Total saponin extract using different ethanol concentrations .... Total saponin extract using different extraction time in 120 minutes .... Extraction methods In herbal science, extract
INTRODUCTION
Research rationale
Gynostemma pentaphyllum, also known as Jiaogulan, is a versatile herb recognized globally as a pharmaceutical food Historical records indicate that the Chinese utilized this plant as early as the 14th and 15th centuries, highlighting its potential as an anti-tumor agent, pain reliever, and antibiotic (Zhu, 1991; Blumert & Liu, 1999) Over the years, G pentaphyllum has gained a reputation for enhancing health and promoting longevity (Linfu et al., 2016), with users reporting relief from various ailments.
General information regarding Gynostemma pentaphyllum has been verified
Research on morphology, classification, growth conditions, cultivation, chemical composition, health effects, and applications of the plant has been conducted in Vietnam, China, and several Western countries, including Croatia, Italy, and the United States (Huyen et al., 2012; Lin-Na & Yong-Xiu, 2014; Gelen et al., 2017; Mastinu et al., 2021) These studies have yielded positive results, revealing the scientific foundations and mechanisms underlying the plant's remarkable effects (Tanner et al., 1999).
Despite global recognition, the use of G pentaphyllum is limited in both geographical distribution and application This plant is primarily popular in Japan, Korea, China, and Vietnam, where it is commonly consumed in the form of infusions However, its presence in markets worldwide remains minimal.
G pentaphyllum can only be found as tea bags and dried plants In a scientfic view, infusion is an extraction method Nevertheless, although it is operationally simple and provides relative adequacy, it is far less specialized compared to other extraction methods The aforementioned information incites requirements for more academic studies on Gynostemma pentaphyllum and utilization matters Furthermore, it is uncertain if the potentials of the subject has been fully reached Therefore, it is safe to assert that a research on the extraction of G pentaphyllum to produce a specialized
4 product, optimizing the benefits from this plant, assures necessity and topicality Simultaneously, it offers solutions for some issues concerning raw material waste and spoilage
Thai Nguyen University of Agriculture and Forestry (TUAF) provides a robust laboratory setting for researching the quantification of saponins, key compounds found in Gynostemma pentaphyllum and Panax Ginseng that contribute to their bioactivities (Tanner et al., 1999) This capability allows for a thorough assessment of saponins in G pentaphyllum, which is essential for evaluating product quality and potential health benefits Additionally, TUAF's faculty of Biotechnology has successfully cultivated Gynostemma pentaphyllum with traits comparable to the wild variety from Ha Giang Province (Bui et al., 2015) Consequently, the study titled “Optimizing the extraction of Gynostemma pentaphyllum to initially produce capsules for storage” is both timely and feasible, aiming to leverage these advantages for specialized product development.
Furthermore, it is going to unravel the capability of G pentaphyllum in bioactivity The success of this study will also pave a way for future researches involving the issue.
Research objectives
This study aims to the following 5 objectives:
• Determine the quality of input G pentaphyllum (leaves and stems)
• Determine the factors affecting G pentaphyllum extraction
• Determine the bioactivity and anti-cancer effects of G pentaphyllum extract
• Retrieve G pentaphyllum extract in the form of capsules for storage
Research questions and hypotheses
The background introduced prior to this section has formulated the primary question for this study, corresponded by hypotheses as follows:
• Question 1 (Primary): Is there a way to utilize (extract) Gynostemma pentaphyllum better than what appears in the market (infusion) nowadays?
This inquiry prompts hypotheses concerning extraction methods, solvents, and operational factors such as time, temperature, and the ratio of raw materials to solvents Consequently, this leads to the formulation of question 2.
• Question 2: What are the optimal conditions for the extraction of
From question 1, the form of utilization is also set as a question for this study Particularly:
• Question 3: What kind of a realistic product is suitable for storing and utilizing Gynostemma pentaphyllum extract?
The secondary questions raised prompt further inquiries and hypotheses related to methodology, implementation, and equipment, which are essential for guiding this study towards obtaining meaningful scientific data and insights.
Limitations
Gynostemma pentaphyllum was chosen for this study due to its established presence in the market and its extensive research documented in various scientific articles Its utilization primarily involves infusion, highlighting its pharmaceutical potential among other plants.
Dried plants remain basic compared to their initial introduction, yet they possess values comparable to valuable pharmaceutical ingredients like red ginseng It would be a considerable loss if this knowledge is not further disseminated and utilized more effectively.
Choosing Gynostemma pentaphyllum opens up opportunities for various materials in scientific research, extraction, and application Additionally, it has influenced the direction of studies, methodologies, and implementations in specific ways.
The Faculty of Food Technology's laboratory at Thai Nguyen University of Agriculture and Forestry is a well-equipped research facility that supports various scientific studies However, for the specific research on Gynostemma pentaphyllum, the laboratory faces limitations in extraction methods and analysis techniques Advanced methods like High Performance Liquid Chromatography could not be utilized due to the absence of control samples, while other sophisticated techniques such as Mass Spectrometry, Infrared Spectroscopy, and Nuclear Magnetic Resonance were entirely unavailable.
Research on Gynostemma pentaphyllum, including its extraction, bioactivity, and health effects, is documented in numerous journals globally, including Vietnam This body of work provides valuable insights and comparative data that enhance the findings of this study However, discrepancies in materials, experimental methods, and interpretations of results present challenges, leading to potential inconsistencies and inappropriate comparisons.
LITERATURE REVIEW
Gynostemma pentaphyllum
Gynostemma is a climbing vine genus in the Cucurbitaceae family, which includes familiar plants like cucumbers and gourds Characterized by palmate leaflets, each leaf typically comprises 3 to 9 smaller leaves.
Gynostemma species are dioecious and require pollination (Fu et al., 2020) Among the various species in the Gynostemma genus, Gynostemma pentaphyllum stands out significantly This species typically reaches an average total length of 8 to 10 meters, showcasing common traits of its genus.
5 pedals with a color of whitish or yellowish The small dark fruit often sizes less than
Gynostemma pentaphyllum is characterized by its 1 cm diameter leaflets that contain seeds and feature dark-green, ovate-lanceolate leaves This plant is classified based on the number of leaves per leaflet, with three main types: 3-leaf, 5-leaf, and 7-leaf varieties This study focuses on the 5-leaf Gynostemma pentaphyllum, which is prevalent and highly valued for its nutritional benefits.
