Analysis of Mogrosides in Siraitia grosvenorii Fruits at Different Stages of Maturity

Abstract

Monk fruit extract has been approved as a natural sweetener by many countries. Its major sweet components, mogrosides, display different sweet intensities and profiles. Therefore, it is important to understand the change of mogroside contents in Siraitia grosvenorii at different maturity stages. In this study, monk fruit cultivars were collected from 4 locations in GuangXi, GuiZhou, and HuNan, at different times. Mogroside IIe, mogroside III, mogroside IIIe, mogroside IV, mogroside V, isomogroside V, and siamenoside I in each sample were quantified using high performance liquid chromatography coupled to triple quadrupole mass spectrometer (LC-MS-MS). As a result, mogroside IIe was the major component at the early maturity stage. It is converted to mogroside III from 15 to 45 days, then continued the glycosylation rapidly to yield mogroside V which kept predominant after 60 days. Highly glycosylated mogrosides, such as mogroside V and siamenoside I, which provide a better taste profile, accumulated and stabilized from 75 to 90 days. It is recommended to harvest the fruit after 75 days of pollination.

With the increasing consumer demands on less consumption of sugar and artificial sweetener, the discovery of natural, high potency, and low-/zero-calorie sweeteners as a solution of sugar reduction has been highlighted in the food and beverage industries. As its intense sweetness, the fruit extract of Siraitia grosvenorii (monk fruit, Luo-Han-Guo) is becoming a second STAR of natural sweetener right after Stevia. It has been used as a traditional medicine in China for centuries. ¹ It has been approved as a sweetener in Asian countries, ¹ United States (GRAS approved), ² Canada, ³ Australia, and New Zealand, ⁴ and currently is under the evaluation by European Food Safety Authority (EFSA). ⁵

Mogrosides, cucurbitane-type triterpene glycosides isolated from S. grosvenorii, are principle sweet components from the fruit. ¹ Among more than 60 mogrosides identified, ⁶ among which several well-studied molecules display a marked difference in sweetness intensity and taste profile. Mogrosides I and II have similar sweetness level as sucrose, while mogrosides IV, V, and siamenoside I are measured 233 to 392, 250 to 425, and 465 to 563 times sweeter than sucrose at various concentration levels, respectively. ^1,7 In our evaluation, siamenoside I presents a better balance in sweetness, bitterness, sweet linger, astringency, and mouthfeel than mogroside V. ⁸

The mogrosides in fruit of S. grosvenorii are proposed to be biosynthesized through mevalonate pathway from acetyl CoA to its aglycone mogrol, followed by glycosylation steps. ⁹ It is verified and widely accepted that mogroside II is predominant in the early stage of immature fruit, while more-glycosylated sweet mogrosides developed and accumulated in the later stages of maturity. ^9,10 However, how does the mogroside profile change in each stage is less discussed. In this study, the fruits of S. grosvenorii from various locations and at different maturity stages were collected. The composition profiles of mogrosides IIe, III, IIIe, IV, V, isomogroside V, and siamenoside I (Figure 1) were described. It would provide a reference for industries when will be the best harvesting time of the fruits to yield products with a better taste quality.

Figure 1

Structures of selected mogrosides.

Siraitia grosvenorii is native to China and grown primarily in the provinces of GuangXi, GuiZhou, and HuNan. The species have 6 major cultivated varieties: Changtan, Lajiang, Qingpi (known as “green peel” or “green husk”), Donggua, Hongmao, and Chashan. Among these cultivars, Qingpi has a shorter maturity period, larger fruit size, better adaptability, and higher yield, so the current available cultivars are dominated by Qingpi or its hybrids. ¹¹ Three monk fruit cultivars from 4 locations at 4 to 8 different maturity stages were collected (Table 1; Supplemental Tables S1-S5).

Table 1

Collection of Monk Fruit Cultivars.

