Trends Sci. 2025; 22(11): 10865

In Vitro and In Vivo Activity Evaluations, LC-MS Profiling, Stability Testing, and Capsule Formulation of a Thai Folk Analgesic Herbal Formula

Tipsuchon Aiamsa-Ard¹, Chaowalit Monton^2,3,*, Jira Jongcharoenkamol⁴,

Thaniya Wunnakup², Abhiruj Navabhatra¹, Jirapornchai Suksaeree⁵,

Natawat Chankana⁶ and Teeratad Sudsai²

¹Department of Pharmacology, College of Pharmacy, Rangsit University, Pathum Thani 12000, Thailand

²Drug and Herbal Product Research and Development Center, College of Pharmacy, Rangsit University,

Pathum Thani 12000, Thailand

³Department of Pharmacognosy, College of Pharmacy, Rangsit University, Pathum Thani 12000, Thailand

⁴Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmaceutical Sciences,

Naresuan University, Phitsanulok 65000, Thailand

⁵Department of Pharmaceutical Chemistry, College of Pharmacy, Rangsit University, Pathum Thani 12000, Thailand

⁶Sun Herb Thai Chinese Manufacturing, College of Pharmacy, Rangsit University, Pathum Thani 12000, Thailand

(^*Corresponding author’s e-mail: [email protected])

Received: 6 June 2025, Revised: 10 June 2025, Accepted: 20 June 2025, Published: 10 August 2025

Abstract

The Thai folk analgesic herbal formula, Ya Ka Sai Sen (YKSS), has been traditionally used among patients in rural Thailand, yet its scientific basis remains unexplored. This study evaluated the in vitro antioxidant, anti-inflammatory and cytotoxic properties, along with in vivo acute toxicity and analgesic activities of YKSS. Stability studies and capsule formulation development were also undertaken. Two batches of herbs, harvested at different times, were decocted to yield eight extracts. Total phenolic and flavonoid contents ranged from 100.50 to 179.89 mg GAE/g and 51.43 to 96.19 mg CE/g, respectively. Antioxidant assays demonstrated DPPH IC₅₀ values of 8.74 - 96.19 µg/mL and FRAP values of 2,148.20 - 3,032.97 µmol Fe(II)/g. However, nitric oxide scavenging was below 50%, while inhibition of nitric oxide production in cell-based assays showed IC₅₀ values of 820.93 - 969.13 µg/mL. Anti-lipoxygenase activity was moderate (IC₅₀: 15.50 - 72.75 µg/mL) and cytotoxicity against HepG2 cells varied between 1 - 10 µg/mL. LC-MS analysis identified N-trans-feruloyltyramine and maltose as major compounds in positive and negative ionization modes, respectively. Stability studies showed some samples degraded after one year under ambient conditions. Acute toxicity testing revealed no adverse effects at 2,000 mg/kg in rats. Analgesic evaluation via acetic acid-induced writhing tests showed significant reductions in writhing episodes, whereas no effects were noted in the hot plate test, indicating peripheral nociception inhibition. YKSS was successfully developed into a capsule formulation, demonstrating antioxidant, anti-inflammatory, and analgesic activities with a favorable safety profile. These findings support further exploration of YKSS as a potential therapeutic agent.

Keywords: Antioxidant, Anti-inflammation, Analgesic, Toxicity, Lipoxygenase

Introduction

Musculoskeletal pain is a prevalent and complex condition that poses significant challenges to both patients and healthcare providers. It is experienced by individuals across all demographics, including age, gender and socioeconomic status, with approximately 47% of the global population affected at some point in their lives. Of these, an estimated 39% - 45% experience persistent symptoms requiring medical intervention. Inadequately managed musculoskeletal pain can lead to a marked decline in quality of life and substantial socioeconomic burdens. Traditional and herbal medicines have long been explored as complementary approaches for pain management, particularly in regions with a strong history of traditional healing practices. There are several methods to treat musculoskeletal pain, incorporating pharmacological, non-pharmacological and interventional therapies within a multidisciplinary framework. These approaches are essential for optimizing patient outcomes, including pain relief, functional recovery and overall quality of life [1].