Figure 2.1 The classification of Gynostemma pentaphyllum (Credit: Ho Trang)
According to Huyen et al (2012) and Lin-Na & Yong-Xiu (2014) although
Gynostemma pentaphyllum, native to China, is also found in Japan, Korea, Vietnam, Thailand, India, and Bangladesh, thriving in forests at altitudes of 300 to 3000 meters This plant prefers humid conditions, well-ventilated soils, and adequate light, and although it is a climbing vine, it does not need a substrate for growth Variations in its living environment lead to notable differences in plant characteristics such as height, color, and leaflet width Notably, research by T Wang et al (2018) highlights the significant influence of light intensity on the synthesis of saponins in Gynostemma pentaphyllum.
Key enzymes involved in saponin synthesis are significantly more active under high light wavelengths, producing up to 40 mg/g, compared to just 18.5 mg/g in darkness Plants with optimal morphology and potent effects are typically harvested from ideal growing regions in East and Southeast Asia, where they also hold substantial monetary value in various markets.
Wild Gynostemma pentaphyllum is known for its high quality, but challenges in transportation, labor, and costs complicate its harvesting, alongside the risk of over-exploitation and inconsistent quality To address these issues, researchers have focused on in-vitro cultivation techniques, which promote uniformity by growing plants from the cells and tissues of a single mother plant Studies, including those by Sahu & Sahu (2013) and Espinosa-Leal et al (2018), highlight the effectiveness of in-vitro methods in producing bioactive compounds in herbal plants, a finding supported by further research (Chandran et al., 2020; Saldarriaga et al., 2020).
The research conducted by Quảng et al in 2019 highlights the potential for standardizing raw materials through in-vitro cultivation of G pentaphyllum, achieving impressive results The study recorded an average of 7.22 shoots per sample for root development and 6.17 shoots per sample for apical bud shooting, demonstrating the effectiveness of in-vitro techniques in enhancing plant propagation.
Gynostemma pentaphyllum is resilient and can thrive in challenging environments Research by Razmovski Naumovski et al (2005) demonstrated its successful cultivation in Australia, yielding results comparable to those of artificially grown plants in China, but with a 2.5-fold increase in extract proportion.
In Vietnam, the cultivation and research of Gynostemma pentaphyllum have significantly increased, particularly at Thai Nguyen University of Agriculture and Forestry, where the Faculty of Biotechnology – Food Technology has successfully planted 5-leaf Gynostemma pentaphyllum on campus A pivotal study by Bui et al (2015) demonstrated effective in vitro propagation techniques, achieving a germination index of 4.36 times after 30 days by creating an optimal growth environment with 0.4 mg Kinetin/L and 0.5 mg BA/L Further research by Ho, Nguyen, and Phan (2018) and Tran et al (2020) in Ho Chi Minh City and Hue City, respectively, has also yielded promising results, highlighting the favorable conditions for scientific exploration of Gynostemma pentaphyllum across Vietnam.
2.1.3 Compositional bioactive compounds in Gynostemma pentaphyllum
Gynostemma pentaphyllum is rich in saponins, a versatile group of bioactive compounds known for their surfactant properties, which enable the formation of foamy structures used in cosmetics and pharmaceuticals More significantly, these saponins offer various health benefits, making them valuable ingredients in medicines, dietary supplements, and functional foods The saponins found in Gynostemma pentaphyllum are particularly noteworthy due to their resemblance to other beneficial saponins.
Panax Ginseng This has been proved by many studies that compared the two herbs (Utama-Ang et al., 2006; Subramaniyam et al., 2011; Liang et al., 2019)
Figure 2.2 Rb1, the saponin present in G pentaphyllum and Panax Ginseng
The extraction and quantification of Gynostemma pentaphyllum have been pivotal in scientific research A study by Xie et al in 2010 identified the highest saponin content at 132.6 mg/g of extract, translating to about 1.3 g per 100g of raw material Furthermore, a 2012 analysis revealed significant variability in saponin concentration, ranging from 24.9 to 681.2 mg/g of extract.
In a study of 11 different samples of G pentaphyllum, the total saponin content was found to be between 2.5 and 68.1 g/100g of raw material Utama-Ang et al (2006) identified key saponins, including ginsenocide Rb1 and Rg1, using gas-chromatographic-mass spectrometry Notably, these saponins are also found in Panax Ginseng (Lee et al., 2016; Mohanan et al., 2018) Liang et al (2019) reported that the saponin content in G pentaphyllum is five times greater than that in Panax ginseng, establishing a strong basis for this study's focus on bioactive compounds and saponins.
Flavonoids are a significant group of bioactive compounds found in notable members of the Cucurbitaceae family, such as Gynostemma pentaphyllum Research by Kao et al (2008) identified eight flavonoid types in this plant using methanol extraction and HPLC-MS analysis A decade later, Wang et al (2018) discovered nine distinct flavonoids through pressure-modified liquid chromatography, with rutin and quercetin standing out as valuable compounds commonly found in various fruits and vegetables The total flavonoid content in G pentaphyllum varies significantly, ranging from 0.38 to 5.23 mg/g of raw material, as reported by Xie et al (2010).
A total flavonoid content of 6.35 mg/g was identified in raw material by a study conducted in 2012 Research by Kao et al (2008), Samec et al (2016), and Huyn et al (2019) consistently highlighted the variability of total flavonoid content in G pentaphyllum from various sources over the past decade.
Figure 2.3 Polysaccharides of G pentaphyllum (Ji, Shen and Guo, 2018)
The polysaccharide profile of Gynostemma pentaphyllum is complex and sophisticated Many compounds were identified in the study of Ji, Shen and Guo
In 2018, a study characterized 18 types of polysaccharides isolated from the herb, identifying significant compounds such as GPMPP, GPA, GPP, CGPP, GPS-2, and GPS-3 These polysaccharides are primarily composed of Xylose (Xyl), Rhamnose (Rha), Arabinose (Ara), Mannose (Man), Galacturonic acid (GalA), Glucose (Glu), and Galactose (Gal) Prior research by Yang et al (2008) on the purification of G pentaphyllum’s polysaccharides also highlighted that the main components included Glucose, Galactose, Arabinose, Rhamnose, Galacturonic acid, Xylose, and Mannose, with respective percentages of 23.2%, 18.9%, 10.5%, 7.7%, 7.7%, 4.7%, and 3.9% The complexity of polysaccharides in this herb underscores their potential significance.
