Nanjing Spring & Autumn Biological Engineering Co., Ltd. (Nanjing, China) provided the lupeol (99% purity). Honeywell Burdick & Jackson^TM (North Carolina, US) provided all the analytical reagents for the HPLC analysis. S.N.P. Scientific Co., Ltd. (Bangkok, Thailand) furnished all disposable accessories for the HPLC apparatus. Manose Health and Beauty Research Center Co., Ltd. (Chiang Mai, Thailand) granted diclofenac diethylammonium, a positive control, and all of the essential chemical reagents used in the in vitro anti-inflammatory assay.

In August 2021, C. adansonii bark (CA), M. siamensis roots (MS), and M. repandus stems (MR) were obtained from Thai traditional medicine and pharmacy stores. These plant materials were verified by one of the authors, Asst. Prof. Dr. Orawan Theanphong, by comparing these crude drugs to genuine specimens stored at the Department of Pharmacognosy, College of Pharmacy, Rangsit University, Thailand, using macroscopic and microscopic techniques documented in the Thai Herbal Pharmacopoeia (Department of Medical Sciences, Ministry of Public Health, Thailand 2018). The specimens that were verified to be factual were given the numbers RSU 0095, RSU 0096, and RSU 0097, respectively.

The individual samples, including CA, MS, and MR, were pulverized to obtain the powdered samples. Each sample was precisely weighed (10 g) into a Soxhlet apparatus thimble. For 3 hours, 300 mL of 100% ethanol was used for extraction. Furthermore, the powdered samples of each plant were allocated and combined evenly in order to create a combination formula (MX). The blend was weighed (30 g) and extracted similarly to each crude powdered substance. The ethanolic extract was reduced to drought in a rotary evaporator. Each sample’s concentrated extract was precisely weighed and reconstituted with small volumes of methanol, then diluted with the same solvent and progressively adjusted to yield a sample solution with a concentration of 10 mg/mL in a stock volumetric flask. This procedure was repeated for each sample in triplicate.

In this study, a protein denaturation assay was conducted to investigate the in vitro anti-inflammatory potential of the dried plant extracts of CA, MS, MR, and MX using the method of Chandra et al. (2012). Each test’s reaction mixture contained 0.2 mL of fresh hen’s egg albumin, 2.8 mL of phosphate buffered saline (pH 6.4), and 2 mL of different doses of the tested extracts diluted in 20% tween to yield final concentrations of 0.25, 0.5, 1, 2, and 4 mg/mL. Similarly, 2 mL of the examined extracts were substituted with double-distilled water and diclofenac diethylammonium to provide the study’s control and positive control, respectively. In an incubator, the reaction mixtures were heated to 70 °C for 5 minutes. After cooling at room temperature, the bioactivity of the mixtures was evaluated at a wavelength of 660 nm using a UV-Vis spectrophotometer (UV-1800, Shimadzu, Japan). Using a formula revealed in a recognized study, the percentage inhibition of protein denaturation was computed from the absorbance of tested samples compared to the control (Chandra et al. 2012). The IC₅₀ value was used to express the potency of the anti-inflammatory effect for all examined extracts and diclofenac diethylammonium as a reference standard. The sample concentration for 50% inhibition of albumin denaturation was estimated by graphing the percentage inhibition versus sample concentration. Also, one-way analysis of variance (ANOVA) and Tukey’s multiple comparison post-hoc test (PSPP, GNU Project) were used to consider the statistical significance at a value of P < 0.05 of the IC₅₀ values across the groups that were tested.

A stock standard solution containing 500 μg/mL of lupeol was prepared by dissolving lupeol (25 mg) in methanol and then placing the solution in a volumetric flask of 50 mL. Standard solutions were prepared by diluting the stock standard solution with methanol. They were made at concentrations ranging from 10 to 400 μg/mL.

For the chromatographic procedure, an HPLC apparatus (1260 Infinity Series, Agilent Technologies, US) with an equipped photodiode array detector (DAD) was employed. OpenLab ChemStation software (Agilent Technologies, US) was used to control the instruments. A 0.45 μm nylon membrane was used to filter the sample and working standard solutions prior to analysis. Isocratic elution of the mobile phase, which included methanol and acetonitrile in a ratio of 90:10, was used to separate the 20-μL samples on the Accucore^TM XL C18 packed column (250 mm × 4.6 mm i.d., 4 µm, Thermo Scientific, US) in the HPLC system. The HPLC instrument’s column chamber was kept at room temperature while the mobile phase flow rate was set at 1.0 mL/minute. DAD recorded a chromatogram of the analyzed materials and collected absorbance data for the lupeol compound at a wavelength of 210 nm. Each injection was given a time limit of 12 minutes for analysis on the HPLC system.