Code	Location	Cultivar	Pollination time	Stage of collection a
GX-1	Changtangou, GuangXi	Qingpi × Changtan	July 24-28, 2017	Days 15, 30, 45, 60, 75, 90 105
GX-2	Changtangou, GuangXi	Qingpi × Hongmao	July 24-28, 2017	Days 30, 45, 60, 75, 90, 105
GX-3	Guzheng, GuangXi	Qingpi	August 7-14, 2017	Days 15, 30, 45, 60, 75, 90, 105, 120
GZ	Deshun, GuiZhou	Qingpi	July 24-28, 2017	Days 30, 50, 60, 75, 90
HN	Yongzhou, HuNan	Qingpi	July 24-28, 2017	Days 30, 60, 75, 90

a

Days after the pollination.

The size and weight of the fruits from all the collections were measured (Figure 2; Supplemental Tables S1-S5). The weight of fruits had a large deviation. The water content in the fruit was much higher at the very early stage, then decreased gradually as more metabolites were biosynthesized. In a separate research, the weight of dry fruits vs fresh fruits was measured at each stage. The weight ratio of dry/fresh increased from approximately 8% at 15 days to 23% at 60 days, then kept slightly varied after 60 days (data not shown). The shape index was determined using the ratio of vertical diameter/horizontal diameter. The size of samples from 30 to 90 days did not have a significant change, and it was smaller at the late maturity stage. As we discussed earlier, the cultivars are all Qingpi or its hybrids. It is not surprising that the fruit shape of all samples kept the consistency from all collection sites.

Figure 2

Measured physical properties of the fruits.

Selected mogrosides were quantified (Figure 3; Supplemental Figures S2-S5) and the yields were calculated based on the dry fruit weight (Supplemental Table S13). Mogroside IIe formed at early stage of maturity and was the primary component from 15 to 45 days. It started to yield higher glycosylated mogrosides at the time of its formation. Mogroside IV and siamenoside I were only detected in a very low level at all stages. Three additional mogroside IV isomers were detected in MRM mode. When semiquantifying using mogroside IV, the yields were all much lower than mogroside IV (data not shown). The yield of siamenoside I maximized on 60 days from all the locations. Mogroside V formed from 45 days, and the yield increased rapidly to keep dominant after 60 days. Mogroside V reached to the maximum from 75 to 90 days, then kept relatively stable after 90 days (Figure 4; Supplemental Figures S6-S10).

Figure 3

Stacked LC-MS chromatogram of GX1 at different stages.

Figure 4

Mogroside contents in the fruits.

It was reported that Changtan is the best in quality among 6 major cultivated varieties. However, it is not adaptable to various environment. ¹² The hybrid across Changtan and Qingpi would keep the high quality of the fruit and improve the adaptability. This could explain that among the collected samples we analyzed, the yields of mogroside V and siamenoside I were higher in GX1 (Figure 5).

Figure 5

Yields of Mog V and Sia I in different cultivars (D75-D90).

The pollination season of monk fruit is usually from July to late September. This brings a question if the climate change during the pollination time would change the yield of mogrosides. In our pretest, we collected additional samples at 45 days from GX1 and GX3, which were pollinated in September. The weight and size of the fruits were much smaller than the fruits pollinated in July, however, the content of the target mogrosides in the fruits from 2 pollination times were similar (data not shown). Further analysis would be required to address the question.

Experimental

Chemicals and Reagents

Seven mogroside reference standards (purity >95%), mogrosides IIe, III, IIIe, IV, V, isomogroside V, and siamenoside I, were provided by Shanghai Standard Technology Co., Ltd. HPLC/MS-grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany). Formic acid (purity >99.5%) from ROE scientific INC and ultra-pure water prepared by a Milli-Q water system (Millipore, MA, United States) were used in the mobile phase. Other reagents of analytical grade used in preparing monk fruit extract were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Instruments

Quantitative determination of mogrosides in S. grosvenorii was performed by LC-MS-MS (Agilent 6410 triple quadrupole mass spectrometer coupled to 1290 LC system). A sonicator (KQ-300DB, Kunshan ultrasonic instruments Co., Ltd., China) and an ultra-high speed centrifuge at low temperature (Sigma 3K15, United States) were used.

