Sono-Maceration – a rapid and inexpensive method for the isolation of ursolic acid from Neolamarckia cadamba leaves

Abstract This article records for the first time the isolation of Ursolic acid from the leaves of Neolamarckia cadamba (Roxb.) Bosser (Family: Rubiaceae) using ultrasonic waves. This bioactive triterpenic acid was isolated without its isomer, oleanolic acid, in a very convenient way with good yield. The structure was identified by means of one dimensional Nuclear Magnetic Resonance (NMR) spectroscopic techniques like 1H NMR, 13C NMR, distortionless enhancement by polarization transfer (DEPT) and two dimensional NMR spectroscopic method for example, heteronuclear single quantum coherence (HSQC). It was also assayed for antidiabetic and antioxidant potencies. About 71.5 mg of pure ursolic acid was isolated from 2.6 grams of ethyl acetate soluble fraction using sono-maceration as an extraction technique. Graphical Abstract


Introduction
Ursolic acid or 3b-hydroxy-urs-12-en-28-oic acid (UA), a pentacyclic triterpenic acid, frequently occurring together with its isomer Oleanolic acid (OA), belongs to the cyclosqualenoid family (Shanmugam et al. 2013;Jesus et al. 2015). They are chemical isomers occuring simultaneously and hence isolated together as a mixture. It has various pharmacological activities (Ovesna et al. 2004). The methods used to isolate UA from plants including soxhlet, batch extraction, precipitation, column chromatography and novel techniques like counter current chromatography and ultrasound assisted extraction (Vetal Mangesh et al. 2014), often result in a mixture of UA and OA, which is difficult to separate. Not only do these techniques require large volume of solvents, higher temperatures and more time, but they also give lower yields. Thus, an efficient, eco-friendly, economically viable and commercial extraction protocol needs to be established. Neolamarckia cadamba (Roxb.) is an underutilised plant from Indian subcontinent (Pandey and Negi 2018) traditionally used in antidiabetic treatment (Atul et al. 2011). Literature study shows more than 50 compounds being isolated from its barks, leaves, roots, flowers and seeds.
In the present research work, an ultrasound-assisted extraction method is described by means of which UA was isolated in a very simple, fast and a convenient way with good yield from Neolamarckia cadamba. This method resulted in an increase in the isolation yield of this bioactive natural product as well as in the decrease of the organic solvent usage and the amounts of time. To the best of our understanding, there were no studies on the isolation of UA from Neolamarckia cadamba. The major advantage of this source is that it is a very large evergreen tree. As a result, plant material is very easily available in bulk quantity. Therefore, UA can be isolated in a very good yield all year round. The study also aims at the screening of isolated UA for antidiabetic and antioxidant potency.

Results and discussion
About 71.5 mg of the compound was isolated from 0.52 g of solid fraction from column chromatography. This isolated compound was characterised by various spectral analyses as follows:

Characterisation of the isolated compound
The isolated compound was obtained as a white amorphous powder, insoluble in water, moderately soluble in chloroform and methanol but completely soluble in chloroform-methanol mixture at room temperature. It gave positive Liebermann-Burchard test giving pink violet ring and Salkowski reaction giving reddish brown ring (Rimjhim et al. 2014 un-hydrogenated olefinic carbon), 126.0 (C-12, hydrogenated olefinic carbon). The 1 H and 13 C NMR data obtained for the isolated compound was in comparison with the data for Ursolic acid reported in literatures. Based on spectroscopic data and reported data (Martins et al. 2013;Raza et al. 2015), the isolated compound was characterised as ursolic acid.

Biological activities of isolated ursolic acid
According to the results obtained from Table S2 (supplementary material), IC 50 of UA was found to be 98.05 mg/mL and 57.48 mg/mL for acarbose. Since almost twice the quantity of UA is required to inhibit 50% activity of glucoamylase enzyme, ursolic acid can be used only as a moderate glucoamylase inhibitor. Also in the alpha-amylase assay, IC 50 of UA was 83.04 mg/mL and 61.94 mg/mL for acarbose. Thus, it can act as a good a-amylase inhibitor. In the DPPH assay, the IC 50 value of UA was found to be 16.69 mg/mL, 5.22 mg/mL and 34.31 mg/mL for ascorbic acid and BHT respectively. Thus, UA showed very good antioxidant potential with respect to both Ascorbic acid and BHT.

Experimental
The experimental section is available online in the supplementary material.

Conclusions
The current work has developed an inexpensive, simple and fast method that incorporates extraction and detection techniques for the isolation of Ursolic acid from methanol extract of leaves of Neolamarckia cadamba in remarkably good yield (13.75%). The results reveal that Neolamarckia cadamba can be a promising candidate for bulk isolation of pure UA. Also, extraction using ultrasonic waves was found to be a very powerful tool for effective extraction of this bioactive phytochemical. To our knowledge, this is the first report of UA from this plant. The instrumental setup has great prospects for commercial developments. Neolamarckia cadamba is, therefore, a high yielding plant source of UA. UA exhibited good antioxidant activity. It can also act as a potent a-amylase inhibitor.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by Non-NET Fellowship of University Grants Commission, New Delhi.