an equilibrium level after a few months with only one exception; the
untreated 321 -65 in 50F storage continued to decrease.

Holding at 45F for 20 days after harvest, the MH -30 treated
potatoes were stored at 58F for 10 days, and the glycoalkaloid content
was increased markedly over that present in replicate lots stored at
42F (Figure 16). It would appear that during the dormant period the
glycoalkaloid synthesis was greatly stimulated by a sudden rise in
temperature. .

After six months of storage (four months of controlled
storage), the average glycoalkaloid level of the entire tuber was
generally low (Figure 17). The MH —30 treated tubers of Russet
Burbank and 709 had higher levels of glycoalkaloid thanthe untreated
tubers. The difference of glycoalkaloid level was more distinct
between the MH -30 treated and untreated Russet Burbank. The

storage temperatures made large differences in the untreated

70

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Kennebec, 709 and 321 -65. The untreated 321 -65 in 50F storage had
the highest glycoalkaloid level. However, among four different
potatoes, 7 09 had the lowest level of glycoalkaloid under all conditions
(Figure 17). This seemed to support the finding that 709 performs
uniquely with the MH -30 and light treatment.

A comparison of the glycoalkaloid content of the surface
layer and of the entire potato indicated that the ratio ranged from
5 -10 to 1. The variation indicated that glycoalkaloid determinations
of the surface layers could not be used as a means of predicting
glycoalkaloid content of the entire tuber.

With the exception of MH —30 treated 709, the glycoalkaloid
content of the tubers exposed to white light for six days, after 2
months storage at 45F, increased from 110 to 330 mg/100 g d.b. 1
over that of the controls (Figure 18). When a similar experiment
was carried out with potatoes stored for five months at 40F, all
light -treated potatoes had higher levels of glycoalkaloid. After
2 months storage, glycoalkaloid content in the MH -30 treated 709
after light treatment was 114 mg/100 g d. b. as contrasted to
137 mg/100 g d. b. in the control. After 5 months storage, the

glycoalkaloid content of the light treated tubers was double that of

the control tubers (194 and 96 mg/100 g d. b. respectively). The

 

1mg/100 g d.b. = mg per 100 g of potato sample on dry basis.

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73

inhibitory effect of MH -30 on the production of glycoalkaloid,
therefore, disappeared during the longer storage. This result
agreed with an early report that the length of inhibition period of
MH -30 was proportional to the MH -30 concentration used (Schoene
and Hoffmann, 1949). The MH -30 treated potatoes after the light
treatment tended to synthesize less glycoalkaloid than did the un-
treated potatoes (Figure 18).

The glycoalkaloid produced in Russet Burbank potatoes

under white light in 100% CO atmosphere increased less than under

2
normal storage conditions (Figure 19a). After exposure to white
light, anthocyanin of Russet Burbank also increased significantly.
The sample solution, from which the glycoalkaloid is precipitated,
became pinkish. The absorption spectra of the solution were taken
with a Bausch -Lomb 505 spectrophotometer after concentration.
The maximum absorption occurred at around 520 to 530 nm. The
anthocyanin was not identified, but the absorbance of each sample
solution was recorded on a quantitative basis. Increase of antho-

cyanin was not affected by CO as much as by MH -30 (Figure 19b).

2
Similar observations were made on the Kennebec variety.
Russet Burbank potatoes exposed to red and UV light for

6 days showed a slight increase in glycoalkaloid, but in the tubers

exposed to far -red light it was slightly below the control (Figure 20a).

b.
0.4)
‘ :1 Control
White Light
0 3 White Light
plus 100%

C02

Glycoalkaloid mg/ 100 g d. b.
Absorbance (530 nm)

 

 

 

Aux": -:-:-:-:

ll.-
.
— ""' — ....

0 , =.‘.-:=: .
Untrea ed MH -30 Untreated MH -30
Treated Treated

Figure 19. ~-Glycoalkaloid and anthocyanin synthesized by potatoes (Russet
Burbank) after treatment with white light or white light plus
100% C02 for 6 days

Da rk
// UV

Red
Far -red
White

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Figure 20. --Glycoalkaloid and chlorophyll synthesized by potatoes (Russet
Burbank) after treatment with different lights for 6 days

RI
‘1

I
' , a i.

Glycoalkaloid mg/100 g d. b.

 

 

 

 

0

75

Similar trends were obtained for the amount of chlorophyll produced
(Figure 20b). The chlorophyll was determined using the chloroform
fraction of these samples. The absorbance of the samples was taken
withva Beckman DB Spectrophotometer at 660 nm after chloroform
was displaced by ethyl ether. This suggests that far -red light may
have a specific effect on the metabolic activities of potato tubers.

It is not clear whether'the high energy of far -red light has anything
to do with this phenomenon.

Change of Sterols During Storag_e_
and Light Treatment

 

 

During dark storage, total sterol1 in potatoes stored at
40F tended to increase during storage (Tables 6, 7). This increase
was primarily due to an increase in [B-Sitosterol. At 50F there was a
markedincrease between the second and third month of storage and
a slight decrease between the third and fourth month of storagé with
the MH -30 treated potatoes (Table 7). No consistent pattern of change
was observed with the untreated potatoes, but the amount of cholesterol
decreased while that of fl-sitosterol and stigmasterol tended to
increase during storage.

Under white light cholesterol and stigmasterol content

increased while IB-Sitosterol decreased (Table 8). It appears that

 

1Total sterol is the sum of cholesterol, stigmasterol and

,8 -sitoste rol.