Regarding pharmacological treatment, non-steroidal analgesics and antipyretics (such as aspirin and paracetamol), non-steroidal anti-inflammatory drugs (NSAIDs) (such as ibuprofen, naproxen, meloxicam, celecoxib, parecoxib, etoricoxib, etc.), opioids and combinations with anticonvulsants, antidepressants, local anesthetics, topical agents, anxiolytics and other medications may be used [1]. However, concerns over adverse effects, including gastrointestinal complications and opioid dependence [2,3], have led to increased interest in traditional remedies with potential analgesic and anti-inflammatory properties. While pharmacological medications can be effective, traditional or folk medicines may provide a safer and more effective alternative for pain management, particularly for long-term use [4-6]. The use of herbs presents a promising and cost-effective alternative for the prevention and treatment of inflammatory conditions. This approach is supported not only by the accessibility and affordability of these natural sources but also by their inherent potential to reduce the risks associated with the adverse effects of conventional treatments [7].

Thai traditional medicines are treatment remedies native to Thailand. It is defined as “The medicinal procedures concerned with examination, diagnosis, therapy, treatment or prevention of, or promotion and rehabilitation of the health of humans or animals, obstetrics, traditional Thai massage and also includes the production of traditional Thai drugs and the invention of medicinal devices, base on knowledge or text that has been passed on from generation to generation” [8]. Thai folk medicine, a branch of traditional Thai medicine, relies on locally practiced knowledge and herbal formulations developed over centuries. This practice has been widely utilized in rural areas for treating musculoskeletal pain and inflammation. This traditional wisdom is derived from experience rather than from systematic medical theory [9].

The Ya Ka Sai Sen (YKSS) formula, developed by Mr. Arj Ramadthong, has been traditionally used as a folk medicine in Buached District, Surin Province, Thailand, for the treatment of back pain, wrist pain, abdominal tightness and as a general tonic. This herbal remedy reflects the deep-rooted wisdom of local traditional medicine and has been relied upon by communities for its claimed therapeutic benefits. This formula is composed of 18 herbal ingredients, as detailed in Table 1. However, the formulation is inherently flexible, allowing for adjustments based on the availability of specific herbs. For instance, as noted in Table 1, Ventilago denticulata Willd. was absent in Batch 1, while Cladogynos orientalis Zipp. ex Span. was absent in Batch 2. The underlying principle of this formulation relies on balancing herbs categorized by traditional knowledge as having “hot” and “cool” tastes. A small quantity of “hot-tasting” herbs is combined with a larger quantity of “cool-tasting” herbs to achieve a therapeutic balance, reflecting the wisdom of folk medicine practices. A high amount of “hot-tasting” herbs should be avoided, as it can be toxic or induce abortion in pregnant users and may cause dizziness, headache, or vomiting in non-pregnant individuals. Additionally, the quantities of individual herbs in the formula are not fixed or measured precisely by weight. Instead, they are mixed based on estimation, leading to variations in the ratio of ingredients between different batches. In this study, the authors intentionally replicated the practical variability observed in traditional use to closely reflect real-world applications of the YKSS formula. The preparation of the YKSS formula begins with thoroughly cleaning all herbs with water. The cleaned herbs are then boiled in water, ensuring that the water level completely covers the herbs. Once the mixture reaches a boil, it is removed from heat and allowed to warm or cool before use. The recommended dosage regimen varies among users. Traditionally, one teacup of decoction is consumed throughout the day as a substitute for water. The herbs are reboiled daily with added water to restore approximately the same volume as the initial preparation. The decoction can be used for four to five consecutive days. This traditional knowledge and detailed preparation method for YKSS were kindly provided by Pra Ajan Dhanes Jattabhayo, a monk of Wat Pacha Ban Kraisorn in Buached District, Surin Province, Thailand, reflecting the cultural and medicinal heritage of the region. However, there is no scientific evidence to support the safety and efficacy of this Thai folk medicine; therefore, pre-clinical and clinical evaluations are required to validate the use of this formula [10].