Gynostemma pentaphyllum is a significant factor contributing to various effects of the plant, thus asserting the benefits and the importance of extracting it
Extraction methods
In herbal science, extraction involves obtaining chemical components from herbs through the use of solvents and the principles of gradients and osmosis Consequently, it is essential to consider the various factors that influence the extraction process and the techniques employed for effective implementation.
The choice of solvent is crucial in any extraction process, significantly influencing the compounds extracted, overall costs, efficiency, and usability Additionally, the selection of the appropriate solvent is determined by the specific compounds desired, particularly in the extraction of pharmaceutical herbs.
Gynostemma pentaphyllum requires a solvent that is both polar and non-polar to effectively extract its bioactive compounds, which possess these dual characteristics (Abubakar & Haque, 2020) Additionally, the solvent should be non-viscous to enhance movement during the extraction process (Abubakar & Haque, 2020) Ethanol, methanol, and water are the most commonly used solvents for extracting G pentaphyllum, owing to their availability and efficiency.
Ethanol (C2H5OH) is a highly versatile and non-toxic alcohol commonly used as a solvent, making it an economical choice for applications ranging from laboratories to industrial plants Its unique structure features a slightly polar alcohol end and a non-polar carbon chain, contributing to its effectiveness in various settings.
Numerous studies have demonstrated the effectiveness of ethanol in extracting G pentaphyllum, with notable success For instance, Xie et al (2010) achieved remarkable results using 100% ethanol, extracting 132.6 mg of saponins per gram of extract and 63.5 mg of rutin equivalent to flavonoids per gram Additionally, efforts to isolate and identify polysaccharides have been undertaken in this research area.
Ji, Shen, and Guo (2018) demonstrated that using ethanol as a solvent for extraction is effective for Gynostemma pentaphyllum This method is not only feasible but also highly productive, as ethanol successfully extracts a majority of the beneficial bioactive compounds present in the plant.
Compared to methanol, water, and even other solvents used for extracting
Gynostemma pentaphyllum extraction using ethanol is advantageous due to its non-toxic nature and effectiveness in purification A study by Jiang et al (2017) demonstrated the efficiency of this method in extracting and purifying Ombuoside, a key compound found in G pentaphyllum.
Like ethanol, methanol (CH3OH) is also an alcohol that has been successfully employed in studying the extraction of Gynostemma pentaphyllum Utama-Ang et al
Research has shown that Rb1 and Rg1 saponins in Gynostemma pentaphyllum closely resemble those in Panax Ginseng when extracted using methanol (2006) Methanol effectively characterizes the composition of G pentaphyllum (Blythe et al., 2017) and demonstrates extraction efficiency comparable to, or even exceeding, that of ethanol However, due to its toxicity, methanol poses significant risks for purification and consumable products (Lachumy et al., 2010), and its cost is higher than that of the more environmentally friendly ethanol Consequently, methanol is primarily recommended for identification, quantification, and comparative studies rather than for producing consumable extracts Notably, several studies have utilized methanol to compare its efficacy with ethanol and other solvents in extracting Gynostemma pentaphyllum (Zhao et al., 2012).
Water is a versatile solvent commonly used in various extraction processes, but it is less efficient than organic solvents like ethanol and methanol due to its higher boiling temperature (Huang & Yuan, 2015) This limitation affects the retrieval and purification of bioactive compounds However, water remains valuable for comparative studies in extraction While pure water has not been utilized in specialized studies on Gynostemma pentaphyllum, other research on similar bioactive compounds has employed it, offering useful comparative insights Notably, the study by Lượng et al (2020) focused on extracting saponins.
Sapindus mukorossi demonstrated an effective extraction process using water, revealing that water serves as a powerful solvent, yielding results with only about a 2% reduction compared to alcoholic solvents.
This efficient extraction method involves placing herbs in a non-reactive container, adding solvent to cover the samples, and sealing the container for 24 hours at room temperature During this period, bioactive compounds are extracted due to the concentration gradient between the samples and the solvent After filtering the fluid, the solvent is evaporated to obtain the extract To maximize extraction efficiency, this process is repeated two to three times.
The submerged extraction method, while beneficial for handling large sample sizes and ease of use, has significant drawbacks It is time-consuming and requires a substantial amount of solvent to achieve efficient results, leading to increased costs Additionally, there is a risk of sample decomposition during the process.
2017) And most importantly, controlling operational conditions is virtually inappropriate Therefore, the submerged extraction method is not suitable for experiments involving optimization In fact, no scientific studies on extracting
Gynostemma pentaphyllum have been found using this technique
2.2.2.2 Ultrasonically assisted extraction method (UAE)
Ultrasonically assisted extraction (UAE) is an efficient method that enhances the extraction of compounds from biomass by using sound waves above 20 kHz to create cavitation bubbles at the interface between the cell wall and the solvent This process damages the cell wall as the vibrating bubbles explode, facilitating the release of internal constituents at a faster rate compared to traditional submerged extraction methods While UAE is particularly effective on a laboratory scale and may not accommodate as much biomass as submerged techniques, it is automated, simple to operate, and cost-effective Additionally, UAE significantly reduces time and energy consumption while improving extraction efficiency.
Research has shown that the ultrasonically assisted extraction method for Gynostemma pentaphyllum yields favorable outcomes A notable study conducted by Tang et al in 2012 investigated the impact of ultrasound on the extraction of Gypenoside III, a type of saponin.
Gynostemma pentaphyllum As a result of 25 minutes ultrasonically processing, the
19 yield of saponins extracted was close to the predicted value To have identified 18 different polysaccharides, the study of Ji et al (2018) on G pentaphyllum utilized
UAE The effect of single-factor in extracting G pentaphyllum in the study of
Kamkayan & Assatarakul (2021) utilized UAE to determine the total phenolic content of Gynostemma pentaphyllum, which was found to be 405.17 mg/100 g dry sample, alongside a total flavonoid content of 2.59 mg Quercetin equivalent/100 g dry sample These findings demonstrate that the ultrasonically assisted extraction method effectively optimizes the quality of the extract.
