25 июня 2016г.
Abstract: Saponins are important bioactive compounds which have a wide array of biological and pharmacological properties. Styrax officinalis is a shrub growing in Syria and producing saponins as secondary metabolites. The first part of the present research study was aimed to describe the morphological characterization of the fruits of Styrax officinalis. The other part of this study covered saponins extraction from the fruits pericarps; their isolation and detection by chromatography methods.
Keywords: Styrax officinalis; Styrax-saponins; Styrax pericarp; saponins extraction.
Introduction: Styrax officinalis L. (Styracaceae) is widespread genus in the eastern Mediterranean countries, Italy, Greece, Turkey, Syria, Palestine and in the south-east Franc (Aliboufier). It is a shrub (up to 6 meters) with small whitish leaves [2] [10] [14]. Fruits Styrax officinalis mature in September-November [3]. Earlier studies on the pericarps reported the isolation of a triterpene sapogenin and a triterpene saponin [1] [8] [17] [11]. These saponins were determined as Styrax-saponins A, B, and C and Styrax-deacylsaponin [17]. The triterpenoid saponins are natural surfactants widely occurring in many plants and show a number of biological properties such as cholesterol-reducing [5], anti-proliferative cancer cell [16], antimicrobial [12], anti-fungal [13], anti-inflammatory, larvicidal, molluscicidal and spermicidal [4] [9] [6]. Due to their spermicidal effects, these saponins and similar compounds have potential as safe, effective, and economical contraceptive agents [7]. The current contraceptives such as oral contraceptive pills dominated the market but locally acting contraceptives are coming back due to the increase in sexually transmitted diseases. Local contraceptives with additional anti-infective properties would be of great value [15].These data promoted us to undertake a detailed evaluation of Styrax officinalis fruits containing saponins and their spermicidal activity starting with saponins extraction.
Purposes: The aim of the study was focused on description of the morphological characterization of the Styrax officinalis dried fruits and the isolation of total saponins from pericarps of Styrax officinalis L..
Materials and Methods
Chemicals: All chemicals used in the study were of analytical reagent grade and of highest quality available and were purchased from reliable firms and institutes.
Procurement of experimental plant: The experimental plant Styrax officinalis L. (Styracaceae) fruits were collected in Syria – Lattakia city, the month of August 2015.
Morphological description of Styrax officinalis dried fruit:
We conducted a study of morphological characters, specific for dried fruit and their parts. Dried fruits are of different sizes and various shapes: round, spherical. Size fruits - Length: 10 to 25 mm; width: 7 to 15 mm; thickness: 10 to 25 mm. Color of fruits range from light brown to grayish (Figure 1).
Dried fruit tough. In one fruit is 1 or 2 sometimes 3 large ovoid seeds (Figure 1). Tissue pericarp is clearly different. It is
consists of 3 layers: light brown outer layer of the fruit - exocarp; the hard shell surrounding
the seed is the inner layer of the pericarp- the endocarp; and a bright, yellowish transparent layer mesocarp between them (Fig. 2).
`Seeds are round, egg-shaped or oval-spherical shape. Their surface is smooth, shiny or matte, the color varies from light brown to brown (Fig. 3, a). The seed shows a distinct attachment scar (hilum) where it was joined to the placental region on the inner ovary wall (pericarp) (Fig. 3, b).
Figure (4) showing the thick, woody seed coat (endocarp). In the inner surface of the seed coat is like a thin membrane with silvery color - tegmen. Endosperm dimensions: length from 4 to 11 mm, thickness - from 1 to 3mm, width - from 3 to 8 mm. Endosperm consists of two cotyledons (Figure 5). This seed is monoembryonic – it develops only one embryo. By cutting the endosperm along, seen one white embryo (Fig. 5).
Saponins extraction from the pericarps:
Air-dried powdered pericarps of Styrax officinalis L. fruits (1055 g) were extracted
with ethanol and methanol in different concentrations. The average number of
extraction´s hours was (9h) in soxhlet apparatus with
average number of cycles per hour (1.4 c.h-1). The extracts were then concentrated on a rotary
evaporator to left a gummy residue which had been measured for humidity content and then stored in air-tight containers in cold room for further studies. Ethyl acetate, n-BuOH and water were used to purify the extraction. The pure saponins precipitate in water as white powder. We scanned this powder under the electron microscope (Figure 6).
And in methanol the pure saponins precipitate as transparent crystals. View of these crystals are shown under the optical microscope in (Figure 7).
Chromatographic purification and isolation:
Thin layer chromatography (TLC): By phytochemical screening, thin layer chromatography (TLC) was carried
out to isolate the saponins with the mobile phase (dichloromethane : methanol : water) in the ratio (40:30:10). Fluorescent spots for saponins were visualized under UV light. TLC was sprayed
with 10 % solution of blood in normal saline.
High Performance Liquid Chromatography (HPLC): Sample was dissolved in HPLC grade methanol. HPLC was performed for sample isolated by TLC using (C 18 column) and mobile phase (methanol 70, water 30) with (flow: 0,5 ml/min and wave: 260 nm).
Results
By extraction we obtained (605 g ) dry raw extract, which composes about (68%) percentage yield from the dry pericarps of Styrax officinalis L. Higher yield was obtained by using methanol as a solution for extraction- yield was (78.7%). Powder with dimensions smaller than (0.25 mm) is not suitable for extraction by soxhlet apparatus (it caused compactness mass which prevents access solution inside the plant mass leading to very law yield 16%). The quantity of pure saponins has not been detected yet.
Chromatographic purification: TLC of extract was carried out to confirm the presence of saponins and to isolate active saponin ingredients from the extracts. The blood hemolysis test on TLC showed clear spots.
HPLC profile of collected saponins spot from TLC was detected at a
wavelength 260 nm. The sharpness of peaks, its retention time (Rt min), height and percent area were recorded for saponins from TLC (Figure 8) and for washed extract (Figure 9). The HPLC chromatogram of extract has shown 4 peaks. As long as total saponins are similar in physical and chemical characteristics, so their separation not simple.
Conclusions:
Extract yield more than 68% from dry fruit pericarp weight. Thus, the Syrian medical plant Styrax officinalis is a promising source for saponins with expected spermicidal activity. That makes them an interesting research subject for pharmaceutical purposes.
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