Tocopherols and Carotenoid in Fruit Pulp Oil of Seabuckthorn
Growing in Dry Temperate Himalayas
V. Singh, R.K. Gupta, R.C. Sawhney* and C. Arumughan**
CSK HP Agricultural University, Hill Agriculture Research and Extension Centre,
Bajaura 175125 (Kullu), H.P., India (E-mail: virendrasingh2003@yahoo.com)
*Defence Institute of Physiology and Allied Sciences (DRDO), Delhi 110054, India
** Agro Processing and Natural Products Division
Regional Research Laboratory (CSIR), Thiruvananthapuram, Kerala, India
ABSTRACT
The chemical studies were conducted on the dynamics of tocopherols and carotenoids in fruit pulp oils of
Hippophae rhamnoides ssp. turkestanica, a selection from Lahaul (semi-arid region) and another selection of
Hippophae rhamnoides ssp. turkestanica from Spiti (arid region) and H. salicifolia, a selection from Lahaul, all
raised at the CSK Himachal Pradesh Agricultural University’s Regional Research Station at Kukumseri (2730 m
asl) in Lahaul valley, a dry temperate region of Himachal Himalayas. Fruit samples were collected for analysis
during first week and third week of September and first week of October 2003, at an interval of 15 days. Fruits
of both forms of H. rhamnoides were reddish-orange, whereas, it was yellow in H. salicifolia. Total tocopherols
ranged from a maximum of 3063 ppm in Spiti form during first week of September, followed by 2786 ppm in
Lahaul form during third week of September and a minimum of 1919 ppm in H. salicifolia during first week of
September. In general, tocopherol content decreased with maturation of fruits. α-tocopherol was the most
dominant followed by α-tocol. Carotenoids were found to be maximum in Spiti form of H. rhamnoides (6207
ppm), followed by Lahaul form (4398 ppm) and least in H. salicifolia (1186 ppm). The carotenoid content was at
peak during third week of September in both forms of H. rhamnoides, where as it decreased with maturation of
fruits in H. salicifolia.
Key words: Seabuckthorn forms, pulp oil, tocopherols carotenoid and dry temperate Himalayas.
Introduction
Seabuckthorn, a nitrogen fixing deciduous thorny plant of 2-4 m height, grows widely in dry
temeperate Himalayas, comprising upper regions of Himachal Pradesh, Uttranchal, Ladakh and
Sikkim and Arunachal Pradesh in north-east India (Singh, 2003). Since ancient times,
seabuckthorn has been traditionally used in traditional medicines in Himalayan countries. Indian
scientists discovered as early as 1962, the anti-tumor effect of seabuckthorn bark (Ambaye et al.,
1962), however, real credit to start the work on the modern medicines of seabuckthorn goes to
Russian scientists during 1950s (Gurevich, 1956). The oil and juice, commercially produced from
its fruits and extracts from leaves, are used in Russian traditional and official medicines for the
wound healing, antibacterial, as anti-ulcer and anti-inflammatory (antiphlogistic) agent and as the
multivitamin product (Eidel’nant 1998). In the official medicine of Russia, the plant extracts are
commonly used as the components of various medicinal, compositions in dermatology,
stomatology, ophthalmology, veterinary and cosmetology etc. A chemical composition and
biological action of active principles from different organs of seabuckthorn have been reviewed in
a number of monographs (Eidel’nant, 1998; Matafonov, 1983). In China, positive effects of
seabuckthorn oil on heart diseases have also been reported (Li and Wang, 1994). Seabuckthorn
fruit oil has also been found to possess curative effect on atopic dermatitis (Yang et al., 2000).
Seabuckthorn oil also has wound healing effects (Varshney et al., 2003).
Studies carried out on biochemical characteristics revealed that fruit, leaves and other
parts of seabuckthorn are very rich sources of variety of anti-oxidants like carotenoids,
tocopherols, flavonoids and other bioactive substances (Yang and Kallio, 2002; Lu Rongsen,
2003) with medicinal properties, therefore it has a strong anti-oxidant and immunomodulatory
properties (Geetha et al., 2002) etc., which make this plant a very promising raw material for the
preparation of products for heath protection and treatment of various diseases. Present paper
describes the carotenoids and tocopherols in pulp (soft part) oils of fruits of three selected forms
of the seabuckthorn.
