Front Cover
 Table of Contents
 Review of literature
 Characteristics of hesperidin
 Quantity in fruit
 Hesperidin content of whole...
 Hesperidin content of juice
 Recovery from oranges
 Effect of cultivar upon hesperidin...
 Effect of recycling hesperidin...
 Maturity vs. recovery of hespe...
 Maturity vs. purity of extracted...
 Recovery of hesperidin filtrate...
 Commercial recovery
 Purification procedures
 General procedure
 Analytical methods
 Chemicla procedures
 Literature cited
 Back Cover

Group Title: Bulletin - University of Florida. Agricultural Experiment Station - no. 684
Title: Hesperidin in Florida oranges
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00027501/00001
 Material Information
Title: Hesperidin in Florida oranges
Series Title: Bulletin University of Florida. Agricultural Experiment Station
Physical Description: 42 p. : ill., charts ; 23 cm.
Language: English
Creator: Hendrickson, Rudolph
Kesterson, J. W
Publisher: University of Florida Agricultural Experiment Station
Place of Publication: Gainesville Fla
Publication Date: 1964
Subject: Hesperidin   ( lcsh )
Oranges -- Composition -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
Bibliography: Bibliography: p. 36-42.
Statement of Responsibility: R. Hendrickson and J.T. Kesterson.
General Note: Cover title.
Funding: Bulletin (University of Florida. Agricultural Experiment Station) ;
 Record Information
Bibliographic ID: UF00027501
Volume ID: VID00001
Source Institution: Marston Science Library, George A. Smathers Libraries, University of Florida
Holding Location: Florida Agricultural Experiment Station, Florida Cooperative Extension Service, Florida Department of Agriculture and Consumer Services, and the Engineering and Industrial Experiment Station; Institute for Food and Agricultural Services (IFAS), University of Florida
Rights Management: All rights reserved, Board of Trustees of the University of Florida
Resource Identifier: aleph - 000929268
oclc - 18354600
notis - AEP0044

Table of Contents
    Front Cover
        Page 1
    Table of Contents
        Page 2
        Page 3
    Review of literature
        Page 4
        Page 5
    Characteristics of hesperidin
        Page 6 (MULTIPLE)
        Page 7
        Page 8
        Page 9
    Quantity in fruit
        Page 10 (MULTIPLE)
    Hesperidin content of whole oranges
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
    Hesperidin content of juice
        Page 17 (MULTIPLE)
        Page 18
    Recovery from oranges
        Page 19 (MULTIPLE)
    Effect of cultivar upon hesperidin yield
        Page 20
    Effect of recycling hesperidin extracting liquor
        Page 21 (MULTIPLE)
    Maturity vs. recovery of hesperidin
        Page 22
    Maturity vs. purity of extracted hespridin
        Page 23 (MULTIPLE)
    Recovery of hesperidin filtrate losses
        Page 24
    Commercial recovery
        Page 25
    Purification procedures
        Page 26
    General procedure
        Page 27 (MULTIPLE)
    Analytical methods
        Page 28 (MULTIPLE)
        Page 29
    Chemicla procedures
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
    Literature cited
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
    Back Cover
        Page 43
Full Text


The publications in this collection do
not reflect current scientific knowledge
or recommendations. These texts
represent the historic publishing
record of the Institute for Food and
Agricultural Sciences and should be
used only to trace the historic work of
the Institute and its staff. Current IFAS
research may be found on the
Electronic Data Information Source

site maintained by the Florida
Cooperative Extension Service.

Copyright 2005, Board of Trustees, University
of Florida



U~~ -i S -S --

~Pl'.- I




INTRODUCTION ------------ -.............-.-.. --- ---

REVIEW OF LITERATURE ---...--.. --------...... ......--

CHARACTERISTICS OF HESPERIDIN ----...........-...- -- -----

Physical ---...................... .------------------------

Chemical ........ .-------. .........------.-------

Medical ..........-....------------------

QUANTITY IN FRUIT ..............----------------

Method of Analysis ----- --- ---..---

Hesperidin Content of Whole Oranges ......------

Hesperidin Content of Juice ........-------------....

Distribution --...............~......... ....---------------.

RECOVERY FROM ORANGES .. .-.._.... ...........----------

Recovery Procedure ---..............---..-----....

Effect of Cultivar upon Hesperidin Yield --........

Effect of Particle Size upon Yield -------..........

Effect of Recycling Hesperidin Extracting Liquor

Effect of Extracting pH -......-...............---

Maturity vs. Recovery of Hesperidin ------........

Maturity vs. Purity of Extracted Hesperidin --..

Maturity vs. Recoverable Hesperidin per Acre --..

Recovery of Hesperidin Filtrate Losses --..-......-

Commercial Recovery -......- ...... ------------

PURIFICATION PROCEDURES ..-....------ --------.............

General Procedure -----------------...................

Effect of Alcohol on Filtration Rate ........---....

Effect of Alcohol upon Hesperidin Recovery ...---

Effect of pH upon Hesperidin Recovery --...-.......-


Bioassay ...--------------....-----

Chemical Procedures --------

SUMMARY .....------ ------ -----

LITERATURE CITED ---........ ---...------------

------- 3

--. --.......-.......- 4

...--..-........------ 6

----..---......-.... 6
..-...........- 6


..--... ..--------... 10

.........----- .- --. 10

.--..------------- 11

.--.. ...--- .---.-- 17

-..........-- ---- 17

---........---_- 1. 19

-..-..... .------- 19

....... -- ----.. .. 20

--.....--... --. .-- 20

-............---- 21

..........-------- 21

..--..-.... ..-- -- 22

-.-........------- 23

............-..-- 23

........---..... 24

-----........-.. 25

--............---. 26

------.- 27

------ 27

----- 27

.-----------------... .. 28

------ 28

------ 29

..............-- 30

----.....----. 35

.----- -- -...-. 36

A contribution from the Citrus Experiment Station.
Fig. 1 (cover).-Hesperidin crystals magnified 120 X.

Hesperidin in Florida Oranges

R. Hendrickson and J. W. Kesterson1

Hesperidin, the principal flavonoid or glucoside of oranges,
is one of at least 137 natural flavonoids that are known to occur
in at least 62 families, 153 genera, and 277 species of plants
(111)2. Five plant families in which hesperidin specifically has
been found are: Umbelliferae, Rutaceae, Labiatae, Lobeliaceae,
and Compositae (45). Hesperidin not only occurs in the sweet
orange (Citrus sinensis), but also in C. aurantium, C. limon, C.
medical, C. reticulata, and quite possibly in other Citrus species
(59). It also is found in the citrus blossom (30) and in the
leaves, twigs, and bark (45).
Although hesperidin is the most often recognized flavonoid
in sweet oranges there are others present also, but in consider-
ably smaller quantity. For example, three related flavones have
been found: 5,6,7,3',4'-pentamethoxyflavone or sinensetin (14),
5,6,7,8,3',4'-hexamethoxyflavone or nobiletin (100), and 3,5,6,7,-
8,3', 4'-heptamethoxyflavone (59). More recently (101) another
flavone called tangeritin, which is usually associated with C.
reticulata, was found in sweet orange along with the previously
mentioned flavones. Dunlap and Wender (28) claim to have iso-
lated from the peel of California 'Valencia' oranges small quan-
tities of the 7-rhamnoglucoside of isosakuranetin (sometimes
called poncirin) (59), and naringin, the major glucoside of
grapefruit. Eriodictyol glucoside was reported also as being
present in sweet orange (107), with certain earlier workers sug-
gesting that it was present or formed by demethylation of hes-
peridin on ripening of the fruit (17, 102). Neither eriodictyol
nor its glycoside was found, however, in a more recent, exacting
investigation of mature 'Valencia' orange peel (56).
Hesperidin belongs to that smaller class of flavonoids that
have shown physiological and biochemical activity; recognition
of this class has increased considerably in recent years. The

1Associate Chemist and Chemist, Citrus Experiment Station, Lake
Alfred, Florida.
2 Numbers in parentheses refer to Literature Cited.

