Isolation and Characterization of Lupeol from Pterocarpus Osun Leaf Extract

Fadeyi AE and Adeniran OI

Published on: 2024-02-24


Pterocarpus osun, an indigenous plant with traditional therapeutic applications, was macerated with n-hexane, ethyl acetate, and methanol to obtain crude extracts. Extensive phytochemical screening led to the isolation of a white solid compound, lupeol, which was analysed using FTIR, 1D and 2D NMR, HR-LCMS, and by comparison with reference spectral data. 1H NMR showed seven methyl singlet’s at 0.72, 0.77, 0.79, 0.81, 0.82, 0.83 and 0.87 correspond to C-24, C-28, C-25, C-27, C-23, C- 26, and C-30 with two olefinic protons at 4.57 and 4.69 ppm representing the exocyclic double bond. The 13C-NMR spectrum revealed 30 carbon signals, including 7 methyl, 11 methylene, 6 methine, and 6 quaternary carbons, which came up at 78.6 ppm. Around 150.98 ppm (quaternary C) and 109.312 ppm (methylene C), respectively, the olefinic carbons of the exocyclic double bond were noted. These are designated as the C-20 and C-29 double bonds of the lupane-type triterpenoid molecule. 1H-1H COSY spectrum served as confirmation for the accurate designation of each proton. Mass spectrometric (MS) showed that lupeol exhibits a parent ion peak at m/z 427 [M+H] [+]. Lupeol is a well-known triterpenoid with pharmacological properties.


Characterization Ethyl acetate HR-LCMS Isolation Lupeol Pterocarpus osun


Various cultures throughout the world have employed plants in conventional healthcare for thousands of years. These conventional medical practices frequently rely on the curative powers of plants to cure a broad spectrum of illnesses. The traditional Indian medical system known as Ayurveda makes substantial use of herbs for healing. There is scientific evidence supporting the therapeutic benefits of herbs like Withania somnifera, Azadirachta indica, and turmeric [1]. Herbal medications, which are more effective, safe, and cheap, are gaining ground in both urban and rural communities [2]. Information from ethnic groups or traditional medicine practitioners has played an important role in the development of novel bioactive medicinal products from herbs as therapeutic agents [3].

Medicinal plants are in high demand in developed as well as developing countries for basic healthcare needs because of their wide biological and medicinal properties, higher safety margins, and lower costs [4]. Over the years, the WHO suggested that countries should get involved in the use of herbal medicine to exploit the secondary metabolites that provide effective and safe solutions for diseases of both non-microbial and microbial origins [5] Medicinal plants have been revealed to have valid utility, and over 80% of people living in rural communities rely on their efficacy for their primary health care [6]. The efficacy of herbal medicines against ill health is possible due to the presence of many bioactive constituents, such as nutrients, phytochemicals, etc., that have pharmacological activities in the bodies of living organisms [7].

A naturally occurring triterpenoid substance called lupeol is present in several plant species. According to [8], the potential therapeutic benefits of this substance, such as its anti-inflammatory, antioxidant, anti-cancer, anti-microbial, and anti-diabetic actions, have attracted interest recently. Typically, extraction and purification procedures are used to isolate lupeol from plants.

Pterocarpus osun (PO), sometimes called African padauk or Osun in Nigeria, is a plant that has a long history of usage in traditional African medicine. Its use dates back many years, and it is largely regarded for its therapeutic qualities. Due to its therapeutic qualities, Pterocarpus osun has historically been utilized throughout West Africa, most notably in Nigeria. The stem bark and leaf of Pterocarpus osun are traditionally used in herbal remedies for a variety of maladies and conditions. The antibacterial, anti-inflammatory, and antioxidant effects of Pterocarpus osun are well-known [9]. It has been administered to treat skin eruptions, dermatitis, and infections caused by fungi on the skin. It is frequently used in remedies or infusions to treat fever, coughing, and other respiratory disorders [10]. The continuous rising interest in the therapeutic properties of Pterocarpus osun, is responsible for this study, the chemical composition of this substance, and its potential pharmacological effects.

From the literature search, there have been no reports of pentacyclic triterpenes being isolated and identified from Pterocarpus osun leaf. However, the current study presents the first isolation and characterization of lupeol from an ethyl acetate fraction of Pterocarpus osun leaf extract.