76

Table 6. --Sterol contents of potatoes (Russet Burbank, Kennebec, 709,
and 321 -65) at different stages of storage at 40F and 50F

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Month Month Month Month
Sample Sterol 1 2 3 4
mg/100 g w. b.
Russet Cholesterol .27 . 17 .20 .33
B b nk Stigmasterol . 43 . 38 . 4O . 37
43‘; a ~sitosterol .65 . 66 .74 . 94
TOTAL 1.35 1.21 1.34 1.64
Cholesterol . 18 . 15 . 16 . 33
Kennebec Stigmasterol . 35 . 34 . 38 . 36
40F -sitosterol .24 . 37 . 43 . 87
. TOTAL .77 . 86 .97 1. 55
Cholesterol . 18 . 13 . 15 .30
709 Stigmasterol . 35 . 38 . 43 . 93
40F -sitosterol . 25 . 37 . 43 1 . 00
TOTAL .78 .88 1.01 2.23
Cholesterol .20 . 18 . 17 . 27
321 -65 Stigmasterol .39 . 38 .37 .32
40F -sitosterol . 48 . 60 . 50 . 87
TOTAL 1.07 1.16 1.04 1.46
R s t Cholesterol . 27 . 20 . 18
us e Stigmasterol .43 .23 .36
Burbank .
50F -81tosterol .65 . 73 .72
TOTAL 1.35 1.16 1.26
Cholesterol . 18 . 18 . 18
Kennebec Stigmasterol . 35 . 35 . 39
50F -sitosterol . 24 . 40 . 72
TOTAL . 77 .93 1. 29
Cholesterol . 18 . 17 . 15
709 Stigmasterol . 35 . 44 . 42
50F -s itosterol . 25 . 52 . 47
TOTAL . 78 1. 13 1. 04
Cholesterol .20 . 20 . 15
321 -65 Stigmasterol . 39 . 37 . 33
50F ~sitosterol . 48 . 54 . 39
TOTAL 1.07 1.11 .87

 

 

 

 

 

 

77

Table 7. -- Sterol contents of the MH ~30 treated potatoes (Russet
Burbank, Kennebec, 709 and 321 -65) at different stages
of storage at 40F and 50F

 

 

 

 

 

 

 

 

 

 

 

 

 

Month 2 Month 3 Month 4
Sample Sterol .
mg/100 g w. b.
Russet Cholesterol . 17 .22 . 33
Stigmasterol . 23 . 40 . 7 1
Burbank .
40F -81tosterol . 29 . 56 . 80
TOTAL .69 1. 18 1. 84
Cholesterol . 28 . 21 . 27
Kennebec Stigmasterol . 38 . 45 . 61
40F -sitosterol . 40 . 60 . 64
TOTAL 1.06 1.26 1.52
Cholesterol . 19 . 16 . 29
709 Stigmasterol . 3.9 . 47' . 69
40F -sitosterol . 38 . 43 . 60
TOTAL .96 1.06 1.58
Cholesterol . 15 . 19 . 28
321 -65 Stigmasterol . 18 . 44 . 68
40F -sitosterol . 22 . 60 . 64
TOTAL .55 1.23 1.60
Russet Cholesterol . 13 . 27 . 27
Stigmasterol . 19 . 45 . 43
Burbank .
50F -s1tosterol . 2 1 . 77 . 45
TOTAL . 53 1. 49 1. 29
Cholesterol . 17 . 35 .27
Kennebec Stigmasterol . 30 . 48 . 53
50F -sitosterol . 27 . 66 . 49
TOTAL .74 1.49 1.29
Cholesterol . 20 . 24 . 17
709 Stigmasterol . 45 . 64 . 51
50F -sitosterol . 33 . 64 . 51
TOTAL .98 1.52 1.19
Cholesterol . 16 . 41 . 12
321 -65 Stigmasterol .28 . 47 .27
50F -sitosterol .21 . 60 . 23
TOTAL . 65 1. 48 .62

 

 

 

 

 

78

under the influence of light, the system was mobilized towards
increasing cholesterol and the unidentified compound of peak 2,
although these compounds did not accumulate to any great extent.
These results seem to be in agreement with the proposed bio-
synthetic pathway where cholesterol is an intermediate for
solanidine biosynthesis. The decrease in fi-sitosterol as well as
total sterol during light treatment is probably due to a shift in
kinetics in favor of synthesis of glycoalkaloid.

Table 8. --Sterol contents of potatoes (Russet Burbank, Kennebec,
709 and 321 -65) with and without white light treatment for

 

 

 

 

 

 

 

6 days ‘

Cholesterol Stigmasterol 3 -s itosterol
Variety Treatment a b a b a b

Concn. % Concn. % Concn. %

Russet Control . 33 20 . 37 23 .94 57
Burbank Light . 37 25 .35 24 . 74 51
Kennebec Control . 32 21 . 36 23 . 87 56
Light . 33 21 .49 32 . 73 47
709 Control . 30 13 . 93 40 1.10 47
Light . 32 20 . 67 42 .60 38
321 -65 Control . 27 18 . 32 22 . 87 60
Light .27 18 .41 28 .79 54

 

 

 

 

 

aConcentration of sterol is expressed in mg/100 g w. b.

b% cholesterol + % stigmasterol + % [B-sitosterol = 100

79

Table 9. -- Sterol contents of Russet Burbank potatoes treated with
white, far -red, red, UV light and in darkness