Florida Agricultural Experiment Stations

function of flavonoids in citrus and other higher plants has not
been established, but it is known that glucosides such as hesperi-
din are synthesized from carbohydrates, the reaction being
favored by low temperature, light, and access to oxygen (88).
Hall (32) has postulated that glucosides, such as hesperidin,
serve as a medium for the translocation of glucose synthesized
in the chlorophyllous tissue. He thought that hesperidin could
combine with glucose to form a soluble easily hydrolyzable com-
pound, that could be held inactive temporarily and metabolically
until brought to that portion of the plant where it would be
stored or utilized. It has been suggested also that flavonoid com-
pounds are the precursors or intermediary metabolites in the
formation of plant pigments such as the anthocyanidins (29).
Possibly their purpose is concerned with regulating photosyn-
thesis (89) or acting as component chemicals or oxidation-
reduction systems (23). Webber and Batchelor (107) suggest
that specific glucosides have taxonomic significance in disting-
uishing Citrus species that have superficial resemblance.
The authors have written this bulletin so that proper at-
tention, recognition, and acquaintance might be given to hesperi-
din as one of the more valuable constituents of sweet orange.
Specific attention has been given to the properties, recovery,
purification, and chemical analysis of hesperidin. Furthermore,
attention has been given to the relationship between hesperidin
and the medicinal properties of bioflavonoids, a term now used
for the vitamin P complex. The latter terminology is found at
times in this bulletin for lack of an adequate substitute.

It was in 1828 that Lebreton (66) first noticed a crystalline
substance when he filtered a macerated tincture of oranges. This
crystalline material was found in members of the family (older
classification system) Hesperides; thus it was given the name
hesperidin. It was proved that this compound was a natural
constituent of oranges, but it was not until 1876 that Hilger
(50) and Hoffman (51) recognized the material as a glucoside.
Hesperidin was found in lemons by Pheffer (82) as early as 1874,
was recognized in citrons by Penzig 1887, and since then has
been acknowledged in numerous other plants. The aglycone,
hesperitin, which is obtained by hydrolyzing hesperidin with
dilute mineral. acid (4),was synthesized by Shinoda and Ka-

Hesperidin in Florida Oranges 5

wagoye (92) in 1928, even though its structure had been estab-
lished much earlier by Tutin (105), who made a comparative
study of hesperitin, eriodictyol, and homoeriodictyol. King and
Robertson (63) determined the position of the sugar molecule
on hesperidin, and it was synthesized finally in 1943 by Zemplen
and Bognar (116). The exact configuration of the sugar portion
of this molecule was more correctly represented as recently as
1959. (31).
In studying the hesperidin content of 'Washington navel'
orange peel, Harvey and Rygg (36) reported the albedo and
stylar end to have higher percentages of hesperidin than the
flavedo and stem end of the fruit. They showed also that the
percentage of hesperidin in the dried peel changed from 3.6
percent in November to 2.0 percent in April. In fresh fruit
storage, however, percentage of hesperidin increased almost 50
percent within 46 days, with the larger increase occurring at
the higher storage temperature. Davis (25) analyzed portions
of freshly harvested mature 'Valencia' oranges and found 0.12
percent hesperidin in the juice, 1.6 percent in the albedo, 1.5
percent in the carpellary membrane, 1.0 percent in the flavedo,
and 4.5 percent in the central vascular tissue. Five seasons of
analyses by Atkins et al. (6) of Florida commercial frozen
orange concentrate on a reconstituted basis indicated the hes-
peridin content to be from 60 to 99 mg per 100 ml for most
samples by the Davis method (25). They observed also that
midseason samples and freeze-damaged fruit samples had hes-
peridin values that averaged higher.
There have been a large number of patents granted on the
recovery, purification, and preparation of useful derivatives of
hesperidin. Higby was granted three patents (47, 48, 49) on the
recovery of hesperidin and flavanone glucosides from citrus peel.
Baier (9) and Burdick et al. (18) similarly have described pro-
cedures for this purpose, while Sokoloff (97) and Sokoloff and
Redd (96) claimed a method for extracting hesperidin-like ma-
terial from citrus molasses. Patents were given to Lautenschla-
ger et al. (65) and Wender et al. (108) for their techniques
of purifying flavonoid compounds. The conversion of hesperidin
to a carboxylate derivative was patented by Ohta (77), to be
an alkylated chalcone by Wilson (113), to a water soluble
alkoxyl substituted chalcone glycoside by Wilson (114), to hes-
peridin methylene carboxy chalcone by Hart (35), to hesperitin

Florida Agricultural Experiment Stations

and a soluble complex by Wilson (115), to a modified rhamnoglu-
coside by Wender et al. (109), to an azo dyestuff by the authors
(41), and to azo dye wood stains by Toulmin (103, 104).

Notable for its fine crystalline form, hesperidin crystallizes
as colorless needles in a beautiful rosette pattern from dilute
acetic acid (see cover). Occasionally it is found as evaporator
scale in the manufacture of frozen orange concentrate (37). It
is insoluble in common solvents, such as benzene, acetone, carbon
tetrachloride, carbon disulfiide, isopropyl alcohol, and water;
sparingly soluble in methanol, ethanol, and glacial acetic acid;
more soluble in trimethyl cyclohexanol, ethyl examine, and iso-
phorone; and freely soluble in dilute alkalies, formamide, and
Hesperidin in solution is tasteless, possessing no bitterness
(60), and in this property is quite different from other flavanone
glycosides such as poncirin of trifoliate oranges, naringin of
grapefruit, and the closely allied isomeric glycoside neohesperi-
din from sour oranges. According to Horowitz and Gentilli (60)
the point of attachment of rhamnose to glucose in the structure
of these compounds is the factor that determines bitterness or
The highest recorded melting point for hesperidin is 261 to
263 C (83), while synthetic hesperidin has been produced with
a melting point of only 256 to 258" C (116). This glycoside is
further characterized by its specific rotation, [a] L-75.0 in
pyridine (116).
The physical location of hesperidin in an orange can be dem-
onstrated by the addition of a ferric chloride solution to the cut
surface (Fig. 2). The usual wine-red color that develops in
hesperidin solutions of great dilution is almost black at higher
Hesperidin, hesperitin-7-rutinoside, hesperitin-1-rhamnosido-
d-glucoside, and C28H34015 are various ways of referring to the
same compound of molecular weight 610.55. It is not to be con-
fused with the isomeric neohesperidin found in sour orange (C.
aurantium), trifoliate orange (Poncirus trifoliata), and the Pon-

Hesperidin in Florida Oranges

Fig. 2.-Demonstration of the location of hesperidin in an orange by
addition of ferric chloride to the cut surface.

derosa lemon (C. limon) (55). Asahina and Inubose (5) greatly
aided establishment of hesperidin's molecular weight by showing
that it can be hydrolyzed with dilute mineral acids to one mole
each of hesperitin, glucose, and rhamnose. Hesperitin can be
decomposed further to phloroglucinol and hesperitic acid.
According to Wawra and Webb (106) there exists in the
fruit an equilibrium between hesperidin and its chalcone, which
is the naturally-occurring compound. This equilibrium can be
shifted by changing the pH of the medium with the chalcone
form favored in an alkaline medium. Shimokoriyama (91) sug-
gests that in nature an enzyme may be involved in the chalcone-
flavanone isomerism. When crystallized the chalcone forms
bright yellow crystals melting from 243 to 257 C, while the

Florida Agricultural Experiment Stations

colorless hesperidin crystals melt at 261 to 262* C. The struc-
tural formulas for hesperidin and its chalcone follow:

CH3--CH2 o H

(Rhamnose) (Hesperitin)


Many of the colored reactions that hesperidin undergoes
will be described later under Analytical Methods.