Materials and Methods
Propagation materials of seabuckthorn of desirable forms were selected from Madhgaon, a low
altitude place in semi-arid region of Lahaul, Shego, a high altitude place in arid region of Spiti,
both forms of H. rhamnoides ssp. turkestanica, and a form of H. salicifolia, selected from Tinu
village of Lahaul. Their seedlings were raised in 1995 at regional research center of our university
at Kukumseri (2730 m asl) in Lahaul valley, a semi-arid region of district Lahaul-Spiti, a dry
temperate zone of Himachal Himalayas (Table 1). All the three selected forms were mainly
characterized by high yield of fruits Fruit samples were collected thrice, i.e. on 5 th September (I
collection), 20th September (II collection) and 5th October 2003 (III collection) and analyzed for
carotenoids and tocopherols in the fruit pulp oils of all the three forms.
Estimation of oil content in fruit pulp (soft part) of seabuckthorn was done following
AOAC (1994). Tocopherols and tocotrienols in pulp oil of seabuckthorn berries extracted by
Christie’s method were analyzed in a Shimadzu make HPLC binary system (LC-10A) equipped
with amino column and UV-visible scanner at 297 nm with a solvent system, hexane:isopropane.
Conditions were standardized using authentic standards. Absorption at 460 nm of properly diluted
pulp oil was measured and the amount of carotenoids were calculated by plotting calibration curve
with authentic standard of β-carotene and expressed in terms of β-carotene. Each analysis was
conducted in triplicate.
Results and Discussion
Recent studies have found strong association between prevention of diseases like cancer and
cardiovascular and intake of fruit and vegetables, rich in anti-oxidants like E and carotenoids
(Halliwell, 1997). The biological properties of seabuckthorn fruit oil is believed to be governed by
the presence of variety of carotenoids and tocopherols (Lapic et al., 1983; Shvenik et al., 1983).
Table 1. Characteristics of 9 yr. old selected forms of seabuckthorn growing at
Kukumseri (2730 m asl)
H. salicifolia
Parameters
Lahaul form
Spiti form
Source
Madhgaon
Shego
Tinu
Altitude (m asl)
2625
3760
3150
Climate
Semi-arid
Arid
Semi-arid
Rainfall (mm/yr)
450
50
150
Fruit weight (g/100)
13.5
18.2
30.0
Fruit colour
Reddish orange
Reddish orange
Yellow
Carotenoids
Carotenoids, a fat-soluble group of naturally occurring plant pigments, are a subclassification of the terpenes. Diets rich in carotenoids are linked with a decreased risk of
heart diseases, cancer, and degenerative eye diseases like macular degeneration and
cataracts (Functional Foods and Nutraceuticals, 2003). Out of 600 carotenoids known in the
nature, 39 have been identified in seabuckthorn fruits. α-carotene, β-carotene, and
cryptoxanthin are the main vitamin A precursors. β-carotene intake is associated with
reduced risk of breast, stomach, esophageal, and pancreatic cancers (Nishino et al., 2000).
Carotenoids are the major source of vitamin A. Nutrient deficiency of vitamin A in humans
causes the growth disturbances, poor eye sight, reduced disease tolerance, besides affecting
the mucus membrane of gastrointestinal tract (Berezovski, 1973, Gotke and Wolf, 1990;
Savinov, 1948). Carotenoids raise physical and physiological capacity of organism to work
in stress conditions (Bogdanov et al., 1986). β-carotene has been used for preparing the drug
capsule for curing bed shores (Qingping et al., 1999). The ability of carotenoids, β-carotene
and cantaxanthin to act as anti-cancerogenic, anti-mutagenic and anti-swelling substances
gave an opportunity of using them for safeguarding from some kinds of skin cancers
(Kartangi et al., 1984; Mashkovski, 1997; Sergeev 1998). Oily substances of carotenoids
(seabuckthorn, dog-rose and etc.) have proven to be effective remedies against burn,
frostbites, ulcers, skin cancers and various gynecological illnesses (Akulinin 1958; Chugaeva
et al., 1964; Gatin 1963; Goodvin 1980; Mashkovski 1997; Rahimv et al., 1983).
