Characterization of natural monatin isomers, a high intensity sweetener from the plant Sclerochiton ilicifolius from South Africa

The objective was to establish the natural occurrence of the various isomers of monatin in extracts of Sclerochiton ilicifolius plant material harvested from different growing regions in South Africa. The natural occurrence of the 2S,4S isomer has been reported as well as the synthesis of the 2R,4R isomer. The 2R,4R is reported as the most intense sweetness however its natural occurrence has not been fully reported, as a result it was not possible to establish whether these isomers are indeed already present in the plant or come from racemisation during the processing of the plant. The presence of the monatin isomers 2S,4S; 2R,4R in aqueous extracts of S. ilicifolius root bark was demonstrated in each sample harvested at two different time points. The 2R,4R, 2S,4S, 2R,4S, and 2S,4R monatin isomers were absent in the aqueous extracts of S. ilicifolius stem and leaf samples, however was shown to be present in the root bark, and root core samples. This report confirms previous findings which suggested that the 2S,4S and 2R,4R monatin isomers occur naturally in S. ilicifolius.


INTRODUCTION
Monatin, (Indol-3-yl)-2-amino-4-carboxy-4-hydroxypentanoic acid (Figure 1), is a naturally occurring high intensity sweetener isolated from the bark of the roots of Sclerochiton ilicifolius, a spiny-leafed hardwood shrub growing in the rocky hills of the Limpopo Province in South Africa (Vleggaar et al., 1992). The same authors also assigned the 2S,4S absolute configuration to the natural levorotatory compound based on NOE NMR experiments on a cyclic derivative and the application of the empirical Clough-Lutz-Jirgenson rule. The crystal structure of synthetic 2R,4R monatin potassium salt dehydrate was only recently determined by single crystal X-ray structure analysis (Amino et al., 2016). The relative sweetness of the 2S,4S monatin isolated from the natural plant was reported in 1992 to be 1200 to 1400 fold more intense than that of sucrose however synthetic 2R,4R monatin, was reported much later to have a more intense sweetness than the 2S,4S isomer i.e. up to 2700 times that of 5% sucrose (Patent No. WO2003059865 A1, 2003). Monatin is compatible with other sweeteners and forms acceptable blends for example with aspartame. Extensive in vivo toxicology studies for 2R,4R monatin have also been completed moving it closer towards commercialization (Brathwaite et al., 2011, Brathwaite et al., 2014. The low concentration of monatin in the root bark has challenged scientists to develop processes to produce natural monatin in sizeable quantities for commercial use. While the synthesis of all four monatin stereo isomers has been reported the natural occurrence of the three remaining stereo isomers was demonstrated in an extract of the plant (Bassoli et al., 2005). However these scientists have referenced that owing to the small amounts available and the impossibility of obtaining a larger sample or information about the exact origin and detailed extraction methodology, it was not possible to establish whether these isomers are indeed already present in the plant or come from some racemisation during the processing of the sample and suggested that this should be further investigated. This study focused on determining the natural occurrence of monatin stereo isomers in minimally processed extracts through appropriate, sustainable, controlled harvesting procedures, extraction and analysis. As a member of the plant family Acanthaceae, S. ilicifolius has been referred to as 'Molomo monate' from the Sepedi name meaning "mouth nice" (Vleggaar et al., 1992).
Based on actual herbarium specimens housed in the South African National Biodiversity Institute (SANBI) in Pretoria, information on plant availability is limited. The "epicentre" of the distribution of S. ilicifolius is the Waterberg area in the Limpopo province of South Africa. The second "main" area of distribution is the Zoutpansberg, a mountain range in northern Limpopo, and a third area in the Blyde River Canyon Nature Reserve near the Abel Erasmus Pass in the Mpumalanga Province.

Chemicals and reagents
The reagents ammonium acetate, sodium bicarbonate and hydrochloric acid were purchased from Saarchem Univar, Marfey"s reagent purchased from Pierce, Rockford, IL, USA, Cat # 48895). HPLC grade acetone and acetonitrile was purchased from Burdick and Jackson (ACS/HPLC grade).

Processing of plant material.
All samples harvested were separately processed. For the 2002 collections, samples were immediately dried in an oven preset at 60°C. After 48 hours the samples were removed for further processing and the root samples were debarked while the root bark retained for further processing. Each sample (10-15 g) was separately ground in a high speed blender. The dried ground root bark was extracted by adding 75 ml of purified water to each of the samples. The mixture was allowed to stand at ambient temperature for 4 hours and shaken manually by hand every 30 minutes. Each of the mixtures was separately centrifuged and the supernatant decanted. The water layer was transferred to freeze drying tubes and separately freeze dried overnight. The dried powder was transferred to pre-weighed labelled vials and stored at 4 C prior to analysis.
For the 2005 collection, the stems, leaves samples and root samples were transferred to trays and immediately dried in an oven preset at 60°C. After 48 hours the samples were removed for further processing. The leaves were stripped and separately retained for further processing. The roots were debarked and the root bark and roots retained for further processing. Each sample was separately ground in a high speed blender. The sample was extracted by adding 200 ml of de-ionized water and the mixture was allowed to stand at ambient temperature for 4 hours and shaken manually by hand every 30 minutes. Each of the mixtures was separately filtered through filter paper, the water layer transferred to freeze drying tubes and separately freeze dried. The dried powder was transferred to pre-weighed labelled vials and stored at 4°C prior to analysis.

Preparation of standards
Monatin synthetic standards, 2S,4S; 2R,4R and mixture of RS/SR stereoisomers used for the characterization in the plant materials were prepared prior at the CSIR as described in literature (Rousseau et al., 2011). The standards were available at the CSIR for this research.

Plant harvesting and processing
A summary of the locations of root samples harvested during September 2002 and that for the recollections from same locations during February 2005 is shown in Table 1.
Even though there was a three year period between the collections, sufficient material was still available for the recollections.

Analysis of separated diastereomers (RR/SS and RS/SR).
The separated 2S,4S and 2R,4R isomers and the mixture of the 2R,4S and 2S,4R isomer standards were analysed using the Marfey"s chiral method. The separated  Table 2. This is the first chromatographic separation of monatin isomers using Marfey"s derivatization procedure as most reported separation of the monatin stereoisomers has generally been done through the use of chiral HPLC (Bassoli et al., 2005, Amino et al., 2016       The results of this study has shown that the 2S,4S and 2R,4R isomers of monatin occur naturally in minimally processed aqueous extracts from the root bark of S. ilicifolius collected from three different sites in the Limpopo Province in South Africa. Previous studies by both Bassoli et al. (2005) and Amino et al. (2016) have shown repeatedly that the sweetness profile between these two enantiomers is vastly different and is analogous to studies in terpenoids where it has been verified that different enantiomers of the same molecule may possess different properties including odor characteristics, odor thresholds and even biological activities (Lina et.al., 2017). This finding was somewhat surprising in that only the 2S,4S isomer of monatin was found in samples in the earlier report by Vleggaar et al. (1992). The study also concludes the 2S,4S and 2R,4R isomers are also much more prevalent in the plant versus the 2R,4S and 2S,4R isomers.

ACKNOWLEDGEMENT
We are grateful to the CSIR for supporting and funding this research.