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High performance liquid chromatography based quantification of reserpine in Rauwolfia tetraphylla L. and enhanced production through precursor feeding

Rohela Gulab Khan, Bylla Prasad, Pendli Sreenu, Korra Rajender, Gandu Rajender, Reuben Christopher

Acta Chromatographica

2022

Vol. 34, no. 2

120--129

Rauwolfia tetraphylla L., is an important medicinal plant in Apocynaceae family and is recognized as an alternative source to Rauwolfia serpentina L., in terms of anti-hypertensive alkaloid production i.e. reserpine. In view of this, the present study is conducted to estimate the reserpine content in different parts (leaf, stem and root) of field grown plants (2 years old), tissue cultured plantlets (R1) (two months old) and cell suspensions cultures (two months old with and without precursor feeding) of R. tetraphylla by using high performance liquid chromatography (HPLC) technique. Overall maximum content of reserpine (in %) was estimated from the root samples. Roots of field grown plants has recorded high percent of reserpine (0.39%) followed by roots of tissue cultured plantlets (0.35%) and root callus based cell suspension cultures (0.38 %) which was fed with precursor amino acid (100 mg/L of tryptophan). In control type of root callus based cell suspension cultures, reserpine content was quantified as 0.14%; by precursor feeding (100 mg/L of tryptophan) it was enhanced to 0.38%. In conclusion, the reserpine content (0.35 and 0.38%) produced by the roots of tissue cultured plantlets (R1) and 100 mg/L tryptophan fed root callus based cell suspensions was comparable to that of the reserpine content (0.39%) of root parts of field grown plants. The present study demonstrates the reserpine production by in vitro cell suspension cultures throughout the year without sacrificing the medicinal plants.

Preparative separation of two alkaloids from devil pepper radix (Rauvolfia verticillata [Lour.] Baill.) by ph-zone-refining counter-current chromatography

Duan W. J., Liu Q., Zhao R. X., Mu Y., Guo L. P., Li D. P., Wang X.

Acta Chromatographica

2018

Vol. 30, no. 2

81--84

A method was developed for the preparative separation of two alkaloids from the crude extract of the radix of Rauvolfia verticillata (Lour.) Baill. in a single run. The two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (5:5:2:8, v/v), where triethylamine (40 mmol/L) was added to the upper organic phase as the stationary phase and hydrochloric acid (10 mmol/L) was added to the lower aqueous phase as the mobile phase, was selected for this separation by pH-zone-refining counter-current chromatography (PZRCCC). For the preparative separation, the apparatus was rotated at a speed 850 rpm, while the mobile phase was pumped into the column at 2 mL/min. As a result, 112 mg of reserpine and 21 mg of yohimbine were obtained from 3 g of crude extract in a single run. The analysis of the isolated compounds was determined by high-performance liquid chromatography (HPLC) at 230 nm with purities of over 91.0%, and the chemical identification was carried out by the data of electrospray ionization–mass spectrometry (ESI–MS) and nuclear magnetic resonance (NMR) spectroscopy. The technique introduced in this paper is an efficient method for preparative separation of reserpine and yohimbine from devil pepper radix. It will be beneficial to utilize medicinal materials and also useful for the separation, purification, and pharmacological study of Chinese herbal ingredients.