Morphological and biochemical comparison of Hippophae rhamnoides, Elaeagnus umbellata and Crataegus oxyacantha intra- and interspecifically

The hilly areas of Pakistan are very rich in medicinal plants. Among these, Hippophae rhamnoides ssp turkestanica L., Elaeagnus umbellata Thunb. and Crataegus oxyacantha L. are native plants of Northern Pakistan. Intraand interspecific comparisons were made to investigate their morphological and biochemical composition using morpho-molecular techniques. The comparisons indicated large genetic and biochemical variation among the populations of each species and between the different species. The sodium dodecyl sulphate polycrylamide gel electrophoresis comparisons using total seed proteins of Hippophae rhamnoides, Elaeagnus umbellata and Crataegus oxyacantha indicated many common proteins and some variable proteins among the species. The biochemical comparison of Vitamin C, fatty oil and phytosterol content was also found to be variable among the species. Vitamin C and fatty oil contents were highest in Hippophae rhamnoides while phytosterol content was highest in Crataegus oxyacantha when compared with the other species tested in this study. Significant variation in morphological characters including plant height, number of branches per plant, number of thorns, plant canopy and berry weight was also observed in these three plant species.


Introduction
Hilly areas of Pakistan including Baluchistan, North Western Frontier Province, Azad Kashmir and northern Areas have a very rich and diverse flora due to their diverse climate, soil conditions and multiple ecological regions. The medicinal plant resources are not only abundant but are also rich in genetic diversity and biochemical composition. The herbs are extensively used locally for treating many diseases; however, their commercial exploitation is limited because of the lack of a scientific basis for their use (Hussain and Khaliq 1996). The farmers in the area are very poor and the cultivated land plots are either very small or unmanageable due to soil degradation and specific topography. Quite recently an initiative has been undertaken to characterise the medicinal plants for genetic and biochemical variation in order to improve medicinal plants for commercial purposes. Three plant species, Sea buckthorn (Hippophae rhamnoides ssp turkestanica L.), Autumn olive (Elaeagnus umbellata Thunb.) and Hawthorn (Crataegus oxyacantha L.), native to Azad Kashmir and northern areas of Pakistan, were investigated intra-and interspecifically for genetic and biochemical composition using morpho-molecular and biochemical techniques Kamal 2002, Ahmad et al. 2003).

Hippophae rhamnoides
Hippophae rhamnoides is a shrub or small tree of the genus Hippophae. The genus belongs to the family Elaeagnaceae that consists of six species and 10 sub-species, among which the most economically important one is Hippophae rhamnoides Linn., commonly known as Sea buckthorn (Rongsen 1992). The only sub-species found in the northern areas of Pakistan is Hippophae rhamnoides ssp. turkestanica, which is widespread in Central Asia and West Asia; that includes Afghanistan, Tajikistan, Turkmenistan, Uzbekistan, Kirghisistan, Xinjiang Province of China and Northern India. It is the only sub-species that can withstand the harsh bio-physical conditions characterised by arid, hot summers and cold winters (Rongsen 1992). Hippophae rhamnoides fruit contains 60 to 80% juice rich in sugar, organic acids, amino acids and vitamins. Vitamin C content is 200 to 1 500mg/100g, which is 5-100 times higher than any other known fruit or vegetable (Ahmad and Kamal 2002). The oil content ranges from 1.5-3.5% in fruit pulp and about 9.9-19.5% in seeds (Rongsen 1992). Oil from the juice and pulp is rich in palmitic and palmitoleic acids, while the oil from the seed contains the essential fatty acids, linoleic acid and linolenic acid. The oil from the seed and juice also contains Vitamin E and carotene (Bernath andFoldesi 1992, Ma andCui 1989).
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of total seed protein/allozymes revealed genetic diversity among the different individuals belonging to Hippophae rhamnoides ssp. turkestanica (Ahmad and Kamal 2002). Four woody species of the Elaeagnaceae family including Hippophae rhamnoides, russian olive, buffaloberry and silverberry have been compared by the Random Amplification of Polymorphic DNA (RAPD) markers technique, which showed phenotypic diversity among these species (Chowdhury et al. 2000).