This study aimed to investigate the in vitro biological activities of YKSS, with a particular focus on its antioxidant, anti-inflammatory and cytotoxicity properties, alongside its in vivo acute toxicity and analgesic effects. The primary objective of this research was to evaluate the biological activities, safety and stability of YKSS, which have not been thoroughly studied despite its widespread use in traditional medicine. To achieve these objectives, in vitro assays were conducted to assess the biological properties and toxicity of YKSS, while in vivo studies were designed to evaluate its toxicity profile and analgesic effects in animal models. These methods were chosen to ensure a comprehensive assessment of the formulation’s therapeutic potential and safety, aligning with the study’s aim of bridging traditional knowledge with scientific validation. Although YKSS has been traditionally used, there has been limited scientific investigation into its biological activities and safety profile. By addressing this gap, our research provides empirical evidence to support the therapeutic potential of YKSS. To the best of our knowledge, this study is the first to comprehensively investigate the biological activities, safety and formulation stability of YKSS, establishing a scientific basis for its traditional use. Additionally, the stability of the formulation was assessed and a capsule formulation was developed to enhance its usability and practicality. The findings from this study contribute valuable insights into the scientific understanding of YKSS, highlighting its potential as a safe and effective alternative for managing musculoskeletal pain and related conditions while preserving the wisdom of traditional Thai medicine.

Materials and methods

Materials

Acetonitrile (LC-MS grade), ferric (III) chloride hexahydrate (FeCl₃·6H₂O), formic acid (LC-MS grade) and water (LC-MS grade) were purchased from Merck KGaA, Darmstadt, Germany. 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), acetic acid, caffeic acid phenethyl ester (CAPE), dimethyl sulfoxide, Folin-Ciocalteu reagent, gallic acid monohydrate, indomethacin, L-nitroarginine (L-NA), lipopolysaccharides (LPS), linoleic acid sodium salt, lipoxygenase from soybean, methylthiazolyldiphenyltetrazolium bromide (MTT) and nordihydroguaiaretic acid (NDGA) were purchased from Sigma-Aldrich, Inc., MA, USA. Tramadol hydrochloride was purchased from Central Polytrading Co. Ltd., Nonthaburi, Thailand. Hydrochloric acid (37%), naphthylethylenediamine dihydrochloride, orthophosphoric acid (85%), sodium acetate, sodium hydroxide, sodium nitroprusside and sulfanilamide were purchased from Carlo Erba Reagents, Cornaredo, Italy. Aluminum chloride and sodium carbonate were purchased from Ajax Finechem Pty. Ltd., New South Wales, Australia. Sodium nitrite was purchased from Loba Chemie Pvt. Ltd., Mumbai, India.

Plant samples and extraction

All herbs composed in the YKSS formula were obtained from Phra Ajarn Dhanes Jattabhayo, the monk at Wat Pacha Ban Kraisorn, Buached District, Surin Province, Thailand. The plant compositions of YKSS, including Thai common name, scientific name, family, part used and mass are shown in Table 1. The first and the second batches were obtained in August 2021 and March 2022, respectively. The first batch contained four samples coded as YKSS 1-1 to 1-4. The second batch contained four samples coded as YKSS 2-1 to 2-4, as shown in the diagram in Figure 1. They were identified by a plant taxonomist at the Department of Pharmacognosy, College of Pharmacy at Rangsit University to ensure the correct plant species. The voucher specimens were coded and deposited at the Drug and Herbal Product Research and Development Center, College of Pharmacy, Rangsit University. They were cleaned with tap water, boiled in 1.5 L of water for 15 min, filtered using cheesecloth and cooled before being freeze-dried by a freeze dryer for 18 - 20 h. The dried extracts were kept in a desiccator until used.