In 1936 Rusznyak and Szent-Gyorgyi (86) postulated the ex-
istence of a new vitamin called "citrin" or vitamin P ("P" for
permeability), that appeared to improve capillary fragility.
Their early work indicated that pure ascorbic acid was ineffective
in certain pathological conditions, characterized by increased
fragility of the capillary wall as in the case of purpura, and that
this condition would readily be cured by the administration of
lemon juice or paprika extracts (2). In 1938 Szent-Gyorgyi
(102) described a procedure for extracting "citrin" from Italian
lemons. The active substance was found to be a mixture of the
flavanone glucosides, hesperidin and eriodictin. It was shown
that this vitamin-like substance was present in citrus fruits
such as lemon and orange. Earlier investigators sometimes
failed to confirm (26, 117) these findings, and some, Higby (46),
claimed crude extracts containing hesperidin to be active and
the purified hesperidin to be inactive. But, later studies (8, 15)
demonstrated that hesperidin and its derivatives do affect capil-
lary fragility and permeability.
The chemical and physical properties of vitamin P complex
are still to be well delineated, since the most active preparations
are known only in the form of concentrates. There has been,
however, considerable attention given to plant distribution of
the vitamin P complex, to the chemistry of certain components,
and to the physiological effects of the complex (84, 88, 90, 93).

Hesperidin in Florida Oranges

Vitamin P substances are widely distributed among plants, with
citrus fruits, rose hips, and black currants considered as being
the best sources (7). The minimum protective dose of vitamin
P is thought to be obtained by consuming as little as 50 to 100
ml of orange juice daily (87). Sokoloff and Redd (95) came to
a similar conclusion about frozen orange concentrate, but empha-
sized that only a very ripe orange contained the maximum
amount and the most active form of vitamin P.
The diagnostic signs and symptoms of vitamin P deficiency
include pain in the legs on exertion, pain across the shoulders,
weakness, lassitude, and fatigue. This deficiency invariably is
associated with much decreased capillary permeability and may
be characterized by the development of spontaneous petechiae
in areas subjected to pressure (88).
In reviewing the diseased states that have been influenced
favorably by therapeutic uses of the vitamin P complex, a very
extensive list can be compiled, such as Table 1. Recent investi-
gators, such as Horoschak (52), have claimed that vitamin P
requires the presence of vitamin C for its activity, and in the

TABLE 1.-A List of Diseased States Responding Directly or
Indirectly to Vitamin P Therapy.

Allergic purpura (76)
Angioneurotic edema (76)
Athletic contact injuries (34)
Beriberi (45, 52)
Bronchial asthma (52)
Bursitis (78)
Carcinoma (52)
Chronic nephritis (70)
Colds (27, 80)
Dermatitis (45)
Diabetes (52, 76)
Diabetic retinopathy (52, 98)
Eczema (52)
Erythema (45)
Essential hypertension (76, 94)
Follicular tonsillitis (11)
Glaucoma (94)
Glomerulonephritis (45)
Graves' disease (45)
Habitual abortion (24, 52)
Haemoptysis (70)
Hematochromatoses (70)
Hematuria (52, 94)
Hemophilia (89)
Hemorrhagic diatheses (70, 94)
Hemorrhagic nephritis (70, 94)
Unpublished reports.

Hemorrhagic telangiectasis (76, 94)
Influenza (11, 12)
Leprosy (52)
Measles *
Ophthalmology (52)
Osteoarthritis (79)
Petechial hemorrhages (70)
Pleurisy (45, 52)
Polio (13)
Polyarthritis (70)
Psoriasis (76, 94)
Radiation hemorrhage (76)
Retinopathies (69)
Rheumatic fever (52, 94)
Rheumatoid arthritis (52, 94)
Rhinitis (11, 12)
Schoenlein-Henoch purpura (45, 70)
Stroke, little (75)
Syphilis (45)
Thrombocytopenic purpura (52)
Thymus involution (73)
Tuberculosis (45, 52)
Ulcerative colitis (70)
Ulcerative lesions (20)
Upper respiratory infection (11)
Vascular purpura (76, 94)

Florida Agricultural Experiment Stations

complete absence of it, vitamin P is inactive. Several mecha-
nisms for the physiological activity of vitamin P compounds
have been suggested by Beiler (10). Martin (72) has con-
tended that the outstanding success of these compounds in the
treatment of numerous medical conditions suggests that there
is no disease state which will not benefit by assuring proper
capillary strength.
Despite the apparent success of these compounds, such as
hesperidin and extracts that relate to vitamin P, Scarborough
and Bacharach (88) pointed out that dosage in some tests sug-
gested a pharmacological action rather than that of a vitamin.
In 1950 (22) a joint committee of the American Society of
Biological Chemists and the American Institute of Nutrition
finally recommended that the term vitamin P be discontinued.
Martin and Beiler (71) found phosphorylated hesperidin to
be a moderately effect antifertility agent. The mechanism
was dependent upon effective inhibition of the activity of hyalur-
onidase, which promoted sperm penetration.

An investigation of the hesperidin content of oranges was
described in 1954 (38) that compared fruits of several cultivars,
parts of fruit, and maturity changes. The specific cultivars
tested for hesperidin included: 'Hamlin,' 'Parson Brown,' 'Pine-
apple,' and 'Valencia.' Each sample was comprised of 16 fruits
and made up of fruits picked at random from 16 positions on the
tree. The trees used for this study were located on the premises
of the Florida Citrus Experiment Station at Lake Alfred, re-
ceived uniform fertilizer and spray practices, and were all on
rough lemon rootstock.

Method of Analysis
The samples were analyzed on a whole-fruit basis or sepa-
rated into the component parts, depending on whether the fruits
were mature enough to give a moderate juice yield. Sample
preparation has been described previously by the authors (38)
and in brief consisted of the following steps. A sample of 16
fruits, or portions of each fruit, was coarsely ground. Then a
100-gram aliquot of the coarsely ground sample was placed in
95 percent ethanol in a Waring blendor and was ground further.
The comminuted sample was extracted three times: first with

Hesperidin in Florida Oranges

ethanol, next with an aqueous calcium hydroxide solution, and
finally with hot water. Between each extraction, residue and
extract were separated by filtering the extract through cheese-
cloth and pressing the final portion through with the help of a
hydraulic press. The three extracts were combined later, and a
new aliquot was taken for analytical purposes.
When other than whole fruit was analyzed, the procedure
was to separate it into albedo, flavedo, juice, seeds, rag, and
pulp. The colored flavedo was first separated from the orange
by means of a potato peeler, after which the orange was halved
and juiced with a hand juice press that folded and squeezed a
half fruit. The seeds were manually separated and the rag and
pulp pulled apart from the albedo. Each of the separated por-
tions was weighed, comminuted, and then analyzed by the pro-
cedure used for whole fruit. The juice was strained and analyzed
The extracts were analyzed for hesperidin by the Davis
method (25) with the following modifications. One-half ml of
extract was added to 24 ml of 90 percent diethylene glycol, and
the increase in color caused by adding 0.5 ml of approximately
4 N sodium hydroxide was read after 30 minutes. Comparison
was made against a standard curve (see Fig. 18) using a Fisher
electrophotometer with a 425 mx filter. All readings were made
at approximately 25" C.

