Tocopherols and Carotenoid in Fruit Pulp Oil of Seabuckthorn Growing in Dry Temperate Himalayas




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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 5th 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)

Parameters

Lahaul form

Spiti form

H. salicifolia


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 sub-classification 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).

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 5th 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 δ- tocotrienol. 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, δ- tocotrienol decreased with maturation of fruits (Table 2).

One study found that a-tocopherol constitutes up to 96% of the total tocopherols in fruit pulp oil (Jablczynska et al., 1994), being clearly the major isomer. In seed, g-tocopherol (up to 40% of total tocopherols) is another dominating isomer, in addition to a-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. a-tocopherol was a major constituent, followed by a-tocotrienol.



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