Elaeagnus umbellata
Elaeagnus umbellata is a rapid-growing large shrub that has been widely planted for shelterbelts, for food, cover for wildlife, for roadside reclamation, and for soil stabilisation. The plant's tolerance to high pH soils, drought, pollutants and it's ability for nitrogen fixation makes this shrub a very successful invasive species. The fruits are 1.25cm to 1.5cm in size, appearing light green in mid-summer and turning to red in the autumn (Dirr 1998). Elaeagnus umbellata fruit contains about 8.3% sugars, 4.5% protein and 1% ash. Its Vitamin C content is about 12mg/100g. The Elaeagnus umbellata berry is an excellent source of minerals, vitamins (A, C, E), flavonoids, essential fatty acids and other bioactive compounds (Graham 1964). Nutritionists have found that the red berries contain high concentrations of lycopene, the pigment that colours tomatoes red. Ripe tomatoes are currently being used as a source of lycopene (7.85mg/100g) but Elaeagnus umbellata contains very high amounts of lycopene, i.e. 53.96mg/100g (Deman 1980).
Lycopene is one of the most potent known antioxidants and has been suggested to prevent carcinogenesis and atherogenesis by protecting critical biomolecules including lipids, low density lipoproteins (LDL), proteins and DNA (Agarwal and Rao 1998). The seeds and flowers of Elaeagnus umbellata are used for treating coughs and essential oil for pulmonary infections. The flowers are also used as an astringent and for the treatment of cardiac ailments (Chopra et al. 1986). Elaeagnus umbellata has been proved to be useful as a deterrent to heart disease and for cervix and gastrointestinal tract cancer (Dirr 1998).

Crataegus oxyacantha
Crataegus, a genus belonging to the rose family, is now recognised to have about 280 species found in northern temperate regions, including North America, Europe and northern Asia. The parts of Crataegus oxyacantha used as medicines are flowers, leaves and berries. In Crataegus oxyacantha these contain a variety of bioflavonoid-like complexes, oligomeric procyanidins (OPCc), vitexin, quercetin and hyperoside (Hamon 1988). Since the 17 th century, Crataegus oxyacantha has been used to treat various heart conditions and today is also believed to lower blood pressure (Occhiuto and Circosta 1986). Clinical trials have confirmed Crataegus oxyacantha to be beneficial for persons with Stage 2 (mild) congestive heart failure. Crataegus oxyacantha improves the blood and oxygen supply to the heart by dilating the coronary vessels (Weihmayr and Ernst 1996).
All of the above-mentioned species are berry-producing plants, having similar morphological characters (plant height, size of berries, stem girth and size of thorns), and they were therefore tested to investigate their evolutionary relationships.

Plant collection
Plant collections -of four populations each of Hippophae rhamnoides, Elaeagnus umbellata and Crataegus oxyacantha) -were made in September 2002. These plants grow as wild plants in Northern Pakistan and Azad Kashmir. Four populations of Hippophae rhamnoides were collected from the district of Skardu, whereas Elaeagnus umbellata and Crataegus oxyacantha plants were collected from the district of Rawalakot Azad Kashmir.

Morphological analysis
Morphological characters -including plant height, number of main branches per plant, number of sub-branches per main branch of the plant, number of thorns per main branch of the plant, stem girth, plant canopy and 100 berries' weight -were compared from five randomly-selected plants (in triplicate for each population) and mean values were taken for analysis using the ANOVA method of Steel and Torrie (1980).

Molecular studies
Extraction of total proteins Total protein was extracted from 1g of seed of 12 plants, including 10 populations of Hippophae rhamnoides and one plant each of Crataegus oxyacantha and Elaeagnus umbellata, with 0.125M tris/borate, pH 8.9 by the method described by Laemmli (1970). Sodium dodecyl sulphate polyacrylamide gels contain 12 wells for electrophoresis; therefore 10 protein samples of Hippophae rhamnoides and one protein sample of each Crataegus oxyacantha and Elaeagnus umbellata were loaded for electrophoresis. All the obtained extracts were kept at 4°C for 24h and then centrifuged at 10 000rpm for 20min. The supernatants were used for electrophoresis.

Determination of protein content
Protein levels of cytosolics were determined using a Spectronic 20-D (Milton Roy Company) spectrophotometer at 595nm, using Coomassie blue G-250 as a protein-binding dye (Bradford 1976). Bovine serum albumin was used as a protein standard.

Protein electrophoresis
Sodium dodecyl sulphate polyacrylamide gel electrophoresis was carried out according to the method described by Laemmli (1970), using 12.5% acrylamide concentration. Before electrophoresis each sample (protein extract) was heated at 100°C for 2min in 10mM Tris HCl buffer (pH 7) containing 2% (w/v) sodium dodecyl sulphate, 2% 2-mercaptoethanol and 5% (w/v) glycerol. A 30µl aliquot of the protein was loaded per well after adding one drop of bromophenol blue and glycerine. A constant current of 250mA was applied for 3h. After the termination of this process the gel was removed from the apparatus and protein sub-units were stained with Coomassie blue R-250, using standard techniques. Finally, the gel was scanned using Jel-Pro-Analyzer version 3.3 (Media Cybermetics, 93-97). The R f values of the protein bands were calculated as follows: R f = distance moved by the protein band/distance moved by solvent front.