 

 

 

 

 

 

 

 

 

 

 

 

 

Content
nght J Sterol Concentration ,7 a
mg / 100 g w. b. 0

Cholesterol . 35 37

Stigmasterol . 31 33

Whlte ,8 -sitosterol . 2 8 30
TOTAL . 94

Cholesterol . 22 3O

Stigma ste rol . 2 5 3 5

Far -red ,8 -sitosterol . 25 35
TOTAL . 72

Cholesterol . 37 37

Stigmasterol . 34 34

Red /8 -sitosterol . 2 9 29
TOTAL 1. 00

Cholesterol . 27 29

Stigmasterol _. 34 36

UV ,8 —,s itosterol . 33 35
TOTAL . 94

Cholesterol . 23 2 1

Stigmasterol . 41 37

Darkness ,8 -sitosterol . 47 42
TOTAL 1. 1 1

 

 

 

 

a% cholesterol + % stigmasterol + % B-sitosterol = 100

80

After the Russet Burbank potatoes were exposed to different
lights, the result showed that the total sterol of potatoes stored in
darkness was the highest and that stored in far-red light was the
lowest among all samples (Table 9). As the tubers were treated with
white, red or UV light, the level of cholesterol increased, while the
level of ,G-sitosterol markedly decreased. Also, the percent of
individual sterols varied in the same manner as obtained from the

white light treatment for all varieties (Table 8).

Flavor of the B5141 -6 Potatoes

 

The B5141 -6 potatoes of both 45F and 65F storage have a
high glycoalkaloid content with no greening (Table 10). The surface
sample, as anticipated, had a much higher level of glycoalkaloid than
did the flesh sample. The greatest difference between B5141 -6 and
Russet Burbank was in the flesh samples, with 35141 -6 having 11
times as much glycoalkaloid as Russet Burbank. In the surface
samples, the difference was only 3 times (Table 10).

In the flavor difference test between B5141 -6 and Russet
Burbank, the surface of both varieties was” excluded. The test
showed no significant flavor difference between the B5141 -6 potatoes
and the control. The majority of the panelists could not distinguish

the control from the B5141 -6. The B5141-6 samples were described

81

as fresh as the control sample, even though the gummy texture of
the B5141 -6 potatoes stored at 65F was noticeable. It appears that
potatoes with a glycoalkaloid level of 8. 3 mg/100 g w. b. were
acceptable to the taste panel under the described experimental con-
dition.

Table 10. --Glycoalkaloid content in surface and flesh of some

potato tubers (B5141-6 stored at 45F and 65F, Russet
Burbank stored at 40F)

 

 

 

 

 

Glycoalkaloid Content
Sample Area
mg/100 g w.b. mg/100 g d.b.
L-45‘=1 71 399
L-65a Surface 69 329
RBT-40b 23 140
L-45‘3L 8. 3 57
L-65a Flesh 8. 3 51
RBT-40b 0.8 6

 

 

 

 

aL -45 and L-65 are B5141 -6 potatoes stored at 45F and 65F
respectively.

bMH -30 treated Russet Burbank stored at 40F is used as

control.

Threshold Level of Glycoalkaloid
Affecting the Potato Flavor

 

 

To facilitate the control of glycoalkaloid concentration,

moisture content and homogeneity of the added glycoalkaloid in the

 

 

 

82

simulated samples, drum -dried potato flakes without any additives

were used as base material. Its moisture content was 8% as

determined by a Cenco moisture balance, and its glycoalkaloid content

was negligible as determined by the bisolvent extraction method. .'
In the first study, surface material from the light -treated F7”

potatoes was used as the source of glycoalkaloid. This material was

 

green in appearance. Four-levels of glycoalkaloid, 3. 8, 6. 8, 11. 4 and y ‘ '=-
17. 0 mg/100 g w.b. with 85% moisture were used, and a paired com- Y
parison test was run under red light. The result (Table 11a) showed
that glycoalkaloid at a level higher than 6. 8 mg/100 g w. b. could be
detected by the panelists, and that all 4 levels were not acceptable to
the panelists. This high rate of rejection gave rise to a thought that
color difference between the samples and control was not masked by
the red light used, and the color difference probably influenced the
panelist' s decision.
In the second series, glycoalkaloid concentrations of 3. 9,
5. 8, 7. 0 and 8. 7 mg/100 g w. b. respectively were used, and the
reference sample dyed green. The panelists indicated a significant
difference in flavor at the 8. 7 mg/100 g level but did not reject any
of the samples (Table 11b). In the third series of glycoalkaloid
concentrations, 7. 7, 9.2, 11. 5 and 15.4 mg/100 g w.b. , the

panelists were able to detect the off-flavor of two high concentrations

83

significantly, and both were considered as being unacceptable

(Table 1 1c).

Table 11. --Paired comparison tests on potato samples using green
potatoes as glycoalkaloid source

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Glycoalkalold Total Number Number
Sample Concentration . . . .
Trials Detection Rejection
mg/100 g w.b.
a. Reference Not Colored
A 3. 8 30 16 20*
B 6. 8 30 23** 23**
C 1 1. 4 30 24** 24**
D 17.0 30 26*** 26***
b. Reference Dyed Green
A 3. 9 30 18 1 1
B 5. 8 30 14 1 1
C 7. O 30 19 15
D 8. 7 30 22** 19
c. Reference Dyed Green
A 7. 7 15 1 1 7
B 9. 2 15 1 1 6
C 1 1. 5 15 13** 13**
D 15. 4 15 13** 12*

 

 

 

 

 

* 5% significance level
** 1% significance level
*** . 1% significance level

Since the presence of green color in the glycoalkaloid donor

seemed to influence the panelists even after the masking technique

 

84

was used, the use of surface material from light treated potatoes as
a glycoalkaloid source was discontinued.