Hesperidin Content of Whole Oranges
The analytical results of measuring fruit diameter, weight,
Brix, acid, and hesperidin content of the whole fruit and its
components for the four cultivars of orange are recorded in
Tables 2 through 5. It was observed in these data that the hes-
peridin content of the developing oranges did not share the
normal growth pattern expected for fresh fruit weight. The
total quantity of hesperidin per fruit increased only in those
months prior to September or alternatively only until fruit
diameter reached approximately 1.8 to 2.0 inches. Beyond this
size there was mostly a dilution of that which had accumulated,
similar to that which had been found for naringin in grapefruit
(62). The net effect of this upon the percentage hesperidin
in 'Pineapple' oranges throughout a season is plotted in Fig. 3,
and was similar to the results obtained with the other three
orange cultivars studied. The very small fruit had a noticeably

TABLE 2.-Monthly Variation in Whole Fruit, Juice and Glucoside Content of
Hamlin Orange.*

Whole Fruit Juice Distribution of Gluocoside-%
Wet Dry Gluceside Glucoside
Diameter Weight Weight Content Brix Acid Content Juice Albedo Flavedo Rag
Date Inches Gms. Gms. Gms. % % by Vol. and Pulp













0.49 1.2

1.5 26

1.8 51

2.2 93

2.5 132

2.6 158

2.8 188

2.8 196

2.9 202

3.0 236

3.0 219

2.9 206

39.7 19.6 39.0

37.1 21.9 38.1

38.5 19.4 39.0

41.1 20.2 35.7

43.8 17.5 35.8

45.9 17.9 33.2

48.3 15.7 33.0

45.0 15.0 36.3




































* Average values for 16 fruit.


TABLE 3.-Monthly Variation in Whole Fruit, Juice and Glucoside Content of
Parson Brown Orange.*

Whole Fruit Juice Distribution of Gluocoside-%o
Wet Dry Glucoside Glucoside
Diameter Weight Weight Content Brix Acid Content Juice Albedo Flavedo Rag
Date Inches Gms. Gms. Gms. 0% % by Vol. and Pulp

4-18-52 0.50 1.3 0.36 0.1

6-1-51 1.5 26 7 0.8

7-1-51 1.9 58 12 1.0

8-1-51 2.4 101 18 1.2

9-1-51 2.6 142 21 0.8 8.3 1.9 0.025 1.6 39.0 17.5 41.9

10-1-51 2.8 186 26 0.9 7.9 1.2 0.027 2.5 44.2 17.9 35.4

11-1-51 2.9 214 31 0.9 8.8 0.8 0.027 2.9 42.0 16.4 38.7

12-1-51 3.0 215 35 1.0 10.4 0.7 0.025 2.4 39.7 20.9 37.0

1-1-52 3.0 252 40 1.1 10.0 0.6 0.028 2.8 49.6 13.2 34.4

2-1-52 3.1 252 42 1.1 11.0 0.5 0.031 2.8 47.0 15.6 34.6

3-1-52 3.1 243 41 1.0 11.2 0.5 0.031 2.7 50.2 12.2 34.9

4-1-52 3.2 260 42 1.2 11.0 0.4 0.058 4.2 50.0 12.7 33.1

Average values for 16 fruit.

TABLE 4.-Monthly Variation in Whole Fruit, Juice and Glucoside Content of
Pineapple Orange.*

Whole Fruit Juice
Wet Dry Glucoside Glucoside
Diameter Weight Weight Content Brix Acid Content
Date Inches Gins. Gs. Gms. % % by Vol.

0.54 1.5

1.4 22

2.1 66

2.4 104

2.6 144

2.8 186

3.1 245

3.2 263

3.2 270

3.3 288

3.2 275

3.1 255

























Distribution of Gluocoside-%

Juice Albedo Flavedo Rag
and Pulp

--- ---- .

1.5 35.4 20.5 42.6

2.4 39.8 18.3 39.5

2.4 38.8 18.4 40.4

2.1 35.7 20.8 41.4

3.2 42.4 18.1 36.3

3.2 41.8 19.0 36.0

3.2 38.0 19.8 39.0 o

3.1 47.3 16.8 32.8









* Average values for 16 fruit.

TABLE 5.-Monthly Variation in Whole Fruit, Juice and Glucoside Content of
Valencia Orange.*

Whole Fruit Juice Distribution of Gluocoside-%
Wet Dry Glucoside Glucoside
Diameter Weight Weight Content Brix Acid Content Juice Albedo Flavedo Rag
Date Inches Gms. Gms. Gms. % % by Vol. I and Pulp

0.50 1.2

1.4 23

1.9 51

2.1 77

2.5 124

2.7 145

2.9 189

3.1 231

2.9 218

3.1 254

3.0 240

3.1 250

3.0 223





































.0 39.7

8.2 49.7

2.3 42.3

8.4 41.5

9.1 48.5

8.1 47.5

8.6 48.4

2.5 44.2










* Average values for 16 fruit.

16 Florida Agricultural Experiment Stations

high hesperidin content, and a remarkably higher value when
considered on a dry weight basis, and would infer the hesperidin
to have considerable physiological importance. Inasmuch as
more than 25 percent of the dry weight of a 1/, inch diameter
fruit was hesperidin, it did not seem logical that it could be
only a metabolic end product. Wawra and Webb (106) stated
that-the hesperidin chalcone was combined with a protein and
could serve as a hydrogen transporter in mammalian tissue, and
believed that it could have an important part in tissue respira-
tion of plants.
When the percentage of hesperidin in whole 'Valencia' fruit
was logarithmically plotted against its equatorial diameter as
in Fig. 4, there appeared to be an inverse linear correlation
between these two variables. Since greater fruit production oc-
curred in the 1951-52 and 1961-62 seasons, it was thought to
have brought about correspondingly lower percentages of hes-
peridin in the fruit, as is shown by the dotted line in this figure.'
The quantities of hesperidin found in each of the four orange
cultivars during one growing season have been compared in

80 40

40 20

6 4

w 2 IQ
I0 0 _

( oW 1.0
1.0 n .8
.6 o

*z Dry Weight -1 960-61
2 O-Wet Weight Fig. 3 -1961 -62 Fig 4
F 2 ig 3

Fig. 3.-Percentage of hesperidin found in 'Pineapple' oranges during
maturation. 0--1951-52 Seson
*4 *--1959-60 \
*--Dry Weight s--1960-6 1
.2 O 0--Wet Weight .2 0 --1961 -62 .

A M J J A S O N D J FMA I 2 3
Fig. 3.--Percentage of hesperidin found in 'Pineapple' oranges during

Fig. 4.-Relationship between fruit diameter and percentage of hesperidin
in 'Valencia' oranges.

Hesperidin in Florida Oranges

Fig. 5. 'Pineapple' orange was the cultivar having the greatest
quantity of hesperidin per fruit; this would help explain the
greater occurrence of hesperidin scale when its juice is con-
centrated (37). The monthly changes in hesperidin content were
similar for all four cultivars, there being a rapid increase in
hesperidin with the maximum quantity per fruit occurring as
early as July in the 'Pineapple' orange. In each case, hesperidin
content decreased after this peak period and eventually returned
to the same approximate value by the end of the season. Simi-
larly, the 'Shamouti' orange of Israel was found by Goren (30) to
attain 700 mg of hesperidin per average fruit early in the grow-
ing season, and to remain constant during further maturation.
He was not able to detect any specific influence of rootstock upon
the hesperidin content of the developing fruit in a study of 20
different citrus rootstocks. According to Lo et al. (68), compo-
sition of fertilizer significantly influenced vitamin P content of
vegetables. They found that compounds of nickel, zinc, and
molybdenum had a physiological function of promoting vitamin
P formation in plant metabolism.