2.2.1.3 Microwave assisted extraction method (MAE)
Encapsulation
Bioactive compounds can have remarkably strong effects even in small amounts, making standardization of herbal extracts essential for safe consumption Encapsulation is a widely used technique to address this issue, offering flexibility with various excipients Research by Uhumwangho and Okor (2007) shows that capsules are more convenient than tablets in terms of research, execution, and cost Additionally, successful encapsulation of G pentaphyllum extracts, as demonstrated by Wang et al (2019), supports the feasibility of this approach.
Research status and usage of Gynostemma pentaphyllum
G pentaphyllum has been extensively studied in China, Japan, and other Asian regions A significant study in 1993 by Tan, Liu, and Liu revealed that the plant's bioactive extract inhibited platelet aggregation, disaggregation, and thrombosis, while also preventing the activities of various coagulants Following this, Li et al (2015) further demonstrated the beneficial effects of G pentaphyllum extract by examining its polysaccharides.
Kao et al (2008) and Wu, Jang and Piao (2014) also well provided information
21 regarding the bioactive composition of G pentaphyllum extract and its benefits In general, the understanding over the subject has been relatively adequate
In a study conducted by Chavalittumrong et al (2007) in Thailand, G pentaphyllum extract capsules were tested on volunteers, revealing that the herb is relatively safe as no clinical incidents were reported Additionally, the capsules led to significant changes in hematology, indicating that the extract has beneficial effects supported by findings from other research.
The results of studying in the world have led to certain practical products Nevertheless, the number of those products is inconsiderable in the world scale
In Vietnam, Professor Pham Thanh Ky is one of the pioneers in studying
Gynostemma pentaphyllum He first discovered this herb on Fansipan Mountain – Lao
Cai Province's research on G pentaphyllum has laid the groundwork for technology transfer in the pharmaceutical industry, leading to various processes adopted by companies (Ky et al., 2010) His findings highlighted the similarity between the saponins of G pentaphyllum and those found in Panax ginseng.
Himalayan ginseng, scientifically known as G pentaphyllum, has been shown to stimulate insulin production, according to a study by the Vietnamese National Institute of Medical Substances in collaboration with the Swedish International Diabetes Federation (Huyen et al., 2012).
Recent scientific research on G pentaphyllum has surged, yielding numerous positive reports and studies that highlight its potential for future development A notable study by Huyen et al in 2013 demonstrated the herb's ability to enhance insulin sensitivity, suggesting its anti-diabetic properties Additionally, Tong et al (2017) established clear methods for evaluating the bioactivity of G pentaphyllum, paving the way for the creation of practical products Consequently, the Vietnamese market has begun to introduce consumable products derived from this herb.
The market for dried leaves, particularly pentaphyllum, in Vietnam is characterized by small, unorganized manufacturers, yet the demand for these products remains significant This presents a substantial opportunity for more specialized and well-organized offerings Below are examples of models currently available in the Vietnamese market.
- Used by infusing in hot water
- Support blood circulation, increase strength
- Used by infusing 20 – 30g in hot water each time
- Support diabetes treatment, reduce cholesterol in blood, increase strength
An exception of casual products is Tue Linh company
This brand provides G pentaphyllum functional tablets that possess anti-diabetes and anti-blood clotting properties, helping to stabilize the circulatory system However, the current popularity of these products is limited, indicating a need for new specialized marketing strategies.
METHODOLOGY
Sample collection and preparation
Fresh Gynostemma pentaphyllum leaves and stems were harvested from the ecological garden at the Faculty of Biotechnology – Food (TUAF) after 18 months of cultivation on December 15th The leaves were then dried at 60°C until they reached a moisture content of 10% before being stored in polyethylene bags under ambient conditions.
The study employed the following chemicals
No Name Formula Usage Origin
2 Methanol (96%) CH3OH Solvent China
4 n-Butanol (99%) C4H9OH Solvent specialized for saponins
5 Petroleum ether C2H5CH(CH3)C2H5 Eliminate lipids China
The study employed the following instruments
1 Universal Oven UN110 plus Germany
In addition, the work also required fundamental laboratory instruments such as Erlenmeyer flasks, test tubes, stirring rods, pipets, and beakers
The study was implemented in the laboratory scale, which is located in the Faculty of Biotechnology – Food Technology, Thai Nguyen University of Agriculture and Forestry, Vietnam.
Experiment design and methods
This research focused on the following 5 contents
• Determine the quality of input Gynostemma pentaphyllum
• Determine the single-factors for the extraction of G pentaphyllum
• Optimize the extraction process and complete the extraction procedure
• Determine the Bioactivity of G pentaphyllum extract including the capability against oxidation and cancer cell proliferation
• Formulate the pharmaceutical flour and complete encapsulation
3.2.2 The input quality of Gynostemma pentaphyllum
After collected, fresh G pentaphyllum is evaluated in terms of input quality
Required criteria include moisture content, ash content, and total saponin content, which is the primary parameter of this study
The study assessed the moisture content of fresh G pentaphyllum samples using the Oven-drying method, adhering to the Vietnamese standard TCVN 4407:2010 with some modifications in calculations Three samples, each weighing 10 grams of fresh G pentaphyllum stems and leaves, were analyzed The drying process utilized natural convection in a Memmert dryer, aiming to reduce the samples' water content to a stable mass Moisture content was determined based on the weight loss during drying, calculated using a specific equation.
The equation defines moisture content (MC) as a percentage, calculated using the total weight of the sample and container (mtotal), the weight remaining after drying (mremaining), and the weight of the sample (msample), which was set at 10 for this study.
Ash is composed of inorganic constituents in plants, including minerals like sodium, calcium, and zinc, as well as salts such as Na\(^+\), K\(^+\), and Ca\(^{2+}\) The ash content is crucial for assessing the quality of input materials due to its implications for potential metal toxicity This study utilized incineration as per TCVN 8124:2009, employing three formulas corresponding to three crucibles, each containing 3 grams of dried G pentaphyllum The crucibles were placed in an incinerator at a temperature of 600 °C for 6 hours to eliminate all organic matter, with the remaining inorganic matter representing the ash content, which was calculated using a specific formula.