Albino sprouts, having an extremely high level of glyco-
alkaloid, were used in subsequent flavor studies. In the first series,
with levels of 5.0, 10. 0, 15.0, 20. 0 and 25.0 mg/100 g w.b. glyco-
alkaloid, significant detection of off ~flavor occurred at the 10, 20

and 25 mg/100 g w.b. levels but not at the 15 mg/100 g w.b. level.

 

No explanation can be given for “the detection at the 10 mg/100 g w. b.
level. Glycoalkaloid at 25 mg/100 g w.b. was rejected (Table 12).
In the second series 20 and 25 mg/100 g w.b. glycoalkaloid were
tasted with single sample procedure. Similar results were obtained,
althoughthe level of rejection was not as high (Table 13a). In the
third series with levels of 10 and 15 mg/100 g w. b. glycoalkaloid,
and the addition of 0.2% salt, significant detection of an off -flavor
occurred at the 15 mg/100 g w. b. level, but it was not rejected
(Table 13b).

An informal tasting with only 3 panelists indicated that a
level of 35 mg/100 g w. b. glycoalkaloid was completely unacceptable
and had a very strong off -flavor. The taste panelists reported that
the off «flavor due to glycoalkaloid was astringent or gave a burning
sensation and had a pronounced aftertaste. Zitnak (1961) reported

the taste of glycoalkaloid as bitter.

85

Table 12. --Paired comparison tests on potato samples using albino
sprouts as glycoalkaloid source

 

 

 

Glycoalkaloid Total Number Number
Sample Concentrat1on . . . .
Trials Detect1on Rejectlon

mg/100 g w. b.

A 5. 0 1 5 4 3

B 10. 0 30 2 1* 8

C 15. 0 30 16 12

D 20. 0 30 2 1* 18

E 25. 0 15 15*** 13***

 

 

 

 

 

 

Table 13. --Single sample procedure tests on potato samples using
albino sprouts as glycoalkaloid source

 

 

Glycoalkaloid
Sample Concentration
mg / 100 g w. b.

Total Number Number
Trials Detection Rejection

 

 

 

 

 

a. Without Salt

 

A ' 20.0 40 26* 21
B 25.0 40 9 35*** 27*

 

 

 

 

 

b. With 0. 2% Salt

 

A 10.0 30 15 3
B 15.0 30 24* 14

 

 

 

 

 

* 5% significance level
** 1% significance level
*** . 1% significance level

86

Talburt and Smith (1967) suggested that potatoes with more
than 100 mg/100 g d.b. (15 mg/100 g w. b.) of glycoalkaloid were not
suitable for human consumption. In this study, none of the varieties
under any of the storage conditions synthesized sufficient glycoalkaloid
in the surface to bring the glycoalkaloid level in the total potato up
to thislevel. However, this level would not be rejected by the con-
sumers on the basis of flavor. Therefore, chemical determination of
glycoalkaloid before a new lot of potatoes is marketed should be
adopted as a safety measure against off ~f1avor or poisoning of pota-

toes due to glycoalkaloid.

 

 

SUMMARY AND CONCLUSIONS

An effective extraction method for glycoalkaloid determina -
tion using a bisolvent mixture, MeOH -CHCl3 (2:1) was developed in

this study. By blending the sample and the solvent mixture at a ratio

of 1 to 20, the sample was dehydrated, and the glycoalkaloid extracted

into the methanolic fraction. The glycoalkaloid so obtained did not
have any polymerized pigment, and a complete recovery of added
glycoalkaloid was obtained.

The glycoalkaloid isolated from the surface sample of
potato tubers was resolved into (X-solanine and CX—chaconine by a
solvent system MeOH -EtOAc-HOAc -HZO (20:30:10:1) on a silica gel
plate. Both glycoalkaloids had identical absorptionspectra after
reacting with the Marquis reagent, the Clarke reagent or 85% H3PO4,
and were determined together quantitatively.

The chloroform fraction of the bisolvent extract contained
sterols of potatoes. After saponification, 6 spots were resolved by
the solvent system benzene -EtOAc (1 :1) on a silica gel plate, but

only 2-spotshad the specific color reaction as the demethyl sterols

such as cholesterol, stigmasterol, B-sitosterol or campesterol

87

 

88

after spraying with the Lowry reagent. They were found to be steryl
glycoside and free sterols.

The steryl glycoside and free sterols were analysed with

 

gas -liquid chromatography. The steryl glycoside had 8 different .
steryl moieties including cholesterol, stigmasterol and ,8-sitosterol, .11
and the free sterols included the above sterols plus two unknowns.

Campesterol was not found in any significant quantity. Presence of v “‘5

cholesterol, stigmasterol and [B-sitosterol was proved by mass
spe ctrome try .