Hesperidin Content of Juice
Since previous work by Maurer et al. (74) indicated a sig-
nificant relationship between naringin content of grapefruit
juice and its maturity, a similar relationship was sought for
oranges. Accordingly, it was noted that there were no seasonal
trends in the hesperidin content of the juices of the four orange
cultivars. Hesperidin content was between 0.015 and 0.034 per-
cent with a few exceptions that occurred at the end of the season.
This was a greater amount than would ordinarily dissolve in
water, but suggested that solubility has had a significant in-
fluence that possibly would be better demonstrated by a more
specific analytical method.

It is shown in Table 2 through 5 that about 75 to 80 percent
of the hesperidin was concentrated in the albedo, rag, and pulp
of oranges. This was in agreement with Braverman (16), who
stated the principal location of the glucosides was in the carpel-
lary membrane, at the boundary between the juice segments and
the albedo. Since this was true, any attempt to obtain a higher
juice yield by increased pressure or deeper burring by juice

18 Florida Agricultural Experiment Stations

1.4 80

1.2\ 40

1.0 -
a- 2
z 0.8 z
Sy 10 -D- Albedo
W 0 B 0- Rag & Pulp
c 0.6 -- Flavedo
S / 6 0D-- Juice
CD 4
< 0.4 O- Pineopple
5D a-- Parson Brown z
s-- Hamlin C
0.2 O-- Valencia U 2
Fig. 5 Fig. 6

A M J J A S O N D J F MA S 0 N D J F M A
Fig. 5.-Quantities of hesperidin found in four cultivars of oranges during

Fig. 6.-Percentage of hesperidin in the components of 'Pineapple' orange
during growth.

extractors can increase the hesperidin content of the juice (37).
Hence, in this study the juice samples were extracted with a
hand juice extractor which required only a moderate amount of
pressure to remove the juice. These samples were found to con-
tain from 1.5 to 6.0 percent of the total hesperidin. The extreme
values were obtained either at the beginning or end of the
usual picking season, with a fairly constant percentage found
in each variety in the other portions of the season.
The seasonal distribution of hesperidin in the component
parts of 'Pineapple' oranges is shown in Fig. 6. Results were
similar for the other orange varieties and illustrated graphically
that picking date was not a variable which influenced the dis-
tribution of the glucoside. The proportion of hesperidin in the
component parts of mature oranges was as follows: juice, 2.3
to 6.3 percent; albedo, 27 to 50 percent; flavedo, 12 to 22 per-
cent; rag and pulp, 33 to 49 percent.
Goren (30), studying the amount of hesperidin in different
tissues of the maturing 'Shamouti' orange (commonly called

Hesperidin in Florida Oranges

'Jaffa' orange (107) ), found a significantly greater amount in
the stylar end than the stem end of the fruit, much as has been
shown for soluble solids (16). He investigated the whole flower,
and separate parts of the flower during development, and found
that hesperidin content continued to increase in the flower dur-
ing development, and just before setting accounted for 37
percent of the dry weight of the whole flower. The greatest
proportion of hesperidin had accumulated in the ovary.

The 'Parson Brown,' 'Pineapple,' and 'Valencia' cultivars of
orange were investigated, so chosen as to represent early, mid-
season, and late fruit. The fruit samples were taken from trees
grown in Lake Alfred on rough lemon rootstock that were re-
ceiving standard fertilizer and spray treatment. A 4/5 bushel-
bag sample of fruit was picked in the first years from numerous
locations on the tree, while in a later phase the 'Valencia' fruit
were harvested from a single tree on each picking, and after
weighing, the sample size was reduced using good sampling

Recovery Procedure
The extraction and recovery of hesperidin from orange peel
residue or young whole fruit embodied the same simple principle
described before. Alkali was used to degrade the pectin in the
cell walls and to dissolve the hesperidin in the aqueous phase.
After a short period of time this liquid was separated from the
peel and acidified. The crystallized glucoside was separated from
the solution after a short holding period. At least four patents
(9, 47, 48, 49) have been granted on this technique, each being
slightly modified versions of the same principle.
In the initial phases of this study (39), the 4/5 bushel-bag
sample of oranges was juiced in a Citro-Mat extractor with 65
pounds per square inch roll pressure. The peel was subsequently
chopped in a Fitzpatrick comminuter having a screen with 1/2
inch openings. A 4,000 gram sample of this chopped peel was
stirred mechanically with 6,000 grams of water over the next 90
minutes, while increments of calcium hydroxide were added to
maintain the pH at approximately 11.0. The alkaline peel slurry
was strained through cheesecloth and additional liquor recovered
by pressing the remaining peel hydraulically at pressures of

Florida Agricultural Experiment Stations

about 300 pounds per square inch. After combining both liquids,
the turbid solution was adjusted to pH 4.7 with concentrated
hydrochloric acid, and 30 grams of standard Super-Cel was
added. After stirring the extract for 90 minutes to aid crystalli-
zation, it was allowed to stand overnight. The crude hesperidin
was filtered, dried, and analyzed. Filtrate losses were determined
by the same method of Davis that was described earlier.
The extraction procedure was modified slightly in a second
study (44) of three-year duration on the recovery of hesperidin
from immature 'Valencia' oranges. Fruit or peel was chopped
in a food grinder and further comminuted in a Waring blendor.
The quantity of water added was dictated by that necessary to
obtain approximately a 0.5 percent hesperidin extract. Whole
fruits were extracted on the first three sampling date of each
season, after which the juice portion was discarded to avoid
troublesome dilution. On each processing date the optimum pH
was sought by duplicating the extraction at pH 10.9, 11.1, and
11.3 with an occasional higher pH when extracting very im-
mature fruit. No filter-cel was used, but sodium bisulfite was
added, equivalent to 300 to 400 ppm of sulfur dioxide, to avoid
slime build-up during the 48-hour crystallization period. Faster
filtration rates were obtained by the prevention of slime forma-

Effect of Cultivar upon Hesperidin Yield
Extraction results for one season are presented in Figs. 7,
8, and 9. Of the three cultivars tested, 'Pineapple' oranges yielded
the highest quantity of hesperidin per ton of peel. When pro-
cessed on their earliest respective maturity dates, the order of
cultivars for best hesperidin yield was 'Pineapple,' 'Parson
Brown,' and 'Valencia.'
Effect of Particle Size upon Yield
When the peel from 'Valencia' oranges picked in November
was divided into three lots and comminuted in a Fitzpatrick
mill through screens having 1/4, 1/', and % inch holes, screen
size had a profound influence on hesperidin yield. The respective
hesperidin yields from each lot were 12.4, 11.3, and 10.2 pounds
per ton of peel and would infer a 20 percent advantage in favor
of the more finely ground peel. As the peel became mature,
this advantage was less pronounced, and a greater difficulty was
experienced in forcing a softer peel through a fine screen.

Hesperidin in Florida Oranges

S 0 N D J

0 N D J

Fig. 7.-Recovery of hesperidin from 'Valencia' orange peel in successive
Fig. 8.-Recovery of hesperidin from 'Parson Brown' orange peel in suc-
cessive months.