In our study, maximum carotenoids was estimated in fruit pulp oil of Spiti form of H.
rhamnoides (6207 ppm) followed by Lahaul form of H. rhamnoides (4398 ppm) during II
collection a minimum of 1186 ppm in H. salicifolia. Content of carotenoids was at peak during II
collection in both forms of H. rhamnoides, whereas it decreased with maturation of fruits in H.
salicifolia (Fig.1).
Red and orange-reddish fruits of H. rhamnoides were more rich in carotenoids than
yellow fruits of H. salicifolia in the present study. Other studies have also found red and
orange-red fruits richer in carotenoids as compared with the less intensely coloured fruits
like yellow and orange-yellow (Lian et al., 2000). Contents of carotenoids were found to
increase with maturation and then decreased in over ripe fruits of H. rhamnoides. However,
carotenoids in present study, decreased in H. salicifolia. In other studies, carotenoid content
has been found increasing with the maturation of fruit (Zhang et al., 1989). Soft part (pulp)
oil of ssp. sinensis growing in Shanxi province of China had a maximum carotenoid level of
21400 ppm (Wei and Guo, 1996) and a minimum value of 21 ppm in north Caucasus
(Shaftan et al., 1986). Generally carotenoid level is maximum in peel oil followed by fruit
pulp oil and seed oil. Our study found 4398-6207 ppm carotenoids in pulp oil of H.
rhamnoides ssp. turkestanica, which has carotenoids in higher sides as compared to above
studies, however it is in lower side in H. salicfolia (1186).
Tocopherols
Vitamin E is an important dietary antioxidant and includes all tocols and tocotrienol
derivatives. Their most important antioxidant function appears to be the inhibition of lipid
peroxidation, scavenging lipid peroxyl radicals to yield lipid hydroperoxides and a
tocopheroxyl radical. The tocopheroxyl radical might be either reduced by ascorbate and
GSH or further oxidized to the respective quinone. Vitamin E deficiency in man causes
defects in development of nervous system in children and hemolysis (Sokol, 1996). Low
intakes of vitamin E and other anti-oxidants results into certain types of cancer and
atherosclerosis (Gey et al., 1991; Knekt, 1993; Rimm et al., 1993). It has been suggested that
supplementation with these anti-oxidants may decrease the risk of these and other
degenerative processes (Blot et al., 1993).
7000
6000
5000
4000
(ppm)
3000
2000
1000
0
Lahaul I
Lahaul II Lahaul III
Spiti I
Spiti II
Spiti III
SBT Forms
H.sal I
H.sal II
H.sal III
Fig.1. Carotenoid accumulation in pulp oil during fruit ripening of 3 forms of seabuckthorn
growing at Kukumseri (2730 m asl.).
Vitamin E was found maximum during I collection in the pulp oil of H. rhamnoides
from Spiti (3063 ppm), which decreased to 2237 ppm during II collection and a minimum of
1968 ppm during III collection. In Lahaul form, it increased from 2675 ppm during I
collection and peaked to 2786 ppm during II collection and declined to 2553 ppm during III
collection. Vitamin E was minimum in H. salicifolia and it decreased from a maximum of
1919 ppm during I collection to a minimum of 1290 ppm during III collection. Therefore,
vitamin E is considerably higher in H. rhamnoides forms than H. salicifolia and in general
decreased with maturation of fruits and (Table 2).
Table 2. Dynamics (ppm) of tocopherol profile in the fruit pulp oils of H. rhamnoides and
H. salicifolia forms
Sample form
αT1
αT3
βT1
βT3
γT1
γT3+δT1
δT3
Total
Lahaul I
1283
518
64
744
66
2675
Lahaul II
1727
361
45
234
419
3
2786
Lahaul III
1206
584
32
176
545
10
2553
Spiti I
1301
628
71
613
284
166
3063
Spiti II
1467
332
20
123
153
137
5
2237
Spiti III
951
427
55
157
349
29
1968
H. salicifolia I
875
98
10
685
30
1919
H. salicifolia II
776
212
35
252
17
1292
H. salicifolia III
221
279
128
649
13
1290
CD (P>0.05)
371
T1= Tocopherol, T3= Tocotrienol, I= Collection of fruits on 5 th September, II=20th September, III=5th October,
