High Performance liquid chromatographic (HPLC) Selection of an Important Medicinal Plant Stevia rebaudiana Bertoni. Based on the Percentage of Sweet Diterpene Glycosides
Neha Sharma1*, Rajinder Kaur2, Nimisha Sharma3, Yashpal Sharma2 and Ravinder Raina2
1ICAR-National Institute for Plant Biotechnology, New Delhi, India
2Dr YS Parmar University of Horticulture and Forestry, Solan India
3Indian Agricultural Research Institute, New Delhi, India
Submission: May 3, 2024;Published: May 27, 2024
*Corresponding author: Neha Sharma, ICAR-National Institute for Plant Biotechnology, New Delhi, India
How to cite this article: Neha S, Rajinder K, Nimisha S, Yashpal S, Ravinder R. High Performance liquid chromatographic (HPLC) Selection of an Important Medicinal Plant Stevia rebaudiana Bertoni. Based on the Percentage of Sweet Diterpene Glycosides. JOJ Hortic Arboric. 2024; 4(3): 555638. DOI: 10.19080/JOJHA.2024.04.555638.
Abstract
Diabetes is the fastest spreading disease that occurs when the blood glucose, is very much high, and being main source of energy, we need to regularly monitor blood glucose. Stevia rebaudiana as a natural sweetener, with its all-medicinal properties, had great potential to keep an eye on blood glucose. Therefore, the aim of this research is to investigate the most efficient S. rebaudiana plants via HPLC among the 84 test plants in their high rebaudioside and stevioside content. A lot of variation was observed among the individuals, as the percentage of rebaudioside-A exceeded up to 9.37% followed by 8.65, 8.62%, and 8.24%. Similarly, in the case of stevioside among 84 individuals the percentage of stevioside varied from 0.23- 8.64%. It would be concluded that HPLC is a fantastic method that offers an accurate analysis that can facilitate the knowledge of more diverse plants that can be used for creating hybrid vigor among the Stevia plants that in turn would help the pharmaceutical industry for making more efficient stevia artificial sweeteners from Stevia
Keywords: Stevia rebaudiana, HPLC, Rebaudioside, Stevioside, Medicinal plant
Introduction
Stevia rebaudiana Bertoni, an incredible member of the family Asteraceae, is one of the most valuable tropical and subtropical perennial medicinal plant [1]. It is at first originated as a South American wild plant [2] and in Paraguay, it had been used as a natural sweetener for centuries [3]. After its Introduction in India at Bangalore, the Institute of Himalayan Bioresource Technology (IHBT-CSIR), Palampur, introduced two accessions of stevia for domestication and cultivation in Himachal Pradesh [4]. Stevia is anti-bacterial, anti-fungal [5], anti-inflammatory, anti-viral [6,7], anticancerous [8] cardiotonic, antihypertensive [9] diuretic and hypoglycemic [10]. It is used as a tabletop sweetener, in soft drinks, baked food items, pickles, fruit juices, jams, jellies, candies and chewing gums. It is recommended for diabetics and has been extensively tested on animals and used by humans with no side effects [11,12].
Stevia is highly distinguished by the presence of sweet diterpene glycosides: rebaudioside-A, rebaudioside-C, stevioside and dulcoside in its leaf tissue. Wide variation in the percentage of these sweet diterpenoid glycosides has been reported in the leaves and other parts of S. rebaudiana and this has been ascribed to both genetic and environmental factors. The stevioside is the major component but it has an unpleasant bitter after-taste. However, rebaudioside-A, normally present in lower amount (25% to 45% of stevioside content) in leaves, does not have bitter after-taste and has a sweetening power 1.2 to 1.6 times higher than stevioside [13]. Usually, stevioside is the dominant glycoside, but types rich in rebaudioside-A have also been reported [14]. The presence of these high potency sweeteners has attracted a huge interest in stevia production. Nearly three decades of breeding and selection have increased glycoside concentration in stevia leaves up to levels of 20 per cent [14]. However, this improvement is based on biochemical selection and measurements are based on High Performance Liquid Chromatography (HPLC) which lead to the selection for plants producing high amounts of glycosides. The objective of this research is to investigate the most efficient plants via HPLC among the 84 test plants in their high rebaudioside and stevioside content in S. rebaudiana. Therefore, HPLC is a fantastic method that offers rapid analysis that can facilitate the knowledge of more diverse plants that can be used for future breeding of Stevia that in turn would help pharmaceutical industry for making artificial sweeteners from Stevia. This study would directly enhance the quality of raw materials in artificial sweetener industry.