Effect of Recycling Hesperidin Extracting Liquor
The quantities of unisolated hesperidin-like material that
were lost in the filtrates of each extraction are shown and added
to the hesperidin recovery results in Figs. 7, 8, and 9. To pre-
vent these losses of from 6 to 8 pounds per ton of peel processed,
to decrease the volume of waste liquor for later disposal, and
to maintain higher sugar concentrations in the extracting liquor,
an attempt was made to reuse the crystallizing filtrate of one
extraction in lieu of diluent water for the next extraction. The
outcome on the many 'Pineapple' orange peel extractions is
shown in Fig. 9. The over-all seasonal effect of recycling the
extracting liquor was an increase in yield of approximately 10
percent. An average of 25 percent more lime and 50 percent
more hydrochloric acid was required when recycling was prac-

Effect of Extracting pH
The influence of pH on the amount of hesperidin extract
from 'Valencia' orange peel throughout one season is shown in

Florida Agricultural Experiment Stations

R Recycled Filtrate
U Isolated
Fig. 9
30 In Filtrate 40 Fig. 10

S N 25_ 50 1O fo Recovery
M pH 10.9

5 O Los

5/20 7/12 9/1 10/13 12/1


Fig. 10.-Effect of maturity and extraction pH upon hesperidin recovery
from 'Valencia' oranges.
15 M2B 10 '-
8 0 N 0 J F 5/20 7/12 9/I 10/13 12/I

Fig. 10. The optimum alkaline pH for extracting hesperidin
varied with fruit maturity. Higher yields were obtained from
immature fruit but required more alkaline conditions to break
down the greater proportion of protopectin at this growth stage.
Experience from three seasons of extracting small 'Valencia'
oranges and 'Valencia' peel indicated that a good extracting pH
was 11.5 for the smallest, most immature fruit, and proportion-
ately lower alkalinity was required when processing fruit of
more advanced maturity until pH 11.1 was reached.

Maturity vs. Recovery of Hesperidin
Since the more immature fruit have been previously shown
by the authors (38) to have a greater percentage of hesperidin
on either a wet or a dry basis, it was not surprising that re-
covered hesperidin was inversely proportional to maturity and
size of fruit, as shown by Figs. 7, 8, 9, and 10. Another com-
parison of maturity versus hesperidin yield is seen in Fig. 11,
where the results of extracting 'Valencia' fruit and peel over

Hesperidin in Florida Oranges 23


100oo 90 'e

o80 70
S0 u. 40 \\@--
- h60
60 570 ( 0
z a--

S40 D--1959- 60
W 9--1960-61
30 0--1961-62 3 --1959-60
S\ \ -----1960-61
20 \ 20 ---1961-62
Fig. II
10 Fi. 10 Fig. 12

6/1 9/1 12/1 3/1 6/1 6/1 9/1 12/1 3/1 6/1
Fig. 11.-Comparison of hesperidin yields in three seasons and at different
stages of maturity.
Fig. 12.-Comparison of the percentage purity of isolated crude hesperidin
samples versus maturity.

three seasons are compared. The curves in this figure were
noticeably similar and presumably could have coincided except
for the advanced fruit maturity in the 1960-61 season.

Maturity vs. Purity of Extracted Hesperidin
Isolated crude hesperidin varied in purity during the season,
but followed a pattern that has been presented in Fig. 12. The
most immature fruit provided the highest purity product, and as
the fruit matured, there was a decrease in purity of recovered
product. The pH conditions also influenced purity of isolated
hesperidin, and it was generally noted that the conditions en-
couraging best yield usually led to a product of highest purity.

Maturity vs. Recoverable Hesperidin per Acre
With sufficiently improved recovery of hesperidin from fruit
picked at some time prior to the normal harvest time, there
would accrue many economic advantages. The external qualities
of the fruit diverted to this purpose would no longer be of any

Florida Agricultural Experiment Stations

importance, and the spray and cultural program would be de-
signed to protect only the bearing tree. In harvesting all the
fruit prematurely from 'Valencia' trees, there would be increased
subsequent blossoming and yield in the next season, according
to West and Barnard (110). There would be the further likeli-
hood of using to advantage physiological sprays, such as 2-4
dichlorophenoxy acetic acid in low concentration during certain
period of the growing season, which Stewart et al. (99) have
found to produce physiologically younger fruit and to reduce
preharvest drop.
Since the yields of recoverable hesperidin per ton of fruit
continually decreased within any season, but the growth rate of
the fruit led to larger quantities of fruit to be processed, there
was a possibility of an optimum processing period. The com-
bined effect of these two variables over the past three seasons
is shown in Fig. 13. Calculations were made on the basis of
there being roughly 70 trees per acre. In each of the three sea-
sons studied an optimum processing period was indicated, when
the fruit had equatorial fruit diameters of 2.1, 1.6, and 1.9 inches,
The green color and the smaller size of immature fruit made
fruit picking more difficult, but not a serious obstacle if the fruit
were at least 2 inches in diameter. The 2-inch size was there-
fore ideal, a smaller size being impractical and too great an
economic disadvantage.
The results shown in Fig. 13 have depended to a great degree
upon yield of fruit stripped from the trees in the three seasons.
The widest difference in this regard have occurred between sea-
sons rather than within a season. Typical of the yearly differ-
ences in fruit yield that occurred were the pick-out results in
September of each year. In this month 193, 220, and 440 pounds
of fruit were picked per tree for the respective 1959-60, 1960-61,
and 1961-62 seasons, which was roughly in proportion to the
hesperidin yields per acre for these seasons. Although not too
noticeable with 'Valencia' oranges, yields of fruit in the 1960-61
season were decreased after hurricane Donna of September
Recovery of Hesperidin Filtrate Losses
Also investigated was the possibility of recovering the 6 to 8
pounds of hesperidin-like material lost per ton of peel processed.

Hesperidin in Florida Oranges



a 140
r 120
a 80

> 60


Fig. 13

6/1 9/1 12/1 3/1 6/1
Fig. 13.-Recovery of hesperidin on
of maturity.

a per acre basis at different stages

Fig. 14.-Change in glucoside content of orange extraction liquor with
addition of neutral and basic lead acetate.

This loss occurred by virtue of a 0.1 to 0.2 percent solubility in
the crystallization liquor. It was shown previously that recycling
of filtrate would diminish these losses. An alternative method
was found in the addition of a concentrated basic lead acetate
solution to increase precipitation of hesperidin so it could be
filtered. The influence of quantity of basic lead acetate added
to 50 ml of an average hesperidin-isolating filtrate is shown in
Fig. 14. Neutral lead acetate was noted to be less efficient, and
similarly, any increase in temperature or decrease in pH was
detrimental to the recovery of hesperidin.

Commercial Recovery
In Florida, the commercial recovery of hesperidin products
from citrus has been divided between two methods. One process
deals with the extraction of hesperidin from citrus molasses with
an organic solvent, while the other process deals with the ex-
traction from citrus peel with an aqueous alkaline solution.
The first method produces a concentrate, which is refined
further to yield a dried therapeutically active product. It is



(D --1959-60O'
a ----IO-1s9661

26 Florida Agricultural Experiment Stations

sold in the pharmaceutical trade and according to one related
patent (97) is a mixture of three substances that are similar
to quercitrin, eriodictyol, and hesperidin. It is stated also that
citrus molasses was found to contain 22 to 28 milligrams of
active flavonoid material per gram of molasses.
At one time the commercial product was analyzed by paper
chromatography and found to contain approximately 5 percent
hesperidin (42). Citrus molasses prepared in the laboratory
from oranges of different cultivars was found by the authors to
contain 1.7 to 2.3 percent hesperidin by the Davis test.
The second commercial method used in Florida resembles
the recovery procedures presented by the authors. Two products
are produced for the trade, a hesperidin complex that has a
minimum analysis of 40 percent hesperidin and a purified hes-
peridin containing a minimum of 90 percent hesperidin. Over
the past six seasons, the industry yields of hesperidin from waste
peel residue have equalled the laboratory results, while product
purity has been in a slightly lower range. Production of purified
hesperidin was related similarly to laboratory results. Plant
production was found to be adversely affected by plain iron
equipment, prolonged storage of wet crude product, and micro-
organisms. Furthermore storage of the dried hesperidin com-
plex or purified product resulted at times in an unexplained loss
of product purity as also happened in the laboratory.