2003. Lahaul (semi-arid) and Spiti (arid) forms, both belong to H. rhamnoides ssp. turkestanica.
In China, the highest vitamin E content in pulp oil has been reported by Lu Rongsen
(1993) in H. rhamnoides subsp. sinensis (2480 ppm). We estimated a maximum of 3063 ppm
vitamin E in pulp oil of Spiti form of H. rhamnoides ssp. turkestanica growing under arid
conditions. Kallio et al. (2002) estimated in the soft part oil, the total value of tocopherols and
tocotrienols was typically 4000-7000 ppm in subsp. sinensis, significantly higher than the
corresponding values in the mongolica and rhamnoides (1000-2000 ppm), all raised under
Finland conditions. Among the tocopherols, in our study, α-tocopherol is the dominant one,
followed by α-tocotrienol. Content of α-tocopherol is at peak during II collection in all the three
forms. It makes 47.2-65% of the total tocopherols in Lahaul form, 42.5-65.5% in Spiti form and
17.1-60.1% in H. salicifolia.
Other components present are β-tocopherol, γ-tocopherol, β-tocotrienol, and δtocotrienol. β-tocotrienol has been found in H. rhamnoides from Spiti. α-tocopherol was
maximum during II collection and a minimum during III collection in both forms of H.
rhamnoides, being maximum of 1727 ppm in Lahaul form (II collection), followed by 1467
ppm in Spiti form (II). α-tocopherol was minimum in H. salicifolia and it decreased from
875 ppm during I collection to 221 during III collection. β-tocopherol was at peak during
first collection in both forms of H. rhamnoides, whereas it was during II collection in H.
salicifolia. α-tocotrienol increased in general with maturation of fruits. However, δtocotrienol decreased with maturation of fruits (Table 2).
One study found that -tocopherol constitutes up to 96% of the total tocopherols in fruit
pulp oil (Jablczynska et al., 1994), being clearly the major isomer. In seed, -tocopherol (up to
40% of total tocopherols) is another dominating isomer, in addition to -tocopherol (Lian et al.,
2000; Yang and Kallio, 2002; Kallio et al., 2002). Dillard et al. (1983) estimated that among
tocopherols, α-tocopherol is the most bioactive (30%), whereas the β-tocopherol has 25-50%
bioactivity and γ-tocopherol has 10-35%. Pulp oil part has generally been richer in α-tocopherol
than the seeds (Yang et al., 2001).
CONCLUSION
Red and orange-reddish fruits of H. rhamnoides were more rich in carotenoids and vitamin E than
yellow fruits of H. salicifolia in the present study. Maximum carotenoids was estimated in fruit
pulp oil of Spiti form (6207 ppm), followed by Lahaul form (4398 ppm) during II collection (20th
September 2003), a minimum of 1186 ppm in H. salicifolia. Content of carotenoids was at peak
during II collection in both forms of H. rhamnoides, whereas it decreased with maturation of
fruits in H. salicifolia. Vitamin E was found at peak in Spiti form (3063 ppm) and H. salicifolia
(1919 ppm) during I collection (5th September). In Lahaul form, it peaked during II collection
(2786 ppm). In general vitamin E content decreased with maturation of fruits. -tocopherol was a
major constituent, followed by -tocotrienol.
References
1. Akulinin I.I. 1958. Sov. Med., Vol 11: p.17-138
2. AOAC 1984. Official Methods of Analysis. Associations of Analytical Chemists.
Arlington, Virginia, USA.
3. Ambaye R.Y., Y.R. Khanolkar, T.B. Panse. 1962. Studies on Tumor Inhibitory Activity of
Indigenous Drugs. Part I. Tumor Inhibitory Acitivity of Hippophae Salicifolia D. Don.
Proceedings of Indian Academy of Science, Vol LVI (2), Sec. B: p. 123-129
4. Berezovski V.M. 1973. Chemistry of Vitamins, 632 p, Moscow.
5. Blot W.J., J.Y. Li, P.R. Taylor, W. Guo, S. Dawsey, G.Q. Wang, C.S. Yang, S.F. Zheng,
M. Gail, G.Y. Li. 1993. Nutrition Intervention Trials in Linxian, China: supplementation
with specific vitamin/mineral combinations, cancer incidence, and disease-specific
mortality in the general population. Journal of The National Cancer Institute, Vol 85: p.