𝑚 𝑠𝑎𝑚𝑝𝑙𝑒 ∗ 100% (2) The crucibles were scaled once after 4 hours, 5 hours, and 6 hours to assure no organic matters remained
The raw material was analyzed for total saponin content at the Life Science Institute of Thai Nguyen University of Agriculture and Forestry The analysis involved ethanol extraction followed by quantification using High Performance Liquid Chromatography (HPLC), with Gypenoside III serving as the standard sample.
3.2.3 The effects of single factors on extracting G pentaphyllum
The extraction method utilized was ultrasonically-assisted liquid-solid extraction, as described by Chemat et al (2017) In this process, 3 grams of dried G pentaphyllum were placed in a 100 ml Erlenmeyer flask, followed by the addition of a solvent The flask underwent ultrasonic processing for a specified duration before being placed in a water bath to control the temperature Consequently, it was essential to identify the optimal extraction conditions for the experiments conducted.
3.2.3.1 The effects of ultrasonic assistance
Ultrasound was employed to support the extraction process The examination of this factor was based on Vinatoru et al (2017) Particularly, the instrument generated
The study utilized 37 kHz soundwaves in degas mode, conducting experiments over a 10-minute duration with recordings taken at 1, 3, 5, 7, and 10 minutes, resulting in five distinct formulas Each formula comprised 3 g of the sample mixed with ethanol solvent at a 1:15 g/ml ratio in an Erlenmeyer flask Following the experiments, the total saponin extract was analyzed to assess the impact of ultrasound on the extraction of G pentaphyllum, with each formula being replicated three times.
3.2.3.2 The effects of different solvents
This study, following Ji et al (2018), utilized ethanol (C2H5OH) and methanol (CH3OH) as the main variables, with water serving as a comparative medium The experiment was structured into three identical trials for each solvent.
This article presents 27 formulas designed to assess the reliability of results under specific operational conditions, which include ultrasonic processing for 5 minutes at a temperature of 60°C, an extraction duration of 60 minutes, and a material-solvent ratio of 1:15 g/ml Additionally, the concentration of solvents used, specifically ethanol and methanol, was maintained at 70%.
3.2.3.3 The effects of solvent concentrations
The study investigated four concentrations: 50%, 60%, 70%, and 80%, using a solvent determined from prior experiments The operational conditions included 5 minutes of ultrasonic processing at 60 °C, a duration of 60 minutes, and a material-solvent ratio of 1:15 (g/ml) The optimal concentration identified in this experiment was utilized in subsequent analyses, following the extraction study by Ji et al (2018).
3.2.3.4 The effects of extraction time
The study examined a 120-minute extraction process, building on the work of Wang et al (2007) with some modifications The solvent and concentration were based on prior research, while the extraction temperature was maintained at 60 °C with a material-solvent ratio of 1:15 (g/ml) Total saponin extracts were recorded every 15 minutes starting from the 45th minute, resulting in a total of six data points.
3.2.3.5 The effects of extraction temperatures
The study investigated extraction efficiency within a temperature range of 60 °C to 80 °C (Ji et al., 2018) Optimal parameters, including solvent type, concentration, and extraction time, were determined based on prior experiments, while maintaining a material-solvent ratio of 1:15 (g/ml) Extraction efficiency was measured at temperatures of 60 °C, 65 °C, 70 °C, 75 °C, and 80 °C, with the optimal temperature selected for subsequent experiments.
3.2.3.6 The effects of material-solvent ratios
There were 3 ratios in examination including 1:10, 1:15, and 1:20(g/ml) This was based on the study of Luong et al (2020) with modifications according to the
In a study examining the extraction of G pentaphyllum, optimal conditions such as solvent type, concentration, extraction time, and temperature were identified based on prior experiments The analysis focused on the total saponin extract to establish the most effective material-solvent ratio for extraction.
3.2.3.7 The quantification of saponins in G pentaphyllum extract
The quantification of saponin extract was conducted following the guidelines from the National Institute of Medicinal Materials (NIMM) as outlined in the Herbal Extraction Techniques (2008) The extract was diluted to a 10% concentration in 50-ml Erlenmeyer flasks, followed by the addition of 5 ml of n-butanol, a selective solvent for saponins and triterpenoid compounds The mixture was agitated for 20 minutes, and the solvent was then removed using a vaporizer at 50 °C and a reduced pressure of 175 mbar The residue was measured to determine the relative proportion of saponin extract from Gynostemma pentaphyllum.
In the equation, %s represents the total proportion of saponin extracts in
Gynostemma pentaphyllum; m is the weight measured from the residue; and M is the initial weight of materials In this study, M is 3 grams as each formula contained 3 grams of G pentaphyllum samples
After conducting batch experiments to identify the optimal single factors, three variables—A, B, and C—were chosen for optimization using Response Surface Methodology (RSM) (Bezzera et al., 2018) The selected factors included extraction time, temperatures, and material-solvent ratios, which were organized in a Box-Behnken design featuring three levels for each factor.
Table 3.1 Coding of factors for optimization
Variable Factor Unit Level -1 Level 0 Level +1
The Box-Behnken matrix produced 17 distinct extraction condition formulas, incorporating various levels of factors A, B, and C Level 0 corresponds to values obtained from single-factor experiments, while level -1 indicates lower values and level +1 signifies higher values Among the 17 formulas, 5 operate at condition level 0 The results from all formulas led to the synthesis of an equation that identifies the optimal conditions for the integrated operation of the three factors.
17 experiments of the Box – Behnken matrix were exhibited as follows:
Table 3.2 Experiment designs for optimization experiments of Box - Behnken
3.2.5 Bioactivity of the G pentaphyllum extract
3.2.5.1 Assessment of anti-oxidation capability
Statistical Analysis
Data from experiments were analyzed using Microsoft Excel and processed with the Duncan comparison method of ANOVA through PASW 18 (SPSS) The optimization experiment employed Design-Expert software for surface response analysis, while numerical and graphical presentations were created using Microsoft Word 2017 and Origin 2019.
RESULT AND DISCUSSION
Determination of input quality
Moisture content, ash content, and total saponin content are essential indicators for evaluating botanical materials, with total saponin content being a key criterion in this research The specific values for G pentaphyllum regarding these parameters were measured and analyzed.