Table 1 Plant compositions of YKSS, including Thai common name, scientific name, family, part used, voucher specimen number and mass used.

Figure 1 Schematic representation of the YKSS samples used in this work.

Determination of total phenolic content

The total phenolic content (TPC) of YKSS extracts was measured using the Folin-Ciocalteu method [11]. Gallic acid standards, ranging from 6.25 to 300 μg/mL, were added in 20 µL aliquots to a 96-well plate (n = 3). Then, 100 µL of 0.2 N Folin-Ciocalteu reagent was added and the mixture was thoroughly mixed. The extracts were incubated for 6 min, followed by the addition of 80 µL of 7.5% sodium carbonate and further mixing. After an additional 1-h incubation at room temperature, the absorbance was measured at 765 nm using a microplate reader (Bio-Rad Laboratories, Inc., CA, USA). A calibration curve was constructed using gallic acid. The YKSS extracts were processed similarly, with a concentration of 0.5 mg/mL used. The TPC of the YKSS extract was then calculated using the gallic acid calibration curve and expressed as gallic acid equivalents (GAE) in mg per g of extract.

Determination of total flavonoid content

The determination of total flavonoid content (TFC) was modified from a previous study [12]. Twenty-five microliters of (+)-catechin hydrate, with concentrations ranging from 4 to 500 µg/mL and 100 µL of water were added to a 96-well plate (n = 3). Next, 10 µL of 5% sodium nitrite was added and the plate was incubated in the dark at room temperature for 5 min. Then, 15 µL of 10% aluminum chloride was added, mixed and incubated in the dark at room temperature for 6 min. Afterward, 50 µL of 1 M sodium hydroxide and 50 µL of water were added and mixed. The absorbance was measured at 510 nm using the microplate reader. A calibration curve of (+)-catechin hydrate was constructed. The YKSS extract was treated similarly to the (+)-catechin hydrate group, with extracts at a concentration of 1 mg/mL. The TFC of the YKSS extract was calculated using the calibration curve of (+)-catechin hydrate and expressed as catechin equivalents (CE) in mg per g of extract.

DPPH radical scavenging activity

The DPPH radical scavenging assay was slightly modified from a previous study [13]. One hundred microliters of the extracts (concentrations ranging from 1 - 500 µg/mL) were added to a 96-well plate (n = 3). Then, 100 µL of 80 µM DPPH methanolic solution was added and mixed. The mixtures were kept in the dark at room temperature for 30 min before measuring the absorbance at 517 nm using the microplate reader. The DPPH radical without the extract was used as the control. The scavenging ability of the DPPH radical and half maximal inhibitory concentration (IC₅₀) were calculated.

Ferric reducing antioxidant power assay

The ferric reducing antioxidant power (FRAP) assay was adapted from a previous study [14]. Twenty microliters of YKSS extracts (at a concentration of 200 µg/mL) were added to a 96-well plate (n = 3). Next, 180 µL of freshly prepared FRAP reagent, consisting of 30 mM acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM hydrochloric acid and 20 mM FeCl₃·6H₂O in a 10:1:1 volume ratio, was added and mixed. The plates were incubated at 37 °C for 15 min, after which the absorbance was measured at 593 nm against a reagent blank using a microplate reader. The reducing power was determined from a calibration curve constructed with ferrous sulfate. The FRAP values were expressed as µmol Fe(II) equivalent per g of extract.

Nitric oxide scavenging activity

The nitric oxide (NO) scavenging assay was adapted from the method described previously [15]. Quercetin served as the positive control. YKSS extracts were prepared in water at concentrations of 500 and 1,000 µg/mL. A 50 µL portion of either quercetin or YKSS extract solution was dispensed into a 96-well plate (n = 3). Following this, 50 µL of 10 mM sodium nitroprusside was added and the mixture was incubated at room temperature for 120 min. After the incubation period, 100 µL of Griess reagent (containing 1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 2.5% phosphoric acid) was added to each well. Absorbance was measured at 546 nm using the microplate reader and the percentage of inhibition was calculated.

