The extraction and fractionation of Ferula samarcandica root exracts

ABSTRACT

This article presents high-quality and modern world-class methods for the fractionation of FERULA SAMARCANDICA root extract. Hexane, chloroform, butanol, ethyl acetate fractions were divided into successful fractions. Sequaterpene compounds, coumarin glycosides were first synthesized from the root part.

АННОТАЦИЯ

В данной статье представлены качественные и современные методы мирового уровня для фракционирования экстракта корня FERULA SAMARCANDICA. Фракции гексана, хлороформа, бутанола, этилацетата были разделены на успешные фракции. Секватерпеновые соединения, гликозиды кумарина впервые были синтезированы из корневой части.

Keywords: coumarins, flavonoids, phenols, terpenoids, steroids, extracts, aromas, hydrochloric acid, phytochemical, cancer.

Ключевые слова: кумарины, флавоноиды, фенолы, терпеноиды, стероиды, экстракты, ароматы, соляная кислота, фитохимия, рак.

Intradaction

Plants have been a constant source of drugs and considerable recent emphasis has been placed on finding novel therapeutic agents from medicinal plants. Many people prefer to use medicinal plants rather than chemical drugs. The Ferula genus from the family of Umbelliferae is a large genus of about 130 species distributed throughout the Mediterranean area and Central Asia [1]. Several species of this genus have been used in folk medicines [2], and investigations on the Ferula species have indicated antinociceptive, anti- inflammatory and antipyretic effects [3], contraceptive action [4,5], and smooth muscle relaxant activity [6,8]. This genus is well documented as a good source of bio- logically active compounds such as sesquiterpene coumarins [9] and sesquiterpene [10]. The Ferula genus has been found to be a rich source of gum resin [1]. The gum resins, which are obtained by incising the roots of several species, are used as spices and drugs in many countries.

Research methods and Experimental part. The roots of Ferula samarcandica (family Apiaceae) were collected in September 2018 from Surkhandarya region, Republic of Uzbekistan. The methods used in the process of investigation on the qualitative analysis as well as separation include thin layer chromatography (TLC), silica gel column chromatography (silica gel: 200-300 mesh, Qingdao Marine Chemical Company, China), sephadex LH-20 column chromatography (LH-20 gel: Amersham Pharmacia Biotech, Sweden), and HPLC with a ODS-A (50 µm) column produced by YMC Co.Ltd (Japan). The fractions were monitored by TLC, and spots were visualized by heating Silica gel plates sprayed with AlCl₃ in EtOH, ammonia vapor, 5% H₂SO₄ in EtOH withheating 105°C (Table 15). Ultraviolet absorption spectra were measured by an UV spectrophotometr (UV-2550 Shimadzu, Japan). ¹H NMR, ¹³C NMR, and 2D NMR spectra were recorded onVarian MR-400, VNMRS-600 NMR spectrometers with TMS as an internal standard.

Table 1.

Chromatography for extraction and fraction of Ferula samarcandica

Extraction and fractionation of Ferula  samarcandica root exracts The roots of F. samarcandica (2 kg) was extracted at room temperature by EtOH (95%, 4×12 L), with 5 times duration of extraction (each 24 h). The crude extract suspended with water (1:1) and fractionated successfully with petroleum ether (19.0 g), chloroform (16.0 g), ethyl acetate (32.0 g) and n-butanol (20.0 g). The sesquiterpene compounds, especially sesquiterpene coumarins were detected from the aqueous extract  by two-dimensional paper chromatography in system solvents (n-hexane: EtOAc 4:1-2:1, CHCl₃: MeOH 9:1). Sesquiterpenes and sesquiterpene coumarin glycosides were detected, using developed TLC plates, examined under UV at 254 nm and after spraying cerium sulfate as developer