Table 4.1 Initial indexes of the raw material
The average moisture content and ash content were 89.56% and 1.16%, respectively Compared to other G pentaphyllum, the samples from China of
Razmovski-Naumovski et al (2005) had 75 – 85% of moisture and 0.9 – 1.4% of ash Whereas, the moisture and ash of G pentaphyllum from Thailand were 86.79% and 1.27%, respectively
The total saponin content in this study's raw material significantly differs from other research, with an average of 15.67% in dried G pentaphyllum In contrast, Z Wang et al (2007) reported a lower yield of 11.29%, while Xie et al (2010) found a maximum of 22.26% in five different commercial samples in China Notably, the yield in this study aligns more closely with in-vitro G pentaphyllum results from China, likely due to variations in saponin quantification techniques, leading to distinct interpretations across studies.
The effects of single-factors on G pentaphyllum extraction
4.2.1 The effects of ultrasonic processing time on extraction
Ultrasonic processing is a supporting factor in botanical extraction as it breaks down fibrous cell walls using vibration Nevertheless, the extreme soundwaves can
35 also damage molecular levels including also include the saponin group That had been well demonstrated through the findings of this experiment, which were in the following table 4.2 and figure 4.1
Table 1.2 Total saponin extract from different ultrasonic processing time
Total saponin extract (g/100g dried material)
Note: In the same column, mean values with different lowercase letters indicate significant differences at p < 0.05
Figure 4.1 The effects of ultrasonic processing time on extracting G pentaphyllum
The application of 37 kHz soundwaves significantly increased the total saponin extract from 6.42 g/100g to 8.13 g/100g within the first 5 minutes, before slightly decreasing to 8.05 g/100g after 7 minutes, and further declining to 7.62 g/100g thereafter This pattern aligns with previous research on ultrasound-assisted botanical extraction, such as Lakka et al (2019), which reported a similar trend in hops extraction, where total polyphenols surged by approximately 17% in 20 minutes but fell by 19% after 30 minutes Liang et al (2019) also noted a decrease in Gypenoside III content from Panax ginseng after extended ultrasonic processing These findings highlight that variations in raw materials and soundwave frequencies can influence optimal extraction times, with 5 minutes at 37 kHz identified as the most effective duration for saponin extraction in this study.
4.2.2 The effects of different solvents
This study compared the effects of methanol and ethanol, with water included to enhance the experimental framework The findings regarding total saponin extract and efficiency are presented in Table 4.2.2 and Figure 4.2.2.
Table 4.3 Total saponin extract obtained from using water, ethanol, and methanol
Type of solvent Total saponin extract (g/100g) Efficiency (%)
Note: In the same column, mean values with different lowercase letters indicate significant differences at p < 0.05
Figure 4.2 Graphic illustration of table 4.2.2
Figure 4.2.2 demonstrates that alcohol solvents are significantly more effective than water for extracting compounds from Gynostemma pentaphyllum This indicates that the traditional water infusion method may not fully utilize the herb's potential The experiment's results and statistical analysis revealed no significant difference in the extraction efficiency of methanol and ethanol, both achieving a maximum saponin extract of 10.97 g/100g, which corresponds to an efficiency of 69.99% Similar findings regarding the effectiveness of methanol and ethanol were reported in studies by Utama-Ang et al (2006) and Xie et al.
(2010) and on extracting commercial G pentaphyllum In botanical extraction, particularly G pentaphyllum, many studies selected ethanol over methanol for that the
Ethanol, recognized for its affordability and non-toxic properties as a solvent, was chosen for further experiments based on the results of this study (Utama-Ang et al., 2006; Ji et al., 2018; Kamkayan & Assatarakul, 2021).
`4.2.3 The effects of ethanol concentration
There were 4 different concentrations including 50%, 60%, 70%, and 80% The results from extracting G pentaphyllum with these solvent concentrations were as follows (table 4.4 and figure 4.3.)
Table 4.4 Total saponin extract obtained from different ethanol concentrations
Note: In the same column, mean values with different lowercase letters indicate significant differences at p < 0.05
Solvent concentration Total saponin extract (g/100g) Efficiency (%)
Figure 4.3 The effects of solvent concentrations on extraction
The results illustrated in Figure 4.3 indicate that increasing solvent concentration enhances total saponin extract, although the rate of increase varies between concentrations A notable rise in total saponin extract was observed when comparing 50% ethanol to 60% ethanol, with an efficiency increase of 5.26% (from 9.91 to 10.73 g/100g) However, the efficiency difference decreased to 3.07% between 60% and 70% ethanol, and further reduced to only 0.6% when moving from 70% to 80% ethanol The extraction with 80% ethanol yielded 11.31 g/100g (72.17% efficiency), which was statistically similar to the 70% ethanol extraction result of 11.21 g/100g (71.57% efficiency) Consequently, 70% ethanol concentration is deemed optimal for further experiments, aligning with findings from Kamkayan & Assatarakul (2021).
After testing various concentrations ranging from 50% to 90%, it was found that a 70% concentration is optimal for extracting G pentaphyllum, as noted by Nizamova et al (2021) This concentration strikes a balance between high polarity and manageable volatility Lower concentrations are less effective due to insufficient affinity for the constituents, while higher concentrations tend to evaporate rapidly, leading to degradation.
4.2.4 The effects of extraction time
Extraction time indicates the duration that the mixture of material and solvent remains in the water bath, with this experiment examining a range of 120 minutes The total saponin extract obtained at various time intervals and their efficiencies are presented in Table 4.5 and Figure 4.4.
Table 4.5 Total saponin extract from different extraction time in 120 minutes
Time (min) Total saponin extract (g/100g) Efficiency (%)
Note: In the same column, mean values with different lowercase letters indicate significant differences at p < 0.05
Figure 4.4 Effect of extraction time
The acceleration rate of extraction from the 45th to the 90th minute was consistent, increasing from 9.98 g/100g to 13.51 g/100g, representing a 63.70% to 86.20% rise This resulted in an average increase of 1.18 g/100g every 15 minutes, equating to a 7.5% efficiency Initially, high osmotic pressure facilitated rapid mass transfer and quick saponin extraction due to the low concentration of solute in the solvent However, after 90 minutes, the acceleration rate ceased as equilibrium was reached between the solvent and the herb's inner matter, halting mass transfer Duncan testing indicated no statistically significant differences in extraction yields at 90, 105, and 120 minutes, confirming that the maximum yield occurred at 120 minutes.