Inhibition of NO production activity

The anti-inflammatory activity was assessed using RAW264.7 murine macrophage cells stimulated with LPS to induce NO production, a key inflammatory mediator. NO suppression was evaluated following the method described previously [16]. RAW264.7 cells (1×10⁵ cells/well) were treated with 100 ng/mL LPS and co-incubated with various concentrations (31.25 - 2,000 μg/mL) of the test samples (n = 3) for 24 h at 37 °C in a humidified incubator with 5% CO₂. Positive controls included indomethacin, L-NA and CAPE. NO levels in the culture medium were quantified by reaction with Griess reagent and absorbance was measured at 570 nm using a microplate reader. The percentage inhibition of NO production was calculated and IC₅₀ values were determined from the inhibition curve.

Anti-lipoxygenase activity assay

The anti-lipoxygenase (LOX) activity assay was modified from a previous study [17]. YKSS extract was dissolved in water to achieve concentrations ranging from 0.01 - 300 µg/mL. NDGA used as a positive control, was prepared and diluted in methanol at concentrations of 0.0000001 - 0.1 mM. Subsequently, 10 µL of each sample was added to a 96-well plate (n = 3). To this, 170 µL of soybean LOX in 0.1 M phosphate buffer (pH 8.0) was added, followed by 20 µL of 2 mM linoleic acid sodium salt in methanol. The absorbance at 234 nm was measured immediately and recorded every 50 s for 5 min to monitor the production of hydroperoxylinoleic acid [18]. Enzyme activity was evaluated to ensure the mean velocity (Vmean) ranged between 80 - 120. The mean velocity was used to calculate the percentage of inhibition using Eq. (1). The blank control consisted of the reaction mixture without the addition of the sample. Finally, IC₅₀ values were calculated from the inhibition curve.

Cytotoxicity test

The mitochondrial-based MTT cell viability test was used to evaluate the possible toxicity of YKSS extract in vitro. In a 96-well culture plate, HepG2 cells were seeded at a density of 1×10⁴ cells per well. They were then incubated overnight at 37 °C with 5% CO₂. YKSS extract at different concentrations (0 - 10 mg/mL) or a positive control of 200 µM hydrogen peroxide were applied to the cells for a duration of 24 h. The cells were then incubated for three more hours after 100 μL of 0.5 mg/mL MTT solution in culture media was added to each well. A microplate reader was used to detect absorbance at 570 nm after the formazan product was dissolved in 100 μL of dimethyl sulfoxide. The percentage of cell viability in relation to the untreated control group was calculated after the experiment was carried out in triplicate [19].

LC-MS analysis

The LC-MS profile of YKSS 2-4 was analyzed. The sample was prepared by dissolving the YKSS 2-4 extract in water to achieve a concentration of 1 mg/mL, followed by filtration through a 0.2-µm PTFE syringe filter before analysis using an LC-MS instrument. The LC-MS analysis was conducted using an Agilent 1290 Infinity LC instrument coupled with an Agilent 6540 series QTOF-MS, equipped with an ESI source and a diode array detector (Agilent Technologies, Inc., CA, USA). Separation was performed on a Poroshell 120 EC-C18 column (4.6×150 mm, i.d., 2.7 µm) maintained at 35 °C. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B), with a flow rate of 200 µL/min. The gradient elution system was as follows: 5% B from 0 to 1 min, increased to 17% B at 10 min and held constant until 13 min, further increased to 100% B at 20 min and maintained until 25 min, then decreased to 5% B at 27 min and held constant until 33 min. The injection volume was 1.0 µL and the total analysis time was 33 min. Chemical constituents in the YKSS extract were identified using Agilent MassHunter Workstation software (qualitative analysis, version B.08.00) and the Personal Compound Database and Library (PCDL). Identified compounds were cross-referenced with the literature using databases corresponding to each plant component. Only chemical constituents with PCDL scores above 80 were selected. Chemical formulas were assigned with a mass error of less than ±5 ppm [20,21]. Mass-to-charge ratios (m/z) and MS analyses were performed in both positive and negative ionization modes.

