Group Title: Citrus Station mimeo report - Florida Citrus Experiment Station ; 56-10
Title: Vitamin P in citrus products
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 Material Information
Title: Vitamin P in citrus products a review
Series Title: Citrus Station mimeo report
Physical Description: 6 leaves : ; 28 cm.
Language: English
Creator: Hendrickson, Rudolph
Kesterson, J. W
Citrus Experiment Station (Lake Alfred, Fla.)
Publisher: Florida Citrus Experiment Station
Place of Publication: Lake Alfred FL
Publication Date: 1955
Subject: Citrus fruits in human nutrition -- Florida   ( lcsh )
Bioflavonoids -- Health aspects -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
non-fiction   ( marcgt )
Statement of Responsibility: by R. Hendrickson and J.W. Kesterson.
General Note: Caption title.
General Note: "October 4, 1955."
 Record Information
Bibliographic ID: UF00072378
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 74710769

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Citrus Station Mimeo Report 56-10
October 4, 1955

Vitamin P in Citrus Products

A Review
R. Hendrickson and J. W. Kesterson

Introduction.- Vitamin P is a term that has been applied to a group of sub-
stances concerned with the maintenance of normal conditions in the walls of the
small blood vessels (22). 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 is invariably associated with much-decreased capillary
permeability and may be characterized by the development of spontaneous petechial
hemorrhages in areas exposed to pressure (24). Vitamin P substances are widely dis-
tributed among plants with citrus fruits, rose hips and black currants considered as
being the best sources. The minimum protective dose of vitamin P is thought to be
obtained by consuming as little as 50-100 ml. of orange juice daily (23). The chem-
ical and physical properties of vitamin P have never been stated, since the most
active preparations are known only in the form of concentrates. It is usually found
in close association with ascorbic acid, yet final evidence is still lacking to con-
firm its requirement as an essential nutritive element. Scarborough and Bacharach
(24) have pointed out that in many of the more recent tests, doses of vitamin P have
been employed of a size that suggest a pharmacological action rather than that of a,-..
vitamin. In 1950 (10), it was finally recommended by a joint committee of the Amer-
ican Society of Biological Chemists and the American Institute of Nutrition that the
term "Vitamin F" be discontinued. It is possible that this will reflect as a dis-
advantage to the citrus industry. It cannot detract from the fact, however, that in
the edible portions of citrus fruits there are certain substances, sometimes called
citrus flavonoids, which have a nutritional effect on the human system that parallels
that of a vitamin.

In deference to the numerous published findings on vitamin P, and for lack of
an adequate substitute, the terminology, vitamin P, has been continued in this paper.

Medical Background.- Whether vitamin P is rightfully a vitamin or not appears
to be trivial after reviewing the numerous therapeutic uses to which vitamin P ex-
tracts have been put. Although the therapeutic action of flavones had been previous-
ly studied, it was not until 1936 that Armentano and Szent-Gyorgi (3) reported that
Hungarian red pepper and lemon juice contained a factor that could increase capillary
strength and reduce capillary permeability in man. Because of its vitamin-like
activity, its apparent effect on permeability and its presence in paprika, it was
provisionally named vitamin P. Since the original observations of Szent-Gyorgi and
his associates, "citrin" (a lemon extract) and many analagous mixtures and similar
compounds have been clinically tested in numerous medical problems.

In reviewing the diseased states in which vitamin P has been found e tive
a very impressive list can be compiled. Table 1 summarizes the medic

Florida Citrus Experiment Station,
Lake Alfred, Florida.

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

Allergic purpura (22)
Angioneurotic edema (22)
Beriberi (15,17)
Bronchial asthma (17)
Carcinoma (17)
Chronic nephritis (20)
Dermatitis (15)
Diabetes (17,22)
Diabetic Retinopathy (17)
Eczema (17)
Erythema (15)
Essential hypertension (2:
Follicular tonsillitis (8
Glaucoma (26)
Glomerulonephritis (15)
Graves' disease (15)
Habitual abortion (17)
Haemoptysis (20)
Hematochromatoses (20)
Hematuria (17,26)
Hemorrhagic diatheses (20
Hemorrhagic nephritis (20

Hemorrhagic telangiectasis (22,26)
Influenza (8)
Leprosy (17)
Measles (*)
Ophthalmology (17)
Petechial hemorrhages (20)
Pleurisy (15,17)
Polio (*)
Polyarthritis (20)
Psoriasis (22,26)
Radiation hemorrhage (22)
Rheumatic Fever (17,26)
Rheumatoid Arthritis (17,26)
Rhinitis (8)
Schoenlein-Henoch purpura (20,15)
Syphilis (15)
Thrombocytopenic purpura (17)
Tuberculosis (15,17)
Ulcerative colitis (20)
Upper respiratory infection (8)
Vascular purpura (22,26)



*Unpublished reports.