The analytical method employed in this study was based on that proposed by Somwong and Theanphong (2021). Prior to performing the analysis, the analytical procedure was verified. The accuracy and precision of the applied analytical method were tested by adding a known amount of lupeol (80–120%w/w) to the three different plant extracts and the mixture. The recovery and the relative standard deviation (RSD) percentages were assessed. Additionally, the specificity of the analytical approach was established to ensure that it was suitable for evaluating the relevant plant extracts.

The concentration of lupeol was measured by using a linear regression equation that was obtained from the calibration curve of working standard solutions. Each sample was analyzed in triplicate and the lupeol concentration was expressed as grams per hundred grams of the crude extract. The contents were displayed together with their standard deviations (SD). The differences in lupeol concentrations across the sample groups were assessed using one-way analysis of variance (ANOVA) combined with Tukey’s multiple comparison post-hoc test (PSPP, GNU Project), with a significance level of P < 0.05 found for all the tested groups.

The anti-inflammatory impact of plant extracts was tested in vitro against the denaturation of egg albumin in the current study. The percentage inhibition of albumin denaturation was computed using linear regression models derived from the inhibition efficiency against sample concentrations as shown in Fig. 1 (A–E). Samples CA, MS, MR, and their mixture formula (MX) displayed inhibitory efficacy on protein denaturation assays, evidenced by the IC₅₀ values shown in Table 1. In that sense, MX was found to be more anti-inflammatory than CA, MS, and MR. When compared to diclofenac, a nonsteroidal anti-inflammatory drug that was used as a positive control, MX, CA, MS, and MR showed differences in their anti-inflammatory activities, which were further investigated statistically. The anti-inflammatory activity of MX, a blend that incorporated CA, MS, and MR equally in a formula, was the strongest. Its inhibitory effect on albumin denaturation was also found to be greater and significantly different (P < 0.05) when compared to the inhibitory effects of each individual sample of CA, MS, and MR. Remarkably, the statistical difference in anti-inflammatory activity between the MX sample and the anti-inflammatory drug diclofenac was found to be insignificant (P = 0.49). Furthermore, the effects of CA and MS samples, which are members of the same medicinal plant family, Capparaceae, were revealed to be identical (P = 0.79). The statistical difference observed in this study among the examined groups revealed that all of the samples were potent differently on this anti-inflammatory examination, with the exception of the pairs of CA and MS, as well as MX and diclofenac. Thus, the studied extracts and the positive control can be arranged from strong to weak anti-inflammatory action in the following order: diclofenac>MX>CA, MS>MR. This finding demonstrated that the extract from the mix of three active plant parts, including C. adansonii bark, M. siamensis roots, and M. repandus stems, had the most potent anti-inflammatory effect. This analysis verifies the conventional use of these plants in Thai herbal medicine by providing scientific evidence why they had never been utilized as a single treatment. They should be utilized as ingredients in herbal pain-relief preparations by combining them with other medicinal herbs, as they had been traditionally. However, an extensive pharmacological and phytochemical examination of the potential anti-inflammatory components in the mixture should be designed and carried out to support the mixture formula as a candidate for an effective alternative combination of an anti-inflammatory pharmaceutical preparation.

Figure 1. 

Plots of various concentrations of different extracts: C. adansonii bark (A), M. siamensis roots (B), M. repandus stems (C), and the mixture formula (D) and the positive control (E) against the percentage of inhibition of the protein denaturation assay. The triangular, diamond-shaped, and square markers on each plot’s lines denoted the three experimental replicates.

This study confirmed the anti-inflammatory properties of C. adansonii extracts, which had an important impact on various anti-inflammatory mediators such as inhibition of 5-lipoxygenase (5-LOX), cyclooxygenase (COX), and myeloperoxidase (MPO), which are key enzymes in the inflammatory process (Rathinavel et al. 2017; Umeti et al. 2019; Thirumalaisamy et al. 2020; Rathinavel et al. 2021). The anti-inflammatory impacts of C. adansonii extracts have been scientifically proven, indicating that the plant should not be limited to folkloric use but should be encouraged to be developed for use in analgesic and anti-inflammatory herbal medicines also. Although there are numerous scientific reports supporting the pharmacological effects of C. adansonii extracts, bioactivity studies of another Capparaceae medicinal plant that is specifically distributed in the Indo-China region, notably M. siamensis, have received limited attention. The plant was found to contain phytochemicals with potential activity in a larvicidal bioassay. Its activities might well be beneficial in preventing the dengue epidemic (Nobsathian et al. 2018). Our study discovered that M. siamensis roots have anti-inflammatory properties. It demonstrated a significant ability to inhibit protein denaturation, similar to the activity of the most well-known plant, C. adansonii bark, indicating that the plant is noteworthy for use in anti-inflammatory herbal treatments in the same manner as C. adansonii bark. This is the first instance that the extract of M. siamensis roots has been proven to have anti-inflammatory properties. Furthermore, this investigation was expanded to include the exploration of another well-known medicinal plant, M. repandus, which is a member of the Euphorbiaceae family and has been used in various traditional herbal recipes for tonics and pain treatment in Thailand (Pongsaree et al. 2020). Due to the activity observed in this study, the extract of the stems of this plant could also be provided as one of the anti-inflammatory herbal treatments. Although this study indicated that the plant has the potential for anti-inflammatory effects, consistent with previous reports (Hasan et al. 2014; 2018), researchers have been urged to conduct additional pharmacological tests to evaluate its importance in herbal medicine, including antioxidant, hepatoprotective, and antibacterial properties (Mondal et al. 2020; Sriset et al. 2021; Zhang et al. 2021). In this comparative study, M. repandus was found to have a less potent anti-inflammatory effect than C. adansonii and M. siamensis in our evaluation of the three pain-relieving medicinal plants. But the results could help support how the plant has been used in the past, which has been in compound herbal recipes for pain relief and reducing inflammation rather than in a single treatment. Even though the traditional uses of C. adansonii and M. repandus have already been shown to have certain effects, this study is the first to show that a mixture of these two plants and M. siamensis has an anti-inflammatory effect that is much stronger than those of either plant alone.