The n-BuOH fraction of F. samarcandica (20 g) was subjected to HPD-300 macroporous adsorption resin (3:2, 7:3, 4:1, 19:1) to yield 6 fractions (Fsb-1-Fsb-6). Similarly fractions (Fsb-3, Fsb-5) ware further subjected to an opening ODS A-120 CC, eluted with a gradient solvents MeOH–H₂O and MeOH (10-90% and 100-0%) to afford five subfractions. All sub-fractions were analyzed by TLC and similar fractions were combined. Fraction Fso-4 and Fso-5 were investigated on Sephadex LH-20 with MeOH–H₂O as a eluent to yield compounds Fsp 21 (15 mg), 23 (50.4 mg) and subfraction of three compounds. Based on TLC analysis, subfractions were selected for HPLC analysis. The n-butanol fraction and all obtained subfractions were analyzed by HPLC using an optimized acetonitrile-water, ranging from 20% acetonitrile to 100% in 60 min at a flow rate of 1 mL/min. Samples were prepared in a concentration range from 1–10 mg/mL in methanol.

Figure 1.  Structures of compounds isolated from the roots of F. samarcandica

The isolation of pure compounds was performed by semi-preparative HPLC (45-70; 25-45%), yielding compounds 24 (11.5 mg), 25 (14.8 mg), 26 (17.2 mg), 27 (17. 5 mg), 28 (13 mg) and 29 (18 mg), 30 (17. 5 mg), 31 (14 mg), 32 (11 mg) 33 (8 mg) and  Phytochemical screening of ethanolic extracts of three medicinal plants was carried out using various chemical assay (such as TLC, HPLC, GC-MS) in order to identify either the presence or absence of secondary metabolites such as alkaloids, coumarins, phenolic compounds, flavonoids, glycosides, quinones, tannins, steroids and triterpenoids. The presence of coumarins, secoiridoids, and phenylethanoids is a characteristic feature of Fraxinus species. The secoiridoids occur mainly in the form of glucosides and esters of hydroxyphenylethyl alcohols. Lignans, flavonoids and simple phenolic compounds are also common, but they appear to have more limited distribution. The occurrence of coumarins distinguishes the genus Fraxinus from the other genera in Oleaceae. Effects of extraction solvent: To find the optimal and effective solvent for the extraction process, various solvents were tested as shown in Figure 2. Extraction with 70% to 90% ethanol, preferably from: 60% to 90%, and most preferably: 70%. Yielded extract of 10g (60%), 13.7g (70%), 12.2g (80%), 10g (90%) from 100 g of dried Fraxinus syriaca plant.  In our experiments, 70% ethanol was used due to the highest yield of extraction and the less toxicity of ethanol compared to the other solvents tested in this study (Fig 1).

Figure 2.