The purification of crude hesperidin has been complicated
by its inadequate solubility in organic solvents and by the slimy
pectinous impurities usually present in crude products made
from citrus. Experience indicated that an economical commer-
cial purification of crude hesperidin would be dependent upon
an alkaline extraction with sufficient alcohol added for proper
filtration followed by neutralization and crystallization steps.
Such a procedure (40) can upgrade hesperidin purity to a per-
centage of 89 to 95. The final product can be further improved
to 99.5 percentage purity by boiling the hesperidin with 8 or 9
volumes of water for 1 hour. Highest purity is obtained by
dissolving hesperidin in formamide or dimethyl formamide and
recrystallizing from the clarified solution by diluting with an
equal volume of water. Pritchett and Merchant (83) have de-
scribed this procedure and indicated it capable of producing

Hesperidin in Florida Oranges

hesperidin with a melting point equal to the highest previously
recorded, namely, 261 to 262* C.
The following is an effective laboratory or commercial pro-
cedure for purifying crude hesperidin from a citrus source, as
well as a discussion of the influence of certain variables.

General Procedure
A sufficient amount of crude hesperidin product is added to
a solution of 0.2 N sodium hydroxide in 50 percent isopropyl
alcohol to yield a 2.0 to 2.4 percent hesperidin solution. The
mixture is then agitated for 1/2 hour. Afterwards, the solution,
containing the dissolved hesperidin, is filtered from the insolu-
bles, acidified to pH 8.5, and allowed to crystallize slowly for
48 or more hours. The crystallized hesperidin is separated by
filtration and dried, while the filtrate is retained and after being
fortified with alkali and alcohol becomes the extracting solution
for the next batch.
Effect of Alcohol on Filtration Rate
The isopropyl alcohol, added to the extracting alkali solution
to speed filtration rate, was noted to have a pronounced effect
upon time of filtration. The degree of improvement brought
about by increasing concentrations of alcohol is shown in Fig. 15.
Effect of Alcohol upon Hesperidin Recovery
The improved filtration speed brought about by increasingly
greater alcohol concentration in the extracting solution was
offset by a decreasing recovery of hesperidin, as shown in Fig.
16. Hesperidin purity was not greatly influenced by alcohol con-
centration, but higher proportions of alcohol were noted to en-
courage a more voluminous crystal that formed more quickly
and was more yellow.
Holding time and percentage of alcohol in the extracting
liquor influenced considerably the quantity of hesperidin-like
material that failed to crystallize from the extracting solution.
The extent of this yield loss is shown in Fig. 17, and is explained
partly by the slightly increased solubility of hesperidin in al-
cohol as against water, and by the more voluminous character
of the crystallized hesperidin with greater proportion of alcohol.
Yield of recoverable hesperidin was further improved by 5 per-
cent with extended crystallization periods, but may be imprac-
tical in some instances.

Florida Agricultural Experiment Stations




z 40

Fig. 15

Filtration Rate

0 6 12 18
Fig. 15.-Effect of isopropyl alcohol



C \ Fig. 16


50 Recovery Rate
60 0

X 40 -

a 0e Recovery Rate

3 60 70 80
upon rate of filtration of extracting

Fig. 16.-Effect of isopropyl alcohol concentration upon the recovery of

Effect of pH upon Hesperidin Recovery

The influence of pH upon hesperidin solubility in the isola-
tion step is seen in Fig. 18. Minimum solubility and maximum
recovery occurred at approximately pH 8.9. Recovery was im-
proved also by longer holding periods, as is evident in the com-
parison between 24 and 48 hours. Over shorter periods of time,
conditions of pH lower than 8.9 encouraged the fastest crystal-
lization, but at the sacrifice of yield of recoverable product. The
longer holding periods are to be recommended if they can be


The absence of an ideal test for hesperidin, hesperidin ex-
tracts, citrin, and similar flavonoids, that would correlate physio-
logical activity with a chemical test or absorption measurement
has been a serious problem to overcome. Under these circum-

Hesperidin in Florida Oranges 29

Extraction Losses 0.9 0-..
80 */ /
i/ / Effect of pH
W 0.8
70 / / \a
50 / y | -- \\

D Fig. 17 / 0.7 Fig. 18
60 / /0 z
S/ 0.6
,O/ 6/ 0 \ ,'
- ,/ 0.5 0 -

w 40 0 0
o // 0.4
4 0/
S30 4 /d ----After 72 Hrs. 0--- After 24 Hrs.
--After 24 Hrs. 0.3 -- After 48 Hrs.

15 20 25 30 35 3 4 5 6 7 8 9 10
Fig. 17.-Effect of crystallization holding time and alcohol concentration
upon hesperidin losses.

Fig. 18.-Influence of pH upon hesperidin solubility in alcoholic extracts
of crude hesperidin.

stances, a great number of analytical procedures, many biologi-
cal, and a greater number of chemical tests have been developed
to measure capillary fragility improvement after dietary
administration of hesperidin or quantity of flavonoid without
resolving the needed inter-relation between the two. This and
the lack of a standard have greatly hindered the exploitation
of flavonoids for therapeutic treatment.

The bioassay of extracts capable of improving capillary
fragility, such as has been claimed for hesperidin (17, 24, 88),
has been studied with guinea pigs, rats, and rabbits (88) ; but
Bacharach et al. (8), who developed a modified guinea pig test,
have claimed that there was not a satisfactory animal test for
vitamin P. Less accepted than animal tests, which are unsuited
to routine analysis, have been the alternative short-cut methods

Florida Agricultural Experiment Stations

of biological assay, such as measuring bleeding time, inhibition
of succinoxidase or hyaluronidase, toxicity protection against
arsenicals, and others mentioned by Scarborough et al. (88).

Chemical Procedures
The chemical determination of hesperidin and other flav-
onoids has been founded mostly upon a number of color reactions.
One of the earliest procedures devised and employed by Armen-
tano et al. (3) was a silver lactate method to determine the
citrin content of human urine. The silver lactate removed
urinary pigments, while a later addition of sodium cyanide and
sodium carbonate developed a brownish-red color. A more re-
cent method, described by Wilson (112), was a boric-citric acid
reaction that produced a brilliant yellow color with flavones,
"citrin," and hesperidin derivatives under anhydrous condi-
tions. A saturated boric acid acetone reagent with added citric
acid developed the color in a few minutes. Horowitz (53) described
how flavanones, such as hesperidin, naringin, and their agly-
cones, can be detected either in solution or on paper chromato-
grams by reduction with sodium borohydride. The purple to
blue-red color formed was the basis for a quantitative determina-
tion of flavanones in citrus bioflavonoids by Rowell and Winter
Chromatographic procedures with their attendant Rf values
and special color tests have offered one method of specifically
identifying flavonoid compounds. The chromatography and
identification of hesperidin and a large number of other flav-
onoids have been discussed by Casteel et al. (21) and Harborne
(33), and the authors have used these techniques for semi-
quantitative analysis (42). Column chromatography, especially
with silicic acid, has been noted by Horowitz and Gentili (57)
as being a very satisfactory method of isolating and separating
the flavonoids of citrus.
A methoxyl test which measures methoxyl content, described
in AOAC (5), has given useful information on product purity
with hesperidin, hesperitin, and certain derivatives of these com-
pounds. The lower alcohols, if used in the purification of these
compounds, were so difficult to remove that they too readily
caused high results. Another method of measuring purity of
hesperidin samples has been the gravimetric procedure (19),
which requires the sample to be dissolved under vacuum in an

Hesperidin in Florida Oranges

ice bath with 4N sodium hydroxide. The hesperidin portion was
recovered later by neutralizing, evaporating to dryness, re-
suspending in water, and filtering with a Gooch crucible. Results
were found to be at least 5 percent low with high purity hes-
peridin, whereas products of lower purity were often 10 percent
low (42). Kwietny et al. (64) obtained an average of 91.4 per-
cent recovery of pure hesperidin using this procedure, but by
certain modifications were able to increase the recovery to 95
One of the most frequently used methods of assaying hesperi-
din and extracts of it has been a simple procedure described
by Davis (25). In this method a small quantity of 4N sodium
hydroxide and sample are added to diethylene glycol, and the
increase in absorbence at approximately 420 mA is determined
by a spectrophotometer. In Fig. 19 are shown the optical density
readings to be expected with hesperidin, hesperitin, naringin,
and naringenin when using a 424 mu filter in a Fisher electro-
photometer with 25 ml cell and 30 minutes reaction time.