Potatoes including two commercial varieties, Russet Bur-
bank and Kennebec, and two clones, 709 and 321-65, were used in
studying the change of glycoalkaloid level during storage and light
treatment. Glycoalkaloid level increased rapidly when potatoes
were subjected to a higher storage temperature at the early stage
of storage. No consistent change in glycoalkaloid level of the
potatoes occurred during Sprouting, but there was a tendency for it
to decrease. Potatoes with low glycoalkaloid level at the beginning
of storage maintained the low level throughout the storage period.

The potatoes were also treated with white, far-red, red
and UV lights. Those stored under white light showed the greatest
increase in glycoalkaloid content, while those held under far -red

light had the lowest glycoalkaloid level. The MH -30 treatment

89

generally decreased the rate of light -induced synthesis of glyco-

alkaloid, particularly during the early months of storage. The

MH -30 treated 709 after2 months storage had a lower glycoalkaloid

level after white light treatment than those held in darkness. r
Monthly analyses of sterols showed that total sterol increased ?

in40F storage, and anincrease with a subsequent decrease of total

 

sterol was observedin the MH -30 treated potatoes stored at 50F. .44:
The change of total sterol was primarily’due to IB-sitosterol. In the i
untreated potatoes stored at 50F, no consistent pattern of sterol
change could be found.
During light treatment, fl-sitosterol, as well as total
sterol, tended to decrease. Stigmasterol increased at the expense
of B-sitosterol. Cholesterol along with an unidentified compound
increased slightly. Therewas no accumulation of any sterol in the
same magnitude as the accumulation of glycoalkaloid during light
treatment.
Based on the above data, it is suggested that these sterols,
cholesterol in particular, are involved inthe glycoalkaloid biosyn-
thesis although they are not directly related.
Using a flavor difference procedure, B5141 -6 potatoes
(45F and 65F storage) which contained 8. 3 mg/100 g w. b. glyco-

alkaloid were compared with a control potato (Russet Burbank at

90

40F storage) which contained 0. 8 mg/100 g w.b. glycoalkaloid. No
difference of flavor was detected by the 24-member taste panel.

A dilution procedure was employed to determine at what
level glycoalkaloid would affect the potato flavor. Both the paired

comparison procedure and the single sample procedure were used.

 

When the green surface material of the light treated potatoes was
used as the source of glycoalkaloid, the detection threshold was at
around 10 mg/ 100 g w.b. , but when the juice of albino sprouts was

used, the glycoalkaloid was not detected until the level was higher

than 20 mg/100 g w.b. Since potatoes with more than 15 mg/100 g

w. b. of glycoalkaloid were thought to be health hazards, it was con-

cluded that potatoes with a glycoalkaloid level high enough to be a

health hazard might not be rejected by the consumers on the basis

of flavor.

PROPOSALS FOR FURTHER RESEARCH

In this study, cholesterol among other sterols was found
to increase during the glycoalkaloid.biosynthesis. According to
the proposed biosynthetic pathway (Figure 2), cholesterol
instead of stigmasterol and ,B-Sitosterol is the intermediate of
glycoalkaloid biosynthesis. The result of this study has shown
that stigmasterol increases at the expense of B-sitosterol.
There is no proof that stigmasterol and B—sitosterol are not
the intermediates of glycoalkaloid synthesis. In addition, there
seems to be an interrelationship among cholesterol, stigmasterol
and /8 -sitosterol.

It is therefore suggested that C14-labelled cholesterol be
incorporated into the potato sprout or a piece of sprouting tuber,
and the glycoalkaloid and sterols extracted with the bisolvent
extraction method a few days later. The radioactivity in the
glycoalkaloid precipitate can be measured, and the radioactivities
in cholesterol, stigmasterol and B—sitosterol can also be
measured if their intensities do not change after separation with

preparative gas -liquid chromatography.

91

 

2.

92

Higher concentration of glycoalkaloid is found in cortical
tissue of potato tubers and sprouts. It is of interest that a
histo -chemical procedure can be designed to locate the glyco-
alkaloid. An attempt has been made to stain the thin slices of
tissue samples with the Clarke reagent in the hope that the color -; “1
reaction between glycoalkaloid and the reagent can indicate the
location of the glycoalkaloid. The color reaction, however, it
took place, but it was not exclusively blue. Presence of antho-
cyanin in this sample was an interfering factor. In pursuit of
such a histo -chemical procedure, effort should be directed
toward a better technique in stabilizing the glycoalkaloid, fixing

the tissue and elminating the interference of anthocyanin.

Since far -red light has caused a. lower level of glycoalkaloid
and chlorophyll in potato tubers in this study, it may be useful
in the conditioning of potatoes. Potatoes stored at low tempera-
ture are high in reducing sugar, which will cause darkening of
potato chips. Consequently, it becomes common practice that
potato tubers are brought to 65 to 70F for a few days in order to
attain brighter final products. It is possible that treatment with
far -red light may diminish the reducing sugar more rapidly and

completely than the high temperature treatment.

 

 

REFERENCES CITED

REFERENCES CITED

Abbott, D. C., K. Field and E. 1. Johnson. 1960. Observations on 7
the correlation of anticholinesterase effect with solanine
content of potatoes. Analyst 85:375.

 

Akeley, R. V., G. V. C..Hough1and and A. E. Schak. 1962. "*4"?!
Genetic difference in potato tuber greening. Am. Potato
J. 39:409.

Alberti, B. 1932. Zum Nachweis von Solanin. Z. Lebensm. -
Untersuch. -Forsch. 64:260.