Stability test

The YKSS extracts were stored in glass bottles protected from light and kept under ambient conditions for one year. At predetermined intervals, samples were collected to evaluate TPC and TFC in comparison to the initial time point.

Animals

For the acute toxicity and analgesic activity experiments, Wistar rats weighing approximately 220 ± 20 g were selected for the acute toxicity test. To evaluate analgesic effects, Swiss albino mice weighing between 20 - 25 g were used. Both rats and mice were obtained from the National Laboratory Animal Center at Salaya, Mahidol University, Nakorn Pathom, Thailand. They were acclimatized for seven days before the experiments. The animals were maintained under standard conditions, including a temperature of 23 - 25 °C, a 12-h light/dark cycle and free access to standard pelleted feed and water, under animal welfare standards. All procedures involving animals were conducted in compliance with the ARRIVE (Animal Research: Reporting of In vivo Experiments) guidelines [22] to ensure rigorous experimental design, methodology and reporting. The protocol for this study was reviewed and approved by the Ethics Committee for Animal Research at Rangsit University (RSU-AEC 003-2024).

Acute toxicity study

The acute oral toxicity study was conducted according to the Organization for Economic Co-operation and Development (OECD) 420: Acute Oral Toxicity - Fixed Dose Procedure [23]. The animals were randomly assigned to control and experimental groups, with five rats in each group. Rats in the control group were orally administered distilled water, while rats in the test group were given the YKSS extract (Batch 2) at a dose of 2,000 mg/kg body weight. The rats were closely monitored for any signs of death or abnormal symptoms, including changes in skin and fur, eyes and mucous membranes, behavior patterns, tremors, salivation, diarrhea, sleep and coma during the first 24 h (day 1). Observations continued once daily for 14 days. Body weight was recorded on day 1 (before YKSS extract administration), day 7 and day 14.

Analgesic activity study

Grouping and dosing

The equivalent animal dosages of YKSS extract (Batch 2) were determined using the body surface area method [24]. The prescribed human dosage of YKSS extract is calculated for approximately one teaspoonful twice daily, with each teaspoonful weighing 1,630 mg, resulting in a total daily dose of 3,260 mg. Based on this and adjusting for body surface area, the calculated therapeutic equivalent dose for mice was approximately 670 mg/kg. However, for the study, three distinct doses were selected for the mouse experiments: a low dose of 400 mg/kg, a therapeutic equivalent dose of 800 mg/kg and a high dose of 1,200 mg/kg. Mice were divided into five groups (n=6 per group). The analgesic efficacy of YKSS extract was assessed with both single-dose administration and repeated administration over seven days. This approach aims to validate the traditional use of YKSS as a pain-relieving agent and explore its potential for long-term use.

Acetic acid-induced writhing test

The acetic acid-induced writhing test was conducted to evaluate visceral pain using the previously described method [25]. Each group consisted of six mice, which were orally administered YKSS at doses of 400, 800 and 1200 mg/kg and indomethacin at 10 mg/kg as a standard drug. All samples were administered to the corresponding groups by oral gavage once daily for 1 day and 7 days. On the day of the test, one h after administering the test sample, all animals received intraperitoneal injections of 0.75% acetic acid (0.1 mL/10 g body weight) to induce writhing. Each mouse was placed separately in a transparent cage and the number of writhes (contractions) was counted over 60 min following the acetic acid injection. The recorded data represented the total number of writhing episodes observed.