Florida Citrus Experiment Station,
Lake Alfred, Florida.


This impressive list of diseased states responding to vitamin P therapy relates back
to its effectiveness in improving capillary fragility and permeability. Recent in-
vestigations claim a relationship between vitamins C and P. Horoschak (17) claims
that vitamin P requires for its activity the presence of traces of ascorbic acid,
and in the complete absence of ascorbic acid, vitamin P is inactive. Several sug-
gested mechanisms for the physiological activity of vitamin P compounds have been
suggested by Beiler (6). The outstanding success of these compounds in the treat-
ment of numerous medical conditions has recently led to the contention that there
is no diseased state in which the capillaries are not detrimentally modified, or
conversely that there are no diseased states which will not benefit by assuring
proper capillary strength (21).

Methods of Analysis.- Considerable difficulty is encountered in chemically
analyzing for vitamin P since a group of substances of a flavonoid nature, as well
as some related structures, have shown vitamin P activity. As a consequence, there
is not available a chemical test that will give a proportionate response to all
active materials. One of the greatest obstacles to date, has been the inability to
relate chemical properties with physiological activity which has prevented the adop-
tion of a standard and handicapped attempts to concentrate the active principle from
natural sources. There are chemical tests, however, that are helpful when analyzing
extracts of a similar nature from one or two related plants.

The first chemical test to be used to any extent was that of Lorenz and Arnold
(20) which was devised to measure the vitamin P content of lemon extracts. This
procedure is based on the quantity of red color formed by heating lemon extracts in
a boiling water bath with potassium hydroxide for five to twenty minutes. We have
found it advantageous to use a 575 millimicron filter with a modern colorimeter,
instead of comparisons against a 0.05N iodine solution, when making this determination,

Another procedure is called the cyanidin reaction, and is characterized by the
development of a red dye when an alcoholic solution of a hydroxy flavonol or flavon-
one is reduced with magnesium and hydrochloric acid (1). It is important to per-
form the reduction at a low temperature and over a 15 to 30 minute period. Our ex-
perience has shown the procedure to be approximately as sensitive as the Davis test
(11). Most processors are familiar with the Davis test (ll) which has been exten-
sively used to test the naringin or hesperidin content of grapefruit and oranges.
This procedure can be modified to give equal sensitivity to naringin and hesperidin
solutions by changing the presently used 420 mu filter to one having a wavelength
of 365 mu.

Still other chemical methods are available.

None of the foregoing tests have correlated completely with a biological or
clinical assays, which are the final criterion of activity. However, there appears
to be some dispute even as to the best method of biologically assaying vitamin P
preparations. The method of Bacharach et al. (5) is probably as well accepted as
any. In their procedure, guinea pigs of a standard weight are placed on a scor-
butogenic diet with supplemental doses of cod-liver oil and vitamin C. The sample
to be tested is fed orally and, at appropriate later intervals, the animal is tested
by shaving an area on the middle of its back. Suction is then gradually applied to
the bare skin and the pressure recorded at which petechiae are first seen. This

Florida Citrus Experiment Station,
lake Alfred, Florida


pressure is sometimes called capillary resistance. Maximum response to different
doses occurred in about four weeks and was usually averaged for 9 or 10 animals.

Scarborough (23) measured the vitamin potency of certain foodstuffs by clinical
assay and in terms of a provisional unit. The provisional unit (P.U.) by coincidence
gives recrystallized hesperidin exactly 100 P.U. per gram. He designated three
groups: good (1000-100 P.U./100 gm), moderate (100-50 P.U./100 gm), and poor (50-0
P.U./100 gm) sources in which each of many foods were placed.

Sources of Vitamin P.- Although at one time vitamin P was closely associated
only with "citrin', an extract isolated from lemons, chemical study of "citrin" show-
ed it to be a mixture of eriodiotin, hesperidin and a quercitrin-like flavone.
Early work was, therefore, concentrated on the flavonones, hesperidin and eriodictin.
It was later shown, however, that vitamin P activity extended to flavones, flavonols
and flavanones, as well as structurally similar compounds, whether combined with
carbohydrate or in the aglycone form (6). Among the list of biologically active
materials besides hesperidin and eriodictin are: rutin, which is obtained from oak
tree bark, tobacco and buckwheat; esculin, which is obtained from the leaves and
bark of the horse chestnut tree; catechin and its epimers which can be found in ma-
hogany wood (6), and other more uncommon but related compounds.