Alfa, J. , and E. Heyl. 1923. Kartoffeln 1922-er Ernte mit
ausserordentlich hohem Solaningehalt. Z. Unters. Nahr.
Genussm. 46:306.

Allen, E. H., and J. Kfic. 1968. OC-solanine and “—chaconine as
fungitoxic compounds in extracts of Irish potato tubers.
Phytopathology 58(6) :7 7 6.

Ardenne, M. Von, G. Osske, K". Schreiber, K. Steinfelder and
R. Tummler. 1965. Uber die sterine von Solanum

tuberosum L. : Sterine und triterpenoid. Kulturpflanze
13:101.

Arnold, W. 1950. Eine verbesserte Methode zur Gewinnung und
quantitativen Bestirnmung des Solanin--des Glykoloids der

Kartoffelpflanze. Pharmazie 5:490.

Arutyunyan, L. A. 1940. Solanine content of potatoes. V0prosy
Pitaniya 9(5):30.

Autenrieth, W. 1928. The Detection of Poisons and Strong Drugs.
Philadelphia, Pennsylvania. Blakiston' 8 Son and Co.

93

94

Baker, L. C., L. H. Lampitt and C. B. Meredith. 1955. Solanine,
glycoside of the potato. III. An improved method of
extraction and determination. J. Sci. Food Agri. 6:197.

Baup, M. 1826. Extrait d' une lettre sur plusieurs nouvelles sub-
stances. Ann. Chim. Phys. 31(2):108.

Barug, R. 1962. Influence of different rates and intensities of light
on solanine content, cooking quality of potato tubers.
European Potato J. 5:242.

Bennett, R. D., and E. Heftmann. 1965. Biosynthesis of Dioscorea
sapogenins from cholesterol. Phytochem. 4:577.

Blanchet, R. 1838. Uber die Zusammensetzung des Solanins.
Liebigs Ann Chemie 7:152.

Bémer, A. , and H.. Mattis. 1923. Uber hohe Solaningehalte bei
Kartoffeln. Z. Unters. Nahr. Gemussm. 45:288.

Béimer, A. , and H.. Mattis. 1924. Der Solaningehalt der'Kartoffeln.
-Z. Unters. Nahr. Gemussm. 47:97. *

Briggs, L.-H., and L. C. Vining. 1953. Solanum alkaloid: X.
Mode of linkages intrisaccharide moiety of solanine and
solasonine. J. Chem. Soc. 1953:2809.

Clarke. E. G. C. 1958. Identification of solanine. Nature 181:
1152.

Clemo, G. R., W..M..Morgan and R. Raper. 1936. Occurrence
of solanine in sprouting potatoes. J. Chem. Soc. 1936:1299.

Conner, H. W. 1937. Effect of light on solanine synthesis in the
potato tuber. - Plant Physiol. 12:79.

Dabbs, D.. H. , and R. J. Hilton. 1953. . Method of analysis for
solanine in tubers of Solanum tuberosum. J. Technol.
31:213.

Desfosses, M. 1821. Examen du principe narcotique de la morelle
(Solarium nigrum), etc. J. de Pharmacie (2) 7:414.

 

" -3.-
y r:

95

Folch, J., M. Lees and G..H. Sloane —Stanley. 1957. A simple
method for the isolation and purification of total lipids from
animal tissue. J. Biol. Chem. 226:497.

Gawecki, K. , and J- Ilecki. 1966. Influence of some factors on
solanine content in ensiled tops of potatoes. Roczniki Nauk
Rolniczych Ser. B 87(3):337.

Gmelin, O. 1859. Uber die Constituion des Solanins. Liebigs Ann.
Chem. 110:167.

Griebel, C. 1923. Solanin reiche gesundheits schadiliche Kartoffeln.
.Z. Unters. Nahr. Genussm. 45:175.

Griebel, C. 1924. Zum Solaningehalt der Kartoffeln 1922 -er Ernte.
-Z. Unters. Nahr. Genussm. 47:436.

Gull, D. D., and F. M. Isenberg. 1958. Light burn and off-flavor
development in potato tubers exposed to fluorescent lights.
Proc. Am. Soc. Hort. Sci. 71:446.

Gull, D. D., and F.-M. Isenberg. 1960. Chlorophyll and solanine
content and distribution in four varieties of potato tubers.
Proc. Am- Soc.-Hort. Sci. 75:545.

Guseva, A. R., and V. A. Paseschnichenko. 1957. Enzymic split-
ting of potato glycoalkaloids. Biokhimiya 22:843.

Guseva, A. R., and V. A. Paseschnichenko. 1958. Potato glyco-
alkaloid biogenesis study with the aid of labelled atoms.
Biokhimiya 23:412. .

Guseva, A. R., V. A. Paseschnichenko andM. G. Borikhina.
1960. Mevalonic acid as precursor of some polyisoprene
compounds. in plant. Dokl. Akad. Nauk. S.S.S.R. 133:228.

Hansen, A.. A. 1925. Two fatal cases of potato poisoning. Science
61:340.

Harris, F. W., and T. Cockburn. 1918. Alleged poisoning by
potatoes. .Analyst 43:133.

Harris, F. W., and F. Cockburn. 1918. Alleged poisoning by
potatoes. Am. J. Pharm. 90:722.

96

Johnson, D. F., -R. D. Bennett and E. Heftman. 1963. Cholesterol
in higher plants. Science 140:198.

Kingsbury, J.' M. 1964. Poisonous plants of the U. S. and Canada.
Prentice —Hall, Englewood Cliffs, New Jersey.