Hot plate test

The hot plate test was employed as an additional technique to evaluate analgesic activity. Using the previously mentioned methodology, it was carried out to measure latency response. Five groups of six mice each were randomly assigned. Excluded mice had a baseline latency time of more than 30 s or less than 5 s. Tramadol (100 mg/kg, the conventional medicine) and YKSS (400, 800 and 1200 mg/kg) were given orally to the mice and their reaction times were recorded at 0, 15, 30, 60 and 90 min. The temperature of the hot plate was kept at 54.0 ± 1 °C. The latency response index was defined as the number of sec that passed between the mice being placed on the hot plate and a discomfort reaction (such as leaping or licking the back of the hind paw) being observed. Complete analgesia was defined as a 45-s cutoff duration. In order to prevent paw damage, the experiment was terminated if the cutoff time was surpassed.

Capsule preparation

The YKSS extract was filled into hard gelatin capsule no. 0. They were evaluated for individual weight and disintegration time. In the case of individual weight, ten capsules were analyzed using an analytical balance (Model: Entris224i-1S, Sartorius AG, Göttingen, Germany). The disintegration time of six capsules was determined using a disintegration tester (Model: BJ-2, Tianjin Guoming Medicinal Equipment Co. Ltd., Tianjin, China), with water at 37 ± 0.5 °C as the disintegration medium.

Statistical analysis

Data were analyzed using an independent sample t-test for comparisons between two groups and one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test to compare differences between more than two groups using IBM SPSS Statistics v. 22 (IBM Corp., Armonk, NY, USA). A p-value of < 0.05 was considered statistically significant at a 95% confidence interval.

Results

Extraction yield, TPC and TFC

The extraction yields, TPC and TFC for each batch of plants obtained from decoction are shown in Table 2. The extraction yields for Batches 1 (YKSS 1-1 to 1-4) and 2 (YKSS 2-1 to 2-4) ranged from 1.26% to 2.44% and 0.88% to 1.02%, respectively. Batch 1, which contained C. orientalis root, yielded higher extraction rates than Batch 2, which contained V. denticulata stem instead of C. orientalis root.

The TPC of Batch 2 was significantly higher than that of Batch 1, with values ranging from 142.76 to 179.89 mg GAE/g of extract for Batch 1 and 100.50 to 156.50 mg GAE/g of extract for Batch 2. The TFC showed a broader range in Batch 1 compared to Batch 2, with values ranging from 51.43 to 96.19 mg CE/g of extract for Batch 1 and 61.90 to 76.90 mg CE/g of extract for Batch 2.

Table 2 Extraction yield, TPC, TFC, DPPH radical scavenging, FRAP and NO scavenging activity of YKSS.

Different letters within the same column indicate statistically significant differences (p < 0.05).

YKSS: Ya Ka Sai Sen, TPC: Total phenolic content, TFC: Total flavonoid content, CE; Catechin equivalent, FRAP: Ferric reducing antioxidant power, NO: Nitric oxide

Antioxidant activities

Antioxidant activities assessed using the DPPH radical scavenging assay, FRAP assay and NO scavenging assay are presented in Table 2. The DPPH radical scavenging assay, expressed as IC₅₀ values, ranged from 36.52 to 68.64 µg/mL for batch, which were comparable to the range of 33.77 to 61.71 µg/mL for Batch 2. The FRAP values, expressed as Fe(II) equivalents, were 2,148.20 to 2,601.14 µmol Fe(II) equivalent/g of extract for Batch 1, which were slightly lower than the range of 2,493.67 to 3,032.97 µmol Fe(II) equivalent/g of extract for Batch 2. For NO scavenging, the IC₅₀ values could not be determined as 50% inhibition was not achieved. Therefore, two concentrations were selected to demonstrate the percentage of NO scavenging. Batch 1 exhibited NO scavenging activity ranging from 27.85% to 37.14% at 500 µg/mL of extract, which increased to 40.98% to 46.52% at 1,000 µg/mL of extract. Similarly, Batch 2 showed NO scavenging activity of 22.51% to 39.21% at 500 µg/mL of extract, increasing to 41.83% to 46.83% at 1,000 µg/mL.