1483-1492
6. Bogdanov N., L.G. Grozdova, M.S. Belakovski. 1986. Problems of Nutrition, Vol 3.
7. Chugaeva V.N., A.I. Gurvich, M.G.Ushakova. 1964. Mater. Conf. on Vitamins from
Natural Raw Mat., p. 172-177, Kuybushev.
8. Dillard C.J., V.C. Gavino, A.L. Tappel. 1983. Relative Antioxidant Effectiveness of Tocopherol and -Tocopherol in Iron-Loaded Rats. Journal of Nutrition, Vol 131
9. Eidel’nant, A. 1998. Seabuckthorn (Hippophae rhamnoides) in Medicine and Cosmetics
Culinary, 376 p, Kron-Press, Moscow
10. Functional Foods and Nutraceuticals, 2003, 64 p.
11. Gatin J.I. 1963. Seabuckthorn, Moscow, 159 p.
12. Geetha S., M. Sai Ram Singh, V. Ilavazhagan G., R.C. Sawhney. 2002. Anti-oxidant and
Immunomodulatory Properties of Seabuckthorn (Hippophae rhamnoides) an in-vitro
study. Journal of Ethnopharmacology, Vol 79: p. 373-378
13. Gey K. F., P. Puska, P. Jordan, U.K. Moser. 1991. Inverse Correlation between Plasma
Vitamin E and Mortality from Ischemic Heart Disease in Cross-cultural Epidemiology.
American Journal of Clinical Nutrition, Vol 53: p. 326 S - 334 S
14. Goodvin T.W. 1980. The Biochemistry of the Carotenoids. Vol 1, Chpman and Hall, 19,
London, NY.
15. Gotke R. K., G.D. Wolf. 1990. Ervebs-Obstbau, Vol 32 (8): p.224-226
16. Gurevich S. K. 1956. The Application of Seabuckthorn Oil in Ophthalmology. Vesttn
Oftalmologii, Vol 2: p. 30-33
17. Halliwell B. 1997. Anti-oxidants and Human Diseases-A General Introduction. Nutr.
Rev., Vol 55: p. 44-52
18. Jablczynska R., U. Krawczyk, K. Minkowski. 1994. Fatty Acids and Tocopherols in Some
Varieties of Hippophaë rhamnoides. Acta Poloniae Pharmaceutica-Drug Research, Vol 51
(3): p. 267-269
19. Kallio H., B. R. Yang, P. Peippo, R. Tahvonen, R. Pan. 2002. Triacylglycerols,
Glycerophospholipids, Tocopherols and Tocotrienols in Berries and Seeds of two
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
subspecies (ssp. sinensis and ssp. mongolica) of Seabuckthorn (Hippophaë rhamnoides).
J. Agric. Food Chem., Vol 50: p. 3004-3009
Kartangi, N., L.A. Knln, A. Stein. 1984. J. of Lipid Research, Vol 25: p. 400-406
Knekt P. 1993. Epidemiology of Vitamin E: Evidence for Anticancer Effects in Human.
In: Vitamin E in Health and Disease (Packer, L. and Fuchs, J. eds.), p. 513-527, Marcel
Dekker, New York.
Lapic A.S., L.D. Ageeva, N.Y. Bakina. 1983. In: Biol. Chem. Pharm. of Seabuckthorn, p.
105-107, Novosibrsk.
Li Y., L. Wang. 1994. Preliminary Analysis of the Clinical Effects of Seabuckthorn Oil
Capsule and Seabuckthorn Saimaitong Capsule (containing seabuckthorn seed oil and
Chinese herbs) on Ischemic Apoplexy. Hippophae, Vol 7 (2): p. 45-47 (In Chinese)
Lian Y. S., S. G. Lu, S. K. Xue, X. L. Chen. 2000. In: Biology and Chemistry of the Genus
Hippophaë, p. 88-91, Gansu Scientific and Technological Publishing House, Lanzhou,
China.
Mashkovski A.D. 1997. Lecarstvennaye Sredstv., Moscow, 625 p.
Matafonov I.I. 1983. Seabuckthorn Action on Animal Organism (Khundanova, L.L. ed.in-chief), Nauka, Sibirsk. Otdel, Novosibirsk, 166 p.