Materials and Methods

Sample collection and preparation: Pterocarpus osun (PO) leaf was harvested in July 2021 from the medicinal garden of Sheda Science and Technology Complex (SHESTCO), in Kwali Area Council of Federal Capital Territory, Abuja. Sample was dropped at the herbarium of Nigerian Industrial Pharmaceutical Research Institute and Development (NIPRID), Idu Industrial Estate, Abuja, and identification number (NIPRID/H/7251) was assigned. The harvested sample was air dried for three weeks, then powdered using harmer mill.

Extraction: 500g of the powdered PO leaf sample was macerated in succession using n-hexane, ethyl acetate and methanol respectively. The solvent was removed from the extract using rotary evaporator then the ethyl acetate extract was subjected to chromatographic separation using thin layer and column chromatography methods. Purification of the white solid compound obtained was done by recrystallization by dissolving it in acetone.

Isolation of compounds: 8 g of the ethyl acetate extract fraction was weighed and dissolved in ethyl acetate, and then 10 g of silica gel was added. This mixture was crushed into a fine powder, and the solvent was allowed to evaporate. The dry column method was used, where the column was packed with silica gel with occasional tapping to allow for even column packing. The prepared sample was loaded on the sand layer on top of the silica gel. The column was eluted starting with 100% n-hexane and hexane/ethyl acetate as a gradient mixture solvent system of increasing polarity (95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 40:60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, 5:95) successively. Several eluates (75 mL) collected were monitored using TLC, and eluate fractions with similar TLC profiles were pooled together as a fraction and evaporated. Further purification of fraction C1 (125 mg) was carried out and subjected to FTIR, 1D, 2D NMR, and LC-MS/MS analyses.

Results and Discussion

Initial phytochemical screening of the leaves of Pterocarpus osun indicated the presence of tannins, flavonoids, alkaloids, steroids, saponins, and phenols in the extract of the plant. Using tetramethyl silane (TMS) as an internal standard, the nuclear magnetic resonance analysis was recorded on the Bruker AVANCE-600 (500 MHz for 1H-NMR and 150 MHz for 13C-NMR in CDCl3). Compound C1 produced a single spot with an Rf value of 0.580 on the TLC plate, and it dissolved in chloroform. The melting point of the white solid material C1 is 214 o C. The FT-IR spectrum of C1 revealed C-H stretching in CH2 and CH3 at 2942 cm -1 and 2357 cm -1, respectively. It also revealed C-H deformation in CH2 and CH3 at 1417 cm -1, C-O stretch in secondary alcohol at 1035 cm-1, and exocyclic CH2 with C-H bonding at 889 cm-1. a total of seven methyl singlets at 0.72, 0.77, 0.79, 0.81, 0.82, 0.83, and 0.87 correspond to C-24, C-28, C-25, C-27, C-23, C-26, and C-30 in the proton spectrum of C1 (1HNMR, 500 MHz, CDCl3), while two olefinic protons at 4.57 and 4.69 ppm reflect the exocyclic double bond. Thirty carbon signals were found in the 13C-NMR spectrum, including quaternary carbons at 78.6 ppm and signals for 7 methyl, 11 methylene, and 6 methine. The olefinic carbons of the exocyclic double bond were seen at 150.98 ppm (quaternary carbon) and 109.312 ppm (methylene carbon), respectively. The identification of each proton was confirmed by the 1H-1H COSY spectrum. Lupeol has a parent ion peak at m/z 427 [M+H] [+], according to mass spectrometric (MS) results. The correlation between protons and the corresponding carbon atoms is displayed by the HSQC.

The structure of compound C1 (Figure 8) was revealed to be a lupeol by 1H-NMR, 13C-NMR, HSQC, and 1H-1H COSY spectrum data and comparison with the data given in the research published by [11,12]. Finally, lupeol was obtained from a Pterocarpus osun leaf. Some of the plant's documented ethnomedicinal usage could be attributed to the presence of lupeol in the leaf extract. Lupeol's pharmacological actions have been reported in scientific journals [13].

Figure 1: 1H NMR spectrum of lupeol.

Figure 2: 13C NMR spectrum of lupeol.

Figure 3: HSQC spectrum of lupeol.

Figure 4: COSY spectrum of lupeol.

Figure 5: LC-MS spectrum of lupeol.

Figure 6: LC-MS chromatogram of lupeol.

Figure 7: FTIR spectrum of lupeol.

Figure 8: Structure of lupeol.




Chromatographic methods was used in isolation and purification of lupeol from the leaf extract of Pterocarpus osun and the isolate was characterised, and structure elucidated using FTIR, 1D, 2D and LC-MS spectroscopic analytical tools. This is the first time of isolating lupeol from Pterocarpus osun. Lupeol has been reported in literatures to have a wide range of biological and pharmacological activities.


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