Sample Preparation

All the fruits were cold-packed during the shipment, then kept frozen at −20°C till sample process. The fruits were freeze dried and portions of the samples were crushed using a disintegrator. Dried powders (0.5 g, <65 mesh) of monk fruits were ultrasonically extracted with 25 mL of methanol-water (80:20 v/v) for 30 minutes (300 W, 40 kHz), centrifuged (12 000 rpm, 5 minutes), then filtered through a 0.22 µm membrane as the test solution.

Each accurately weighted standard was dissolved in methanol-water (80:20 v/v) to give the stock solutions. Working standard solutions containing 7 reference standards were prepared by diluting the stock solutions with methanol-water (80:20 v/v) solvent to a series of proper concentrations. The solutions were stored at 4°C.

Optimization of the Extraction

The extraction methods, extraction solvent, and extraction time were optimized using mogroside V and siamenoside I as target compounds. Two methods (ultrasonic extraction and reflux extraction) were compared by using 80% methanol for 30 minutes. No significant difference was shown in 2 extraction methods (Supplemental Table S6). Thus, the ultrasonic extraction was chosen in the following experiments. The sample solutions (GX1) were prepared in duplicate with 4 different solvents, including different mixtures of water and methanol (Supplemental Table S7). The MeOH-H₂O (v/v, 80:20) was chosen as the optimized extraction solvents because of the highest value achieved. The experiment investigated the extraction time (Supplemental Table S8). The result showed that 30 minutes was optimal for sonication.

LC-MS Conditions

The amount of 1 µL of sample was loaded on an Eclipse plus C₁₈ column (1.8 µm; 2.1 × 50 mm, Agilent), and eluted with a gradient of mobile phases 0.1% formic acid in water (A) and acetonitrile (B): 0 to 3 minutes: 15% to 21% B; 3 to 10 minutes: 21% to 22% B; 10 to 13 minutes: 22% B; 13 to 17 minutes: 22% to 24% B; 17 to 20 minutes: 24% to 40% B; and 20 to 21 minutes: 40% to 95% B. The flow rate was 0.3 mL/min.

The MS detection was operated in the negative ion mode with multiple reaction monitoring (MRM). To obtain the best MRM conditions, stock solutions of 7 standards were tested separately in negative ionization mode. The precursor ions, 2 product ions, fragmentor, and collision energy were optimized (Supplemental Table S9). Analysis conditions for MS-MS were capillary voltage, 3500 V; gas flow rate, 10 L/min; gas temperature, 325°C; nebulizer, 35 psi; sheath gas flow, 6 L/min; and sheath gas temperature, 300°C.

Method Validation

Mogroside IIe, mogroside III, mogroside IIIe, mogroside IV, mogroside V, isomogroside V, and siamenoside I are quantified using LC-MS conditions described in the “LC-MS Conditions” section (Figure 3; Supplemental Figures S1-S5). The calibration curve of 7 standards (Table S10) was studied by injecting each working standard solution. The result showed good linearity of 7 target standards within the test range.

Recovery was determined by analyzing spiked samples. A known amount of the standards (mogroside V and siamenoside I) was added into a certain amount of samples (0.25 g, GX1 60 Day), and then extracted and analyzed with the same procedures. Six replicate extractives at medium concentrations were used to calculate the extraction recovery rates for evaluating the method accuracy. The recovery is calculated using the following formula: Result (%) = (Found amount – Original amount)/Spiked amount × 100. The overall recoveries were between 92.05% and 104.51% for the reference compounds, with RSDs less than 5% (Supplemental Tables S11-S12).

Supplemental Material

Supporting information - Supplemental material for Analysis of Mogrosides in Siraitia grosvenorii Fruits at Different Stages of Maturity

Supplemental material, Supporting information, for Analysis of Mogrosides in Siraitia grosvenorii Fruits at Different Stages of Maturity by Bin Wang, Zhou Yang, Zhenqiang Xin, Gil Ma, Yong Qian, Tianpei Xie and Indra Prakash in Natural Product Communications