Nishino H., et al. 2000. Cancer Prevention by Carotenoids. Biofactors, Vol 13: p. 89
Qingping G., J. Zhaoxiang, X. Xiaoyang, D. Haiyan. 1999. Assaying of β-Carotene in Bed
Sore Tissue Regeneration Soft Capsule. In: Proceedings of Intenational Workshop on
Seabuckthorn, p. 10-12, Beijing, China.
Rahimv I.F., L.D. Lebedeva, N.D. Khasanshina. 1983. In: Biol. Chem. Pharm. of
Seabuckthorn, p. 109-120, Novosibirsk.
Rimm E.B., M.J. Stampfer, A. Ascherio, E. Giovannucci, G.A, Colditz, W.C. Willett.
1993. Vitamin E Consumption and the Risk of Coronary Heart Disease in Men. New
England Journal Medicine, Vol 328: p. 1450-1456
Savinov B.G. 1948. Carotene (provitamin A) and obtaining of its preparations. Kiev. 229
p.
Sergeev A.V. 1998. Prob.Nur., Vol 3: p. 27-30
Shaftan E. A., N. S. Mikhailova, A. V. Pekhov, N. F. Dyuban'kova. 1986. Comparative
Study of Chladone and Carbon dioxide Extractions from Air-dry Pulp of Fruit of
Hippophae rhamnoides of Caucasian and Siberian origin. Rastit. Resur., Vol 22 (1): p.
60-66
Shvenik L.A., G.P. Kukina, V.L. Salenko. 1983. In: Biol. Chem. Pharm of Seabuckthorn,
p. 102-105, Novosibirsk.
Singh V. 2003. Geographical Adaptation and Distribution of Seabuckthorn (Hippophae
L.) Resources. In: A Multipurpose Wonder Plant-Botany. Vol. I. Botany, Harvesting and
Processing Technologies (V. Singh, Editor in Chief), p. 21-34, Indus Publishing
Company, New Delhi, 518p.
Sokol R.J. 1996. Vitamin E. In: Presnet Knowledge in Nutrition (Ziegler, E.E. and Filer,
L.J. eds), p. 130-136, ILSI Press, Washington DC.
Varshney A.C., A. Kumar, S.P. Tyagi, V. Singh. 2003. Therapeutic Evaluation of
Seabuckthorn Oil in Cutaneous Burn Wound Healing in Bovine: A Clinicohaematological Study. In: 1st Congress of the International Seabuckthorn Association, 1419 Sept. 2003, Berlin, Germany.
Wei C. Y., W. Y. Guo. 1996. A Preliminary Study on the Changing Pattern of Oil Content
of Hippophaë rhamnoides ssp. sinensis in Shanxi province. Hippophaë, Vol 9 (2): p.3743
Yang B. R., H. Kallio. 2002. Composition and Physiological Effects of Seabuckthorn
Lipids. Trends Food Sci. Technol., Vol 13: p. 160-167
Yang B., O.K. Kalimo, R.L. Tahvonen, K. Kalimo, L.M. Mattila, J.K. Katajisto, H. P.
Kallio. 2000. Effect of Dietary Supplementation with Seabuckthorn (Hippophae
rhamnoides) Seed and Pulp Oils on the Fatty Acid Composition of Skin
Glycerophospholipids of Patients with Atopic Dermatitis. J.Nutr.Biocm., Vol 11: p. 338340
41. Yang B., H. Kallio, P. Peippo, R. Tahvonen, P. Ruilin, Z. Xuren, F. Yuhua, H. Yicai.
2001. Tocopherols in Seeds and Berries of two subspecies of Seabuckthorn (Hippophae
rhamnoides ssp. sinensis and mongolica). In: Proceedings of International Workshop on
Seabuckthorn, p. 151-153, Feb.18-21, 2001, N.Delhi.
Zhang W., J. Yan, J. Duo, B. Ren, J. Guo. 1989. Preliminary Study of Biochemical Constituents
of Berry of Seabuckthorn Growing in Shanxi Province and their Changing Trend. In: Proc. of
Int. Symp. Seabuckthorn (Hippophae rhamnoides L.), p. 96-105, Oct 19-23, Xian, China.