60 .S

o0 0 0
x 0.9-
SFig. 19 7n 0.7
a W
_ 30 0

S0.5 I


0 0.04 0.08 0.12 0.16 60
Fig. 19.-Davis test optical density readings
hesperidin and related flavonoids.

Fig. 20.-Ultraviolet optical density readings
hesperidin and related flavonoids.


versus concentration for

versus concentration for

Florida Agricultural Experiment Stations

1.9 1.9

1.7 1.7 I Juice + Cu
2 + 001% Hesperid
1.5 1.5 41 3 +0.02%
> ()D Untreated 4 0.03%
I -
S1.3 ( Copper Added 1.3 5 -0.02%
w G Cu 4 I Hr. Air 2 6 -0.03%
a 1.1 Cu + 2 Hr. Air z-
Z 1.1 7 -0.04%

0.5 0.5

0.1 0.
250 300 350 250 300 350
Fig. 21.-Effect of copper and aeration upon the ultraviolet absorption
of an orange juice.

Fig. 22.-Influence of small increments of hesperidin on the ultraviolet
absorption of orange juice.

Horowitz et al. (54) pointed out that the Davis method was not
too suited for hesperidin estimations since hesperidin absorbs
only feebly at wavelengths greater than 400 m/. It was suggest-
ed that hesperidin and the afore-mentioned flavanones could be
estimated better by measuring height of the absorption band
occurring at 285 mM in neutral solution.
An ultraviolet estimation of hesperidin and similar flav-
anones was described by the authors (42) earlier, wherein the
bioflavonoid was dissolved in neutralized dimethyl formamide.
Fig. 20 shows the typical absorption curves obtained with 0.002
percent solution of hesperidin, hesperitin, naringin, and narin-
genin in a 10 mm cell. Readings were made with a DU Quartz
spectrophotometer. The method was later modified for determin-
ing the hesperidin content of orange juice and peel extracts (43).
In this modified procedure, copper and aeration were used to
avoid an ascorbic acid interference, and the sample was finally
diluted with isopropanol for UV analysis (Fig. 21). A typical


Hesperidin in Florida Ora


( Commercial Conc.
( Pulp Washing Extract
) Immature Valencia
Commercial Conc.
Pilot Plant


,nges 33

() Orange Peel Vinegar
( Alkaline Peel Extract
) Orange Juice Vinegar
) Hesperidin Rec. Filtrate
) Peel Oil Emulsion



0.3 5


250 300 350

Fig. 23.-Ultraviolet absorption curves for orange juice from different

Fig. 24.-Ultraviolet absorption curves for various products from orange
peel and juice.

absorption curve for an orange juice sample is shown in curve 1
of Fig 22. Curves 2, 3 and 4 of this figure demonstrate the effect
from adding known increments of hesperidin to the juice sample.
When the hesperidin increments were added to the blank, how-
ever, instead of the juice sample (curves 5, 6, and 7), the 286 mu
absorption was eliminated to varying degrees. By adding the
known hesperidin increments to the blank sample, the plotted
readings from the DU spectrophotometer reflect the difference.
That hesperidin increment which best eliminated the 286 mp ab-
sorption was taken to be its hesperidin analysis. In the analysis
of orange juice samples, this procedure found an average of
only 26 percent of the hesperidin indicated by the Davis test, and
suggested the latter method to give erroneously high results in
this application. Figures 21, 22, 23, and 24 show the typical 286
mT UV absorbence expected for hesperidin. Ultraviolet spectral
studies of isoflavones and flavanones have been reported by
Horowitz and Jard (58), wherein UV techniques were used to

250 300

Florida Agricultural Experiment Stations

70 70

60 60

o50 050 *
t- 4 0 40
40- / / y 40
o ")
30 30
SFig. 26
20 Fig. 25 a20
020 020


0.04 0.08 0.12 0.006 0.018 0.030
Fig. 25.-Optical density readings by the Arcangeli-Trucco determination
for hesperidin and related flavonoids.

Fig. 26.-Optical density readings by the azo coupling method for hesper-
idin and related flavonoids.

differentiate molecular structure. The infrared spectra of hes-
peridin and many other naturally-occurring flavonoids have been
described also by Inglett (61).
One of the first chemical procedures devised and used to any
extent for measuring vitamin P content of food extracts was
that of Lorenz and Arnold (70). The method was used fairly
extensively by Chinese workers (67, 68); however, it was found
unsuitable for hesperidin, failing to develop the typically red
color produced by heating lemon extracts with potassium hydrox-
ide (42).
Another colorimetric method that was used in some of the
earlier investigations is one described by Arcangeli and Trucco
(1). Sometimes called the cyanidin reaction, this determination
reduces the flavanone with hydrochloric acid and magnesium in
methanol. Other details and modifications were described by the
authors (42), and Fig. 25 graphically presents sensitivity and
absorbency of hesperidin and other flavanones tested.

Hesperidin in Florida Oranges

An azo coupling method devised by the authors (42) was
found to have the advantage of being equally sensitive to hes-
peridin, naringin, and naringenin, as is shown in Fig. 26. This
colorimetric procedure depended on the quantity of color de-
veloped by coupling p-nitroaniline diazo into the respective
The significance of the many methods described in varying
detail lies in the difficulty of finding a specific analytical tech-
nique and one that will correlate with biological activity or
therapeutic effect.

Besides describing the physical and chemical nature of hes-
peridin, a discussion was included of the therapeutic usefulness
of it and its derivatives to show their potential value. Although
their conclusion is controversial, some investigators believe that
combinations of hesperidin and ascorbic acid have supplemental
therapeutic value in virtually all diseased states and specific
action in some.
It was recognized that between 70 and 80 percent of the total
hesperidin content was concentrated in the albedo, rag, and
pulp of the oranges studied, while a linear semi-logarithmic re-
lationship was noted between percentage of hesperidin in whole
fruit versus average equatorial diameter. An unusually high
hesperidin content was found in very small immature fruit, but
the quantity found in the juice portion of the fruit was always
small and unrelated to maturity.
The optimum recovery of hesperidin from the peel of the
orange was related to maturity of fruit, which in turn influenced
the degree of alkalinity needed. The highest yields per ton of
peel were obtained from 'Pineapple' oranges, but the maturity
of the fruit was an important factor. Higher yields were ob-
tained also by chopping the peel finer and by recycling the
hesperidin-saturated filtrate in the subsequent extraction. An
economic advantage was revealed in the recovery of hesperidin
from immature 'Valencia' oranges. When fruit production was
taken into account, peak yields of hesperidin per tree occurred
as the fruit reached 2 inches in equatorial diameter. Purity of
extracted hesperidin was, for the most part, related to and pro-
portional to yield.

Florida Agricultural Experiment Stations

The critical factors, such as alcohol concentration, hesperidin
concentration, crystallization time, and pH were determined
for the purification of crude hesperidin. Improved recovery was
shown to be possible without serious loss of purity by recycling
the alcohol phase. Other methods were described also for isolat-
ing a more chemically pure product.

The chemical and biological methods of analysis for hesperi-
din and similar flavonoid materials having therapeutic activity
were described as well as certain limitations of the methods.
There was no ideal method that was specifically suited to routine
work, and capable of being related to therapeutic activity.

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Hesperidin in Florida Oranges 37

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Florida Agricultural Experiment Stations

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42 Florida Agricultural Experiment Stations

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