42 with 13.70 g total saponin extract /100g (87.42% efficiency), 90 minutes was concluded as the most appropriate time for extracting G pentaphyllum in this study
Research on G pentaphyllum has focused on optimizing extraction methods, revealing varying optimal times Ji et al (2018) found that a 40-minute immersion with a material-solvent ratio of 1:25 and enhanced ultrasonic processing was effective In contrast, Wang et al (2007) concluded that extraction could be achieved in just 10 to 15 minutes at a high temperature of 95 °C, using a more generous solvent ratio of 1:67.
4.2.5 The effects of extraction temperatures
This experiment examined the range of 60 o C to 80 o C in Gynostemma pentaphyllum extraction The results were showed in table 4.6 and figure 4.5
Table 4.6 Total saponin extract from using different temperatures (60 – 80 o C)
Temperatures Total saponin extract (g/100g) Efficiency (%)
Note: In the same column, mean values with different lowercase letters indicate significant differences at p < 0.05
Figure 4.5 The effects of extraction temperatures on extracting G pentaphyllum
Extraction at 60 °C yielded a total saponin extract of 13.51 g/100g, achieving an efficiency of 86.24% This increased to 13.84 g/100g (88.33%) at 65 °C and peaked at 14.29 g/100g (91.20%) at 70 °C, demonstrating a direct correlation between temperature and kinetic energy According to Jawade & Chavan (2013), higher temperatures enhance the kinetic energy of matter, which increases mass transfer rates and particle diffusivity, allowing the solvent to dissolve more saponins However, at 75 °C and 80 °C, extraction efficiency dropped significantly to 13.65 g/100g (87.10%) and 12.56 g/100g (80.15%), respectively, due to ethanol's boiling point of 78.37 °C, resulting in solvent loss.
44 finding was agreed by Che Sulaiman et al (2017) when reviewing herbal extraction
Ji et al (2018), who used the temperature range of 80 o C to 100 o C, also concluded that this range was only proper to water extraction
As the temperature that yielded the maximum extraction efficiency, 70 o C was selected for subsequent extraction experiments
4.2.6 The effects of solvent – material ratios on extraction
In this experiment, 3 different ratios including 1:10 g/ml, 1:15 g/ml, and 1:20 g/ml were investigated in the extraction of Gynostemma pentaphyllum The results were exhibited via table 4.7 and figure 4.6 as follows
Table 4.7 Total saponin extract from using different solvent amount (1/10 – 1/25)
Material-solvent ratio Total saponin extract (g/100g) Efficiency (%)
Note: In the same column, mean values with different lowercase letters indicate significant differences at p < 0.05
Figure 4.6 The effects of material-solvent ratios on extracting G pentaphyllum
The 1:10 ratio yielded a significantly lower total saponin extract of only 11.21 g/100g, achieving 71.57% efficiency, primarily due to insufficient ethanol coverage in the 100 ml Erlenmeyer flask Fang et al (2020) noted that a limited solvent volume results in reduced osmotic pressure and extraction efficiency In contrast, the 1:15 ratio produced 14.29 g saponin extract/100g (91.16% efficiency), while the 1:20 ratio yielded 14.49 g/100g (92.47%), with no statistical difference between these two results The choice of material-solvent ratios significantly influences extraction conditions, as demonstrated by Z Wang et al (2007), who achieved over 90% efficiency using a 1:67 ratio at 95 °C for just 10 minutes.
46 the other hand, Xie et al (2010) and Wang et al (2020) needed 30 – 50 minutes and
To effectively extract G pentaphyllum, a material-solvent ratio of 1:30 at temperatures between 60 and 70 °C is recommended This study demonstrates that ultrasonically-assisted extraction with 70% ethanol at 70 °C for 90 minutes, using a 1:15 ratio, is adequate to achieve extraction equilibrium.
Response surface optimization
To perform response surface methodology (RSM), a total of 17 experiments were conducted to extract Gynostemma pentaphyllum under varying conditions of time, temperature, and material-solvent ratios, with the findings detailed in Table 4.3.1.
Table 4.8 The responses of 17 experiments according to the Box-Behnken design
Table 4.9 ANOVA analysis of response surface optimization
Source Sum of Squares df Mean Square F-value p-value
Figure 4.7 Specific solution and desirability of optimization
The surface response optimization yielded 100 solutions for enhancing the saponin extraction from Gynostemma pentaphyllum These solutions typically involve extending the extraction time to over 100 minutes and adjusting the material/solvent ratio, while slightly lowering the extraction temperature The predicted maximum yield of saponin extract is noted.
15.01 g/100g dried materials, corresponding to a high efficiency of 95.79% This is comprehensible as the longer the extraction proceeds, the more efficient the extraction process is (Raghav & Kumar, 2018; Q.-W Zhang et al., 2018) Furthermore, the more amount of solvent also ensures the stability of extraction, preventing solvent loss and degradation (Fang et al., 2020) This issue is also fortified by the reduced temperature, which avoids evaporation as the boiling point of ethanol is only 78.37 o C (Che Sulaiman et al., 2017) Solution 10 was selected with the optimized time, temperature, and material-solvent ratio of 101 min, 69 o C, and 1/18, respectively
Bioactivity of the G pentaphyllum extract
Free radicals play a crucial role in the human body due to their oxidation capabilities However, they can accelerate aging and trigger undesirable reactions in organs, resulting in inflammation and molecular damage The application of RDSC has shown promising results in addressing these issues.
Gynostemma pentaphyllum extract in this study are shown in table 4.4.1 as follows
Table 4.10 Absorbance obtained from anti-oxidation capability experiment
Concentration Tested Formula %RSA IC50 (mg/ml)
Note: In the same column, mean values with different lowercase letters indicate significant differences at p < 0.05
Figure 4.8 Linear fitting and indexes (the intercept, slope, and R square)
The extract demonstrates significant antioxidant efficiency, achieving a 48.23% reduction in free radicals with just 0.5 mg extract equivalent/ml Additionally, an increase in extract concentration correlates with enhanced radical scavenging activity.