 

Kline, E... H. Elbe, N. Dahle and S. Kupchan. 1961. Toxic effects
of potato sprouts and solanine fed to pregnant rats. Proc.
Soc. Exptl. Biol. Med. 107:807.

Kdnig, H. , and A. Staffe. 1953. Action of solanine upon blood
picture and blood catalase of sheep. Chem. Abstr. 47:7671.

Kuhn, R. , and I. L6w. 1954. Die Konstitution des Solanins.
Angewandte Chemie 66:639.

Kuhn, R., I. Léiw and H. Trischmann. 1955. Die Konstitution des
O(-Chaconine. Chemische Berichte 88:1690.

Kuhn, R. , and I. Law. 1955. Die Alkaloidglykoside der Blatter von
Solanum aviculare. Chemische Berichte 88:289.

Lampitt, L. H., J. H. Bushill, H. S. Rooke and E- M. Jackson.
1943. Solanine, glycoside of the potato. 11. Its distribu-
tion in the potato plant. J. Soc. Chem. Ind. 62:48.

Larsen, E. C. 1949. Investigation on the cause and prevention of
greening of potato tubers. Idaho Univ. Agr. Expt. Sta.
Bull. 16.

Lepage, M. 1964. Isolation and characterization of an esterified
form of steryl glucoside. J. Lipid Res. 5:587.

Liljemark, A. 1960. Greening and solanine development of white
potato in fluorescent light. Am. Potato J. 37:379.

Lowe, H. 1929. Poisoning by bittersweet (Solanum dulcamara).
Analyst 54:153.

Lowry, R. R. 1968. Ferric chloride spray detector for cholesterol
and cholesterol esters on TLC. J. Lipid Res. 9:397.

97

. Meyer, G. 1895. ‘ Uber Vergiftungen durch Kartoffeln. I. - Uber den
Gehalt der Kartoffeln an Solanin and Uber die Bildung
desselben wahrend der Keimung. Arch. Exp. Path. Pharma.
36:361.

Morgenstern, F. Von. 1907. Uber‘den Solaningehalt der Speise-
und Futter -Kartoffeln und fiber den Einfluss derBodenkultur
auf die Bildung vonSolanin in der Kartoffelpflanze. Land-
wirschaftlichen Versuchs -Station 65:301.

Moskaleva, V. E., and O. V. Pogorelova. 1969. Localization of
solanine in Solanum tuberosum sprouts. Rast. Resur.
5(1):31.

Orgell, W..H., and E. T.- Hibbs. 1963. Cholinesterase inhibition
in vitro by potato foliage extracts. Am. Potato J. 40:403.

Orgell, W., H. , and E. T. Hibbs. 1963. Human plasma Cholinesterase
inhibition in vitro by extracts from tuber -bearing Solanum
species. »Proc. Am. Soc- Hort. Sci. 83:651. 8

Osske, O. , and K. Schreiber. 1965. Sterine und Triterpenoide VI.
24-methylene lophenol, ein neues 4(X-methyl Sterin aus
Saccharum officinarum L. und Solanum tuberosum L.
Tetrahedron 21:1559.

Paquin, R. , and M. Lepage. 1963. Separation de la solanine, de la
chaconine et de la solanidine par chromatographic sur
couches minces. J. Chromatography 12:57.

Paseschnichenko, V. A. 1957. Solanine and chaconine content of the
potato during the growth period. Biokhimiya 22:981.

Paseschnichenko, V. A., and A. R. Guseva. 1956. The quantitative
determination of the glycoalkaloids of the potato and method
for their separation. Biokhimiya 21:585.

Patt, P. , and W. Winkler. 1960. ~ Zur Darstellung und Bestimmung
von Solanin durch Ionenaustausch. Arch. Pharm. 293:846.

Pfankuch, E. 1937. Diephotometrische Bestirnmung von Solanin.
Biochem. Z. 295:44.

98

Pierzchalski, T., and A. Mrozowska. 1968. -Polarographic
determination of glycoalkaloids. Chem. Anal. (Warsaw)
13(2):367.

Pope, L. R. 1969. Processing characteristics of selected potato
clones. , M. S. Thesis. > Michigan State University.

Prelog, V. , and S.‘ Szpilfogel. "1942. Uber Steroideand Sexual
Hormone: Uber das 2 -Athyl —5 -methyl pyridine, ein -
Dehydrierungs Produkt des Solanidins. .Helv. Chim. Acta ;
2521306. |

Prelog, V. , and O. Jeger. 1960. The solarium group in The
Alkaloids--Chemistry and physiology VII. P. 343.
ed. Manske R- H. F. N.Y.

 

Rees, H. H., L. J. Good and T. W. Goodwin. 1968. Isolation of
a new sterol from potato leaves. - Phytochem. 7(10):1875.

Reuling, Von L. G. W. 1839. Uber Solanin. Liebigs Ann. Chem.
30:225. -

Rooke, H. S., J. H. Bushill, L..H. Lampitt and E. M. Jackson.
1943. Solanine, glycoside of the potato. 1. Its isolation
and determination. J. Soc. Chem. Ind. 62:20.

Rozanski, A. 1966. Gas chromatographic determination of
campesterol, fi-sitosterol and stigmasterol. Anal. Chem.
38(1):36.

Riihl, R. 1951. Pathology and toxicology of solanine. Arch.
wPharm. 284:67.