Biochemical analysis
Fruits/berries were collected from the same plants for biochemical analysis (i.e. four populations each of Hippophae rhamnoides, Crataegus oxyacantha and Elaeagnus umbellata were selected for biochemical analysis).
Vitamin C content of fruits Vitamin C was determined using the phenol indophenol dye method described by the Association of Official Analytical Chemists (1984). Ten grams of the fresh berries/fruits were blended with metaphosphoric-acetic acid-extracting solution, which acts as a stabilising medium for Vitamin C. Five millimetres of the filtrate extract was then titrated against standard indophenol dye to the pink end point. The experiment was repeated three times.

Lipid content of berries
Oil from the berries/fruits of different plants was extracted according to standard methods described by the American Association of Cereal Chemists (1983). Fruits were dried in an oven at 105°C for 6-12h to constant weight. Ten grams of dried samples were extracted for oil in Soxhlet apparatus (30-40°C) for 6h using diethyl ether as solvent. The solvent was removed under vacuum and the residual oil was dried over anhydrous Na 2 SO 4 . The experiment was repeated three times. Analytical grade chemicals (Merck) were used for extraction of oil.

Sterol determinations
Sterol estimation was carried out by the Lieberman-Burchard method (Said et al. 1995). One gram samples of oil -obtained after Soxhlet extraction -were diluted with chloroform to 10ml. Three ml of diluted sample solutions were taken and their absorbance was determined after adding 2ml of chloroform and Lieberman-Burchard reagent, containing 0.5ml of sulphuric acid dissolved in 10ml of acetic anhydride. Lieberman-Burchard reagent reacts with the sterol to produce a characteristic green colour, the absorbance of which was determined on a Spectronic 20-D (Milton Roy Company) spectrophotometer at 640nm. Ten mg of standard cholesterol was dissolved in 10ml chloroform. Choles-5-en-3-β-ol was used as standard cholesterol, the minimum purity of which was 95%.

Morphological analysis
Seven morphological parameters were investigated; results were analysed statistically and are compared in Table 1. The populations of Hippophae rhamnoides, Crataegus oxyacantha and Elaeagnus umbelatta indicated intra-and interspecific variability (P ≤ 0.01). The plant height was the highest among the Hippophae rhamnoides populations while it was the lowest among the Crataegus oxyacantha populations. The average number of main branches per plant was highest in Elaeagnus umbellata (6), variable in Hippophae rhamnoides (2-5) and lowest in Crataegus oxyacantha (3). Similar variability in Hippophae rhamnoides plant height was recorded in earlier investigations (Sabir et al. 2003). Rousi (1971) and Yao and Tigerstedt (1994) reported Hippophae rhamnoides as extremely variable in height, from a small bush less than 50cm to a tree more than 20m high, whereas Elaeagnus umbellata was found to be a large, spreading, spiny-branched shrub often obtaining 3.5 to 5.5m in height and 3.5 to 5.5m in width (Dirr 1998 (20). Variation in plant canopy was also found to be highly significant among the populations of Hippophae rhamnoides, Crataegus oxyacantha and Elaeagnus umbellata. Elaeagnus umbellata had the highest plant canopy (145cm) followed by Crataegus oxyacantha (79.8cm), while in Hippophae rhamnoides it ranged from 35cm to 50cm. The number of thorns ranged from 50-67 per main branch among Hippophae rhamnoides populations. In Crataegus oxyacantha the number was also found to be in the same range (66). The overall comparison was highly significant, indicating large variability among the individual populations. However, in Elaeagnus umbellata, no thorns were recorded on the main branch but thorns were present on the lateral branches. When stem girth was compared, no significant variation was found among the populations compared, indicating the similar nature of these plant species. The comparison of 100 berries' weight indicated significant variation between the species. The weight of 100 berries was the highest (3.961g) in Crataegus oxyacantha, intermediate (1.971g) in Elaeagnus umbellata and the lowest (0.56g) in Hippophae rhamnoides. The observation on Hippophae rhamnoides berries differs from earlier observations (Yao 1994), in which the weight varied from 4-60g/100 berries among different strains within natural populations, and exceeded 60g in some Russian cultivars.