Materials and Methods
The estimates of Rebaudioside-A and Stevioside were made based on High Performance Liquid Chromatography (HPLC) grade chemicals procured from standard firms such as Sisco Research Laboratories Pvt. Ltd., Maharashtra, India, Sigma (Sigma-Aldrich, Bangalore, India) and Chromadex, Irvine, USA. Plant material i.e. leaves were collected from the field grown 84 individual plants and their fresh weight was taken individually on the day of collection. The collected leaves kept at room temperature for drying for few days till they left around 70% of their moisture. They were then oven dried (60°C) and weighed thrice at regular intervals till their dry weight became constant. The dried leaf samples were then powdered by grinding them in pestle and mortar. The powdered samples were weighed (300 mg), transferred to thimbles and refluxed with dichloromethane on a boiling water bath to remove colour by using soxhlet apparatus individually. Non-polar compounds and colours were removed through extraction with dichloromethane. The boiling point of dichloromethane is 39.75°C so during dichloromethane reflux, the water bath was continuously switched on and off and the process was continued till the color was completely removed.
The residual samples in thimbles were then refluxed with methanol in soxhlet apparatus to extract its sweetness. The process is continued till the samples in thimbles become tasteless. Methanol was used for final sweet compounds extraction. The extract was then distilled off to recover methanol. The concentrated extract was dissolved in 15 ml acetonitrile: water (78:22) mixture and these plant extracts were then filtered through Millipore filter paper using Millipore filter assembly and filtered samples were kept in volumetric flasks and the samples were then analysed by HPLC. Before injecting the solutions of reference compounds and samples, HPLC column was run in HPLC grade methanol for few hours and then with acetonitrile: water (78: 22) solvent mixture.
HPLC Conditions
Instrument: Waters HPLC unit with Waters HPLC pump 515 and dual-absorbance detector 2487
Mobile phase: Acetonitrile: water (78:22)
Flow Rate: 1.0 ml/min.
Column: 5 μm spherisorb NH2 column (4.6 mm x 250 mm).
Volume injected: 20μl
Detection: 210 nm
Percentages of rebaudioside-A and stevioside were calculated using the following formula:
Using the above formula rebaudioside-A and stevioside content of all the 84 test plants were estimated.
Result and Discussion
The percentage of rebaudioside-A and stevioside was obtained on high performance liquid chromatography (HPLC) for all the 84 test plants. Standard curve for rebaudioside-A and stevioside were prepared using standard rebaudioside-A and stevioside (Table 1, Figure 1a and b). All the 84 individuals were scored for their percentage of rebaudioside-A and stevioside (Table 2) which is based on the peaks obtained. Percentage of rebaudioside-A and stevioside was calculated on the basis of the formula given in section of Material and Methods. Rebaudioside-A ranged from 0.00 to 9.37% whereas stevioside ranged from 0.23% to 8.64% in the test plants (Table 2) (Figure 2).
On observing the data, it was clearly seen that there was lots of variation among the individuals, as the percentage of rebaudioside-A exceeded upto 9.37% (13) followed by 8.65 (77), 8.62% (69), 8.24% (45). Similarly, in case of stevioside among 84 individuals the percentage of stevioside varied from 0.23 (81) -8.64 (71). Depending on the analysis the scientists can use the most divergent stevia individuals and looking at their variation hybrid vigour can be created.
Conclusion
The chromatographic results obtained in this study have clearly demonstrated that HPLC is a promising technique to discover the best divergent Stevia rebaudiana with high rebaudioside content and low stevioside content (because of mild bitterness) for the material selection process to create hybrid vigour. Additionally, the study also added to our knowledge that leaf parts of Stevia rebaudiana can produce the finest possible products due to its highest content of bioactive compounds. Therefore, HPLC is a fabulous method that offers accurate analysis that can facilitate the quality herbal products in pharmaceutical industry and hence would help the health-conscious people or people suffering from diabetes. This study would definitely boom in pharmaceutical industry for making artificial sweeteners from Stevia. This study would directly enhance the quality of raw materials in artificial sweetener industry.
Declarations
Conflict of interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
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