Vitamin P has been shown to be widely distributed over the entire plant kingdom.
Scarborough (23) evaluated a number of foods as follows: good source-grape, lemon,
orange, plum, prune and black currant; moderate source--grapefruit, apricot, plum,
cherry, blackberry, and bilberry; poor source--tomato, lettuce, cabbage, cauli-
flower, turnip, carrot, parsnip, and beet root.

Preparation of Vitamin P Extracts.- The isolation of "citrin", one of the earl-
iest vitamin P extracts, is based on its property of being precipitated by lead salts
in neutral solution and by alkali in anhydrous alcoholic solution. The isolation of
"citrin" from lemons results in two active components (27)--a crystalline "citrin"
which separates and supposedly contains mostly hesperidin and some eriodictin, and a
solution that contains a higher quantity of eriodictin and a smaller amount of hes-
peridin. "Citrin" is usually prepared from lemon peel, but some highly active con-
centrates have been prepared by this method using orange meal as a starting point.

Since hesperidin has been definitely shown to have vitamin P activity (15,23)
and to be especially ich in citrus fruits it is of special interest to the citrus
processor. Hendrickson and Kesterson (12) have already shown that highest concen-
tration of hesperidin occurs in young fruit and is more concentrated in the albedo,
rag and pulp of mature fruit. There is evidence, however, that vitamin P is fairly
uniformly distributed through the peel and the fruit (4) and that maximum vitamin P
is found in very mature fruit (25), possibly brought about by the demethylation of
hesperidin to eriodictin (9). The optimum method for isolating hesperidin as well
as the expected yield for numerous citrus varieties at various degrees of maturity
has been reported by Hendrickson and Kesterson (14) and a forthcoming paper (13) on
the purification of hesperidin will be presented at this year's Horticultural meeting.

It is of interest at this point to note that Higby (16) reported that crude hes-
peridin possessed vitamin P activity, while the purified product became inactive.

Florida Citrus Experiment Station,
Lake Alfred, Florida.


It was later shown by Scarborough (23) that pure hesperidin is active; however, most
investigators seem to feel that hesperidin's poor solubility explains its low activ-
ity as a concentrate. Numerous attempts have been made to make a more water-soluble
derivative from hesperidin (16,28).

At the present time, two vitamin P products derived from citrus are being mar-
keted nationally--"Hesper-C" by National Drug Company, each capsule of which is
claimed to contain 100 mg. of hesperidin concentrate and 100 mg. ascorbic acid. The
second product is called "C.V.P." and is put out by U. S. Vitamin Corporation.
Each of these capsules is claimed to contain 100 mg. of citrus flavonoid and 100 mg.
vitamin C. Daily dose is listed as being roughly six capsules in divided dosage.
Chromatographic analysis of these products showed the active ingredients of "Hesper-
C" to be mostly hesperidin, while "C.V.P." contained both naringin and hesperidin,
with naringin predominating. Other flavonoids are probably present in smaller quan-
tity in each product, but fail to show up for lack of a specific chromogenic agent.
The product "C.V.P." is claimed to be water soluble and more active than rutin or
hesperidin (7).

Summary.- In summary, therefore, "vitamin P" appears to be an elusive sub-
stance, difficult to analyze, but of considerable therapeutic use. It is associated
with hesperidin, for which the main source is citrus fruits, and would appear now,
after 19 years since it was first discovered, to require still further research.
Of greatest significance to the citrus grower and processor, however, is the use of
hesperidin as a "vitamin P" source and the suggestion by Martin (21) that combin-
ations of hesperidin and ascorbic acid should be regarded as supplemental therapy
of value in virtually all diseased states and specific in action with respect to
Literature Cited

1. Arcangeli, E. F. and F. S. Trucco. Colorimetric determination of the vitamin P.
Ann. Chim. Applicata Z4: 20-3. 1944.

2. Armentano, P., E. B. Iatz and I. Rusznyak. Determination of citrin in the urine.
Orvosi Hetilap 82: 1016-19. 1938.

3. Armentano, P. L., A. Bentsath, T. Beres, I. Rusznyak, and A. Szent-Gyorgi. The
effect of flavones on capillary permeability. Deut. Med. Wchnschr. 62: 1326-28.