Molecular studies
In order to observe variability at a genetic level, the gene products (seed proteins) were compared from 10 populations of Hippophae rhamnoides and one species each of Crataegus oxyacantha and Elaeagnus umbellata, by the fractionation of total seed proteins in sodium dodecyl sulphate polyacrylamide gel electrophoresis. In the absence of standard protein markers, comparisons among different genotypes were made on the basis of banding pattern or electrophoretic mobility within the gel. Photographs were taken by placing the measuring scale along one side of the gel, starting from the top of the wells where protein was loaded, in order to note the distance covered by each protein band. A total of 64 protein bands appeared in the gel with different R f values showing the great genetic variability among the populations. The R f values of different protein bands are shown in Table 2. Table 2 showed that only two bands with R f values of 0.34 and 0.43 were found to be common in all the populations of Hippophae rhamnoides compared. The result, therefore, indicates that the populations are related, with ample variability that may have occurred due to their adaptability under variable microclimatic conditions. Such variation in seed protein banding patterns has been found in earlier investigations (Ahmed et al. 2003). Ahmed and Kamal (2002) (2002) reported the maximum R f value of proteins to be 0.98, whereas the maximum value in the present investigation was lower (R f =0.86). Isozyme analysis has also shown large genetic diversity at the species, sub-species, and population level in Hippophae rhamnoides (Yao and Tigerstedt 1993).
The Crataegus oxyacantha and Elaeagnus umbellata seed proteins were also loaded along with the Hippophae rhamnoides in the sodium dodecyl sulphate polyacrylamide gel electrophoresis, in order to make a biochemical (evolutionary) comparison between these berry-producing plants and Hippophae rhamnoides. Table 2 shows the R f values of protein bands extracted from seeds of Crataegus oxyacantha and Elaeagnus umbellata. Both Crataegus oxyacantha and Elaeagnus umbellata showed two bands with R f values of 0.34 and 0.43, which were common with the R f values of Hippophae rhamnoides populations. Hippophae rhamnoides and Elaeagnus umbellata belong to the same family (Elaeagnaceae) and the resemblance of their banding pattern could be explained by this taxonomic relationship. However, the resemblance in the banding pattern of Crataegus oxyacantha with Hippophae rhamnoides and Elaeagnus umbellata was not expected. This does not necessarily mean that the same proteins exist in their seeds. It may mean that different proteins with similar charges have produced similar banding patterns.

Biochemical analysis
When the populations of Hippophae rhamnoides were compared biochemically on the basis of Vitamin C, fatty oils and phytosterol contents, a wide range of variation was observed among the populations (Table 3). The average concentrations of Vitamin C were found to be 196.25mg/100g in Hippophae rhamnoides, 109.62mg/100g in Crataegus oxycantha and 11.47mg/100g in Elaeagnus umbellata (as shown in Table 4). The concentration of Vitamin C in Hippophae rhamnoides was highly variable among the populations as reported earlier (Karhu et al. 1999) but the concentration in our investigation were low. Rongsen (1992) reported Vitamin C concentrations from 200-1500mg/100g in Hippophae rhamnoides fruits. The lower concentration of Vitamin C in the present investigation may be due to the specific geographic nature of the area, where a short reproductive season prevails (Yao and Tigerstedt 1995).
The average concentrations of oil in the pulp were found to be 3.85g/100g in, Hippophae rhamnoides, 0.34g/100g in Crataegus oxycantha, and 1.62g/100g in Elaeagnus umbellata (Table 4). The range of fatty oil content -from 3.4 to 4.5% -found in the present investigation was the highest reported so far. Rongsen (1992) reported oil contents from 1.5-3.5% in fruit pulp. The higher concentrations of oil in ssp. turkestanica may be very important in regard to its use in medicines.
Phytosterols are plant sterols with structures related to cholesterol, which are capable of lowering plasma cholesterol in humans. Elevated blood cholesterol is one of the well-established risk factors for coronary heart disease  (Thurnham 1999). Phytosterols are the major constituents of the unsaponifiable fraction of Hippophae rhamnoides oils. The major phytosterol in Hippophae rhamnoides oil is βsitosterol, with 5-avenasterol second in quantitative importance. Other phytosterols are present in relatively minor quantities. The total concentration of phytosterol is quite high in Hippophae rhamnoides and may exceed that of soybean oil by 4-20 times. The average concentrations of sterol were found to be 14.2mg/g in Hippophae rhamnoides, 115.2mg/g in Crataegus oxyacantha and 1.45mg/g in Elaeagnus umbellata (Table 4).

Conclusions
This investigation is based on morphological, molecular and biochemical characterisation of berries/fruits of Hippophae rhamnoides, Crataegus oxyacantha and Elaeagnus umbellata. Intra-and interspecific variability among the plants will help to breed better varieties, using conventional methods of breeding. The high concentration of oil found in Hippophae rhamminodes and Elaeagnus umbellata will have commercial importance and will help the local community in marketing their farm produce. Due to the high content of Vitamin C these berries can be used in making fruit juices and beverages. As Hippophae rhamnoides and Crataegus oxyacantha oil are concentrated sources of phytosterols, which compete with cholesterol in terms of absorption into in the body, use of oil from these plants might be helpful in preventing heart diseases.