Results and Discussions. Compound Fs-22 was obtained as an amorphous, white solid and its empirical formula was determined  as C₂₄H₃₀O₆ by HR-ESI-MS (m/z 449.17355 [M+Na]⁺, calcd 449.17000. The ¹³C NMR spectrum of Fs-22 displayed 24 carbon signals, with nine being typical of an umbelliferone skeleton and ascribable to a sesquiterpene moiety (Table 16). The ¹H NMR spectrum of Fs-22 showed signals due to five aromatic protons at δ_H 6.28 (1H, d, J = 9.6 Hz, H-3), 7.91(1H, d, J = 9.6 Hz, H-4), 7.56 (1H, d, J = 8.6 Hz, H-5), 7.01 (1H, dd (J = 8.6; 2.4 Hz, H-6), and 7.07 (1H, d, J = 2.4 Hz, H-8), typical of an umbelliferone moiety The sesquiterpene moiety showed four sharp singlet signals, at δ_H 1.22 (3H, s, H-12ʹ), 1.09 (3H, s H-13ʹ), 1.22 (3H, s, H-14ʹ), 1.03 (3H, s, H-15ʹ) and an oxygenated methylene group at δ_H 4.52 (1H, dd, J = 10.4; 1.6 Hz, H-11ʹaxi) and 4.29 (1H, dd, J = 10.4; 6.4 Hz, H-11ʹeq).  The HSQC experiment allowed the identification of 10 methines, of which five, at δ_C 113.2 (C-3), 145.7 (C-4), 130.3 (C-5), 114.3 (C-6), 102.4 (C-8) were characteristic for the umbelliferone unit, and three, at δ_C 78.8 (C-3ʹ), 65.9 (C-5ʹ), and 61.1 (C-9ʹ), were attributable to the sesquiterpene moiety. Further HSQC correlations were indicative of two aliphatic methylenes at δ_C 39.7 (C-1ʹ), 27.3 (C-2ʹ), 61.1 (C-7ʹ), an oxygenated methylene at δ_C 66.8 (C-11ʹ), characteristic for C-11ʹ usually involved in the linkage with the coumarin moiety, and four methyls at δ_C 25.4 (C-12ʹ), 28.0 (C-13ʹ), 15.8 (C-14ʹ) and 17.7 (C-15ʹ). The HMBC correlations between the proton signal at δ_H 1.22 (Me-12ʹ) and the carbon resonance at δ_C 2.45 (C-7ʹ) and between the protons at δ_H 4.52 and 4.29 attributed to H₂-11ʹ with the same carbon C-9ʹ revealed the location of a tertiary methyl group (Me-12ʹ) at C-8. The correlation of Me-12ʹ with the quaternary carbon at δ 75.6 allowed the occurrence of a tertiary alcoholic function to be deduced at C-8ʹ. A further HMBC correlation between the singlet methyl at δ_H 1.03 (Me-15ʹ) and the carbon resonances at δ_C 39.7 (C-1ʹ), 41.2 (C-10ʹ), and 61.0 (C-9ʹ) revealed that the singlet methyl should be placed at C-10ʹ.

References:

1. Fernch, D., Ethnobothany of the Umbelliferae. In ‘‘The Chemistry and Biology of the Umbelliferae,’’ ed. Heywood, V. H., Academic press, London, pp. 385–412 (1971).
2. Uphof, J. C. Th., ‘‘Dictionary of Economic Plants’’ 2^nd ed., Verlag von J. Cramer, Lehre (1968).
3. Valencia, E., Feria, M., Diaz, J. G., Gonzalez, A., and Bermejo, J., Antinociceptive,      antiinflammatory and antipyretic effects of lipidin, a bicyclic sesquiterpene. Planta Med., 60, 395–399 (1994).
4. Singh, M. M., Agnihotri, A., Garg, S. N., Agarwal, S. K., Gupta, D. N., Keshri, G., and Kamboj, V. P., Antifertility and hormonal properties of certain carotene sesquiter- penes of Ferula jaeschkeana. Planta Med., 54, 492–494 (1988).
5. Parkash, A. O., Pathak, S., and Mathur, R., Postcoital contraceptive action in rats of hexane extract of the aerial parts of Ferula jaeschkeana. J. Ethnopharmacol., 34, 221–234 (1991).
6. Aqel, M. B., al-Khalil, S., Afifi, F., and al-Eisawi, D., Relaxant effects of Ferula Sinaica root extract on rabbit and guinea pig smooth muscle. J. Ethnopharmacol., 31, 373–381 (1991).
7. Aqel, M. B., al-Khalil, S., and Afifi, F., Effects of a Ferula sinaica root extract on the uterine smooth muscle of rat and guinea pig. J. Ethnopharmacol., 31, 291–297 (1991b).
8. al-Khalil, S., Aqel, M., Afifi, F., and al-Eisawi, D., Effects of an aqueous extract of Ferulas ovina on rabbit and guine a pig smooth muscle. J. Ethnopharmacol., 30, 35–42 (1990).
9. 9Abd El-Razek, M. H., Ohta, S., and Hirata, T., Terpenoid coumarins of the genus Ferula. Heterocycles, 60, 689– 716 (2003).
10. Lhuillier, A., Fabre, N., Cheble, E., Oueida, F., Maurel, S.,  Valentin,  A.,  Fouraste´,  I.,  and  Moulis,  C.,  Daucane sesquiterpenes from Ferula hermonis. J. Nat. Prod., 68, 468–471 (2005).