4. Bacharach, A. L. and M. E. Coates. The vitamin P activities of citrus fruits,
rose hips, black currants and of some fruit products and concentrates. J. Soc.
Chem. Ind. 63: 198-200. 1944.

5. 'Bacharach, A. L., M. E. Coates and L. R. Middleton. A biological test for vita-
min P activity. Biochem. J. 36: 407-12. 1942.

6. Beiler, J. M. Biochemistry of the synergists; ascorbic acid and hesperidin.
Exp. Med. Surg. 12: 563-69. 1954.

Florida Citrus Experiment Station,
Lake Alfred, Florida.


Literature Cited (cont.)

7. Bio-flavonoid. Excerpt from Amer. J. Dig. Dis. 21: April 1954.

8. Biskind, M. S. and W. C. Martin. The use of citrus flavonoids inisspiratory
infections. Am. J. Dig. Dis. 21: 177. 1954.

9. Bruckner, V. and A. Szent-Gyorgi. Chemical nature of citrin. Nature 138: 1057.

10. Chem. and Eng. News. Term "vitamin P" recommended to be discontinued. 2827.
Aug. 14, 1950.

11. Davis, W. B. Determination of flavanones in citrus fruits. Anal. Chem. 19:
476-8. 1947.

12. Hendrickson, R. and J. W. Kesterson. Hesperidin, the principal glucoside of
oranges. Fla. Agr. Exp. Sta. Tech. Bul. 545: 5-43. 1954.

13. Hendrickson, R. and J. W,.Kesterson. Purification of crude hesperidin. (Forth-
coming) Proc. Fla. State Hort. Soc. 68: 1955.

14. Hendrickson, R. and J. W. Kesterson. Recovery of citrus glucosides. Proc. Fla.
State Hort. Soc. 67: 199-203. 1954.

15. Higby, R. H. The chemical nature of hesperidin and its experimental medical
use as a source of vitamin P. A review. J. Am. Pharm. Assoc. 30: 629-635.

16. Higby, R. H. The chemical nature of vitamin P. J. Am. Pharm. Assoc. 32: 74-77.

17. Horoschak, S. Clinical applications of the synergists; ascorbic acid and hes-
peridin. Exp. Med. Surg. 12: 570-97. 1954.

18. Lo, Teng-Yi. Vitamin P content of Kweichow vegetables and fruits. J. Chinese
Chem. Soc. 3: 196-203. 1942.

19. Lo, Teng-Yi and Sham-Ming Chen. Vitamin P content of vegetables as influenced
by chemical treatment. Food Research 11: 158-161. 1946.

20. Lorenz, A. J. and L. J. Arnold. Preparation and estimation of crude citrin
solutions from lemons. Food Research 6: 151-56. 1941.

21. Martin, G. Hesperidin and ascorbic acid--naturally occurring synergists. 5.
Conclusion. Exp. Med. Surg. 12: 597-8. 1954.

22. Merck Index of Chemicals and Drugs. 6th Ed. 1163 pp. 1952. Merck & Co., Inc.

23. Scarborough, H. Observations on the nature of vitamin P and the vitamin P
potency of certain foodstuffs. Biochem. J. 39: 271-78. 1945.

Florida Citrus Experiment Station,
Lake Alfred, Florida.


Literature Cited (cont.)

24. Scarborough, H. and A, L. Bacharach. Vitamins and hormones. Vol. VII: 1-55.
1949. Academic Press, Inc.

25. Sokoloff, B. and J. Redd. The vitamin P content of frozen orange concentrate.
Proc. Fla. State Hort. Soc. 62: 165-170. 1949.

26. Sokoloff, B. T. and J. B. Redd. Study on vitamin P. Part III: Vitamin P ther-
apy. Fla. So. Col. Monograph 3: 3-54. 1949.

27. Szent-Gyorgi, A. Method for producing citrin. Ztschr. Physiol. Chem. 255:
126-131. 1938.

28. Wilson, C. W. Alkylated chalcone derivative and methods of preparing the same.
U. S. Patent No. 2,425,291. August 5, 1947.

29. Wilson, C. W. A study of the boric acid color reaction of flavone derivatives.
J. Am. Chem. Soc. 61: 2303. 1939.

Florida Citrus Experiment Station,
Lake Alfred, Florida.

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