Anti-inflammatory activity

Anti-inflammatory activity was assessed using in vitro NO production inhibition and anti-LOX assays. As shown in Figure 2, YKSS extracts demonstrated low potency in inhibiting NO production. Batch 1 of YKSS inhibited NO production with IC₅₀ values ranging from 820.91 to 905.06 µg/mL, while Batch 2 exhibited IC₅₀ values between 883.71 and 969.17 µg/mL. Among the extracts, YKSS 1-2 was the most potent, exhibiting the lowest IC₅₀ value.

Figure 2 Inhibition of NO production by YKSS samples, expressed as IC₅₀ values. Data are presented as mean ± SEM (n = 3). Comparisons made with positive controls: Indomethacin, L-NA and CAPE. Different letters signify significant differences (p-value < 0.05).

In the case of anti-LOX activity, YKSS extract demonstrated strong inhibition of LOX, an enzyme that regulates inflammatory responses. Batch 1 of YKSS exhibited IC₅₀ values ranging from 16.92 to 56.70 µg/mL, while Batch 2 showed IC₅₀ values between 15.50 and 72.75 µg/mL. YKSS 1-4 from Batch 1 and YKSS 2-2 from Batch 2 exhibited the lowest IC₅₀ values, indicating that they possess the most potent LOX inhibition activity.

Figure 3 Inhibition of LOX by YKSS samples, expressed as IC₅₀ values. Data are presented as mean ± SD (n = 3), with comparisons to the positive control, NDGA. Different letters signify significant differences (p-value < 0.05).

Cytotoxicity

Cytotoxicity was evaluated by treating HepG2 cells with the samples and measuring cell viability. Cell viability significantly decreased upon treatment with YKSS extracts, depending on the sample: YKSS 1-3 exhibited decreased viability at a concentration of 0.00001 mg/mL, YKSS 1-1 and 2-1 at 0.001 mg/mL and most other samples at 0.0001 mg/mL (Figure 4). According to ISO 10993-5:2009, a sample is considered toxic if cell viability is less than 70% compared to the control group. Although YKSS extracts caused a significant reduction in cell viability, the concentrations at which toxicity (viability < 70%) was observed were relatively high: 1 mg/mL for YKSS 1-3; 5 mg/mL for YKSS 1-2, 1-4 and 2-2; and 10 mg/mL for YKSS 1-1, 2-1, 2-3 and 2-4.

Figure 4 The cytotoxicity of YKSS samples, represented by cell viability, at different concentrations (mg/mL) after 24 h of treatment. The data are presented as mean ± SEM (n = 3). Symbols *, ** and *** indicate significant differences compared to the non-treated group, with p < 0.05, p < 0.01 and p < 0.001, respectively.

LC-MS profile

Total ion chromatograms (top) and base peak chromatograms (bottom) obtained in positive and negative ionization modes are shown in Figure 5. The YKSS 2-4 extract composed of several classes of compounds, including alkaloids, anthraquinones, benzaldehydes, chalcones, coumarins, disaccharides, fatty acids, flavanone glycosides, flavonoids, glycosides, organic acids, phenolic acids, phenolic amides, phenolic compounds, phenylpropanoids, quinones, resveratrols, steroids, stilbenoids and terpenoids.

The YKSS 2-4 extract comprised 66 compounds previously reported in the literature: 13 compounds identified in positive ionization mode and 53 compounds identified in negative ionization mode (Table 3). In the positive ionization mode, N-trans-feruloyltyramine was the most abundant compound (percent peak area (PA) = 88.38%), followed by derrisscandenon E (percent PA = 4.01%), isodomesticine (percent PA = 2.29%) and others. In the negative ionization mode, maltose was the most abundant compound (percent PA = 26.84%), followed by gluconic acid (percent PA = 19.26%), citric acid (percent PA = 14.32%) and others.

Figure 5 Total ion chromatograms (top) and base peak chromatograms (bottom) obtained in positive and negative ionization modes.

Table 3 LC-MS profiles of YKSS 2-4 in positive and negative ionization modes.