Sachse, J ., and F. Bachmann. 1969. Uber die Alkaloidbestimmung
in Solanum tuberosum L. Z. Lebensm. -Unters. und—
Forchung. 141:262.

Schoene, D. L. , and O.. Hoffman. 1949. - Maleic hydrazide, a unique
growthregulant. Science 109:588.

Schreiber, K. 1957. Solanum alkaloids: V. Isolation of A5-
tomatodin -3 -fl -ol and yamogenin from potatoes. Angew.
Chem. 69:483.

99

Schreiber, K., G. Osske and G. Sembdner. 1961. Identifizierung
von -Sitosterin alsHauptsterin des Kartoffelkéifers.
Experientia Basal 17 :463 .

Schreiber, K., and G. Osske. 1962. Sterine und Triterpenoide.
II. lsolierung von Cycloartenol aus Blattern der‘Kultur-
kartoffel S. tuberosum L. Kulturpflanze 10:372.

Schreiber, K. , and G. Osske. 1963. Sterine und Triterpenoide.
III. lsolierung von 40(-methyl -5 CX-stigma -7 -24(2 8) -dien-
3 -ol aus Solanum tuberosum sowie iiber die Identitat dieser
Verbindung mit (XI -Sitosterin. Experientia, Basel 19:69.

Schreiber, K3, and G. Osske. 1964. Sterine und Triterpenoide.
V. Uber die 4a-Methyl —Sterine der-Kartoffelpflanze
Solanum L. Tetrahedron, London 20:2575.

Schreiber, K. , and H. Rbnsch. 1965a. Solanum alkaloide -LIV.
Syntheses von solanidin und 22 -iso —solanidin aus Tomatid-
5 -en -3B-ol. Tetrahedron 21:645.

Schreiber, K. , and H. Rénsch. 1965b. Solanum alkaloid -XLV.
Syntheses von Tomatid -5 -en —3/8-ol und Tomatida -3, 5 -diene.
Liebigs Ann. Chem. 681:196.

Schwarz, J. J., and M- E. Wall. 1955. Isolation of the sterols
of the white potato. J. Am. Chem. Soc. 77:5442.

Schwarze, P. 1962. - Methoden-zum Solaninnachweis und zur
Solaninbestimmung in Kartoffelzucht Material. Der
Ziichter 32:155.

Schépf, C., and R.. Hermann. 1933. Solanidine. Ber. Deut.
Chem. 66B:298.

Simek, J. 1962. Following up the solanine alkaloid content in
potato tuber. Prumysl- Potravin. 13:380.

Smerha, J. , and S. Ceskoslow. 1948. Experiments on the detoxi—
cation of the solanine sprouting potato tubers. Chem.
Abstr. 43:8538.

100

Soltys, A. , and K. Wallenfels. 1936. Solanine and solanidine.
Ber. Deut. Chem. 69B:811.

Stanko, S. A. 1962. Effect of light on the formation and composi-
tion of anthocyanins in potato tubers during vernalization.
Dokl. Akad. Nauk. S.S.S.R. 1462480.

Street, H. E., A. E. Kenyon and G. M. Watson. 1946. The nature
and distribution of various forms of N in the potato. Ann.
Appl. Biol. 33:1.

Sturckow, B. , and I. L5w. 1961. The effect of some solanaceous
alkaloidal glycosides on the potato beetle. Entomol. EXpl.
et Appl. 4:133.

Szendey, G. L. -1957. Paper chromatography separation and
identification of the alkaloidal glycosides from Solanum
aviculare. Arch. Pharm. 290:563.

Talburt, F. W., and O. Smith. 1967. Potato processing. The
AVI- Publishing Company, Inc. , West Port, Connecticut.

 

Thompson, N. R. 1969. Personal communication.

Turapin, M. L. 1951. Solanine in potatoes. Chem. Abstr. 45:
10429h.

Uhle, F. C., and W.VA. Jacobs. 1945. XXIV. The octahydropyrro-
coline ring system of the tertiary basis: conversion of
sarsasapogenin to a solanidine derivative. J. Biol. Chem.
160:243.

Vecher, A. S. 1967. Solanine content in the most important and
new potato varieties in the BSSR. Beloruss S. S.R. 11(4):
362.

Wackenroder, H. 1843. Uber die Darstellung und fiber einige
Eigenschéften des Solanins. Arch. Pharm. 33:59.

Wells, W. W. ,and M. Makita. 1962. The quantitative analysis of
fecal neutral sterols by gas —liquid chromatography. Anal.
Biochem. 4(3): 204.

101

Wierzchowski, P., and Z. Wierzchowska. 1961. Colorimetric
determination of solanidine and solanine with SbCl3.
Chem. Anal. 6:579.

Wolf, M. J., and B. D. Duggar. 1946. Estimation and physiological
role of solanine in the potato. J. Agri. Res. 73:1.

Zitnak, A. 1961. The occurrence and distribution of free alkaloid
solanidine in netted Gem potatoes. Can. J. Biochem. and
Physiol. 39:1257.

Zucker, M. 1963. The influence of light on synthesis of protein and
of chlorogenicacid in potato tuber tissue. Plant Physiol.
38:575.

Zwenger, C. 1859. Uber Solanin. Ann. Chem. Pharm. 109:244.

Zwenger, C. , and A. Kind. 1861. Uber das Solanin und dessen
Spaltungsprodukte. Liebigs Ann. Chem. 118:129.