The IC50 value was determined to be 0.71 mg/ml, as indicated by the equation y = 0.45745 + 0.0603x (see appendix 1) Utilizing the same RDSC technique, Xie et al (2010) reported that their GP4 samples exhibited an anti-oxidation capacity of approximately 49% at a concentration of 0.4 mg/ml, aligning closely with the findings of this study Additionally, a radical scavenging activity of 57% was observed at a concentration of 1.5 mg/ml In contrast, Xie et al (2012) noted that the anti-oxidation capacity varied significantly, ranging from less than 20% to around 70%, depending on the sample's origin Notably, a capacity between 30% and 50% was the most common, with 10 out of 16 cases falling within this range.
Table 13 below shows the results obtained from testing Gynostemma pentaphyllum extract on the most common cell lines representing liver cancer (Hep-
G pentaphyllum from Thai Nguyen University of Agriculture and Forestry demonstrates promising anti-cancer effects against various tumor cell lines, including Hep-G2, a common primary liver cancer At a concentration of 49.57 µg/ml, the extract inhibited 50% of Hep-G2 cells While it also exhibits some anti-blood cancer effects, these are relatively insignificant However, the extract did not show any inhibitory effect on the metabolism of the lung cancer cell line SK-LU-1.
Table 2 Cell proliferation inhibitory effect of G pentaphyllum extract
Cell proliferation inhibitory effect (IC50)
G pentaphyllum extract 49.57 àg/ml - > 100 àg/ml
Gynostemma pentaphyllum has demonstrated effectiveness against the Hep-G2 cell line and liver cancer, as confirmed by various studies Notably, gypenoside Rg3, found in this plant and also in Panax ginseng, has been shown to inhibit Hep-G2 cells, with research by Shi et al (2015) revealing varying IC50 values from six different G pentaphyllum samples The most effective sample achieved a 50% inhibition at a concentration of just 12.85 µg/ml, while another sample required 143.44 µg/ml for the same effect Additionally, for HL-60 cells, which represent leukemia, the dammarane saponins in G pentaphyllum play a significant role in its effects, although minor polysaccharides have shown negligible impact, as indicated by studies including Ky et al (2010).
Pharmaceutical flour quality and encapsulation
4.5.1 Mixing ratio of G pentaphyllum extract and glucose
After evaporating ethanol to obtain the extract, it was combined with deionized water and glucose to produce the pharmaceutical flour The tested ratios and descriptions are summarized below.
Table 4.12 Mixing ratios and descriptions
Glucose 65 g The mixture had a dark green color with strong herbal odor of bitterness The phrase was semi-solid and viscous
Glucose 70 g The mixture still had an unusual green color The phrase remained viscous
The mixture had a light green color with no apparent odor The phrase of the mixture was virtually solid pieces
Glucose 80 g The green color was insignificant
The phrase was solid pieces with white glucose flour covering the surface
As a result, te ratio between the extract and the glucose was 45 ml/75 g
4.5.2 The effect of drying temperatures
The results on drying temperatures are showed in table 4.5.1 as follows
Table 4.13 Total saponin extract of G pentaphyllum extract flour after drying
Temp ( o C) Time (min) Saponin extract (g) Saponin extract (%)
Note: In the same column, mean values with different lowercase letters indicate significant differences at p < 0.05
As the temperature increased from 50 °C to 90 °C, the saponin extract generally decreased However, the efficiency of drying showed that saponin extraction rates improved with rising temperatures from 50 °C to 70 °C.
70 o C resulted in the highest rate of extraction with 1.46% saponins corresponding to 0.095 grams out of 10 grams Statistically, the saponin extract of 60 o C resembled both
50 o C and 70 o C However, its efficiency is much higher than 50 o C while slightly lower than 70 o C The point 90 o C remarked the lowest numbers with only 0.087 g out of 10 g
The saponin extraction rate increased to 1.37% with 53 grams of flour, primarily due to the water content in the mixture While higher temperatures facilitate water evaporation, they can also harm bioactive compounds like saponins (Cvetanović et al., 2019) Consequently, a drying temperature of 70 °C for 75 minutes was found to be optimal for the pharmaceutical flour produced.
Table 4.14 Indexes of a Gynostemma pentaphyllum capsule
Weight per capsule Moisture content Total saponin extract
The G pentaphyllum flour was dried and encapsulated in 00 gelatin capsules, with each capsule containing approximately 750 mg Experiments to assess total moisture content and total saponin extract were conducted in triplicate For the moisture content analysis, each replication involved 3 grams (equivalent to 4 capsules) dried at 105 °C until weight stabilization was achieved.
The extraction of 3 grams of flour using 70% ethanol yielded a total moisture content of approximately 8.61%, while the total saponin extract was measured at 1.45% The process for producing pharmaceutical flour from G pentaphyllum, aimed at preserving the herb's bioactivity, is illustrated in the accompanying diagram (Figure 4.9).
Figure 4.9 Production process of pharmaceutical capsules from G pentaphyllum
This study investigates the use of ultrasonically-assisted solid-liquid extraction to retrieve saponins and assess the bioactivity of Gynostemma pentaphyllum The analysis of raw G pentaphyllum leaves revealed a moisture content of 89.50%, an ash content of 1.16%, and saponins comprising 15.67% Key parameters for optimal extraction included a 5-minute ultrasonic processing time at 37 kHz, the use of 70% ethanol as the solvent, and a total extraction duration of 90 minutes.
At a temperature of 70 °C and a material-solvent ratio of 1 – 15 g/ml, the maximum saponin extract obtained was 14.49 g/100 g of dried material, achieving an efficiency of 92.47% Utilizing Response Surface Methodology, optimal conditions were identified as 101 minutes, 69 °C, and a 1:18 material-solvent ratio, leading to an expected saponin extract of 15.01 g/100 g with an efficiency of 95.79% The extract demonstrated significant potency as a DPPH scavenging agent, with an IC50 value of 7.21 mg/g, and showed promising effects against liver cancer and potential against blood cancer in MTT assays, indicating its valuable future applications.
The production of pharmaceutical flour involved mixing 45 ml of extract glue (10%) with 75 grams of glucose and drying the mixture at 70 °C for 75 minutes This process yielded a greenish flour containing 1.45% total saponin extract and 8.61% moisture, which aligns with the Vietnamese Standard TCVN I-4:2017 for herbal pharmacy Following encapsulation, the production process was successfully completed, establishing an effective method for storing the optimized extract.
Gynostemma pentaphyllum, initially laying a foundation for future research on the subject and realistic applications
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