Ground samples of CA, MS, MR, and MX were extracted individually with absolute ethanol using the Soxhlet device. After evaporating the extracts from each trial, they were accurately weighed to obtain the crude extracts, providing an extraction yield of 3.68, 6.92, 6.68, and 9.47%w/w, respectively. The extracts were further divided to prepare the sample solutions for the HPLC analysis, which was performed according to the method developed by Somwong and Theanphong (2021). The HPLC method was validated using the lupeol compound and standard-spiked technique prior to the chemical analysis of the plant extracts. The chromatograms obtained from that HPLC system showed the peak of lupeol at 9.6 minutes of retention time, and the signal of that marker was clearly separated from the other interferences in the plant extracts, as shown in Fig. 2 (A–D). As a result, the method was shown to be selective and competent for determining lupeol in these plant extracts. Furthermore, the analytical method used in this study is accurate and reliable, as evidenced by the recovery and relative standard deviation percentages of the verification performed on all plant samples, which showed acceptable values in the ranges of 91.71–101.88 and 0.15–1.73%, respectively. The validated method was then used to determine the concentration of lupeol in all plant extracts, and the percentage of lupeol in each sample was calculated and shown in Table 2. Among the three plants, CA had the highest lupeol yield (8.38 ± 0.26%w/w), while MR had the lowest lupeol content (0.04 ± 0.01%w/w). Lupeol was found in all plant extracts, but the amount of lupeol in each extract varied greatly. The significant difference in lupeol content in extracts of three plant species, as well as their mixture, was investigated in our study. One-way ANOVA revealed that there were significant differences in lupeol content between CA, MS, MR, and MX extracts (P < 0.05, F = 905.84, df = 3, 8). The results of Tukey’s post-hoc test revealed that the amount of lupeol in the tested samples varied significantly, with the exception of the content between the two pairs of CA and MX, as well as MS and MR, which revealed no noticeable difference in the content of lupeol compound at the significant values of 0.78 and 1.00, respectively. The lupeol yields in CA (8.38 ± 0.26%w/w) and MX (8.60 ± 0.50%w/w) were found to be uniform because the amount of lupeol in CA was found to be predominant, whereas the lupeol content in MS and MR extracts was found to be 100–200 times lower than that in the CA extract. Thus, the observed yield of lupeol in the combination most likely corresponded to the estimated yield of that constituent in CA. This discovery showed that the amount of lupeol in the CA extract affected the quantity of lupeol found in the mixture, and that the CA extract could have played a significant role in the formulation of these selected plants. In addition, our results indicate that lupeol is present in sufficient quantities in the examined plants and their mixtures to serve as a chemical marker for the HPLC analysis of their crude drug quality control. Based on the reviewed analytical method for exploring the active ingredients in CA, this study could help to show that the pentacyclic triterpene lupeol is a noteworthy chemical marker for the analysis because of its abundance in this plant extract (Tchimene et al. 2016; Thirumalaisamy et al. 2020; Rathinavel et al. 2021). In the extracts of MS and MR, a small amount of lupeol was found, which shows that the chemical analysis of the molecule in these plants is ordinary, as revealed in previous reports (Nobsathian et al. 2018; Mondal et al. 2020). However, this is the first study to quantify and confirm that their extracts contain the active compound lupeol, which makes them valuable for anti-inflammatory usage. Furthermore, this finding could facilitate the investigation of additional phytochemical components and their relevant medicinal effects in plant extracts.

Figure 2. 

The HPLC chromatograms demonstrate the peak for lupeol compound at a retention time of 9.6 minutes from different extracts: C. adansonii bark (A), M. siamensis roots (B), M. repandus stems (C), and the mixture formula (D).

Comparing the amount of lupeol expressed in CA, MS, and MR extracts with their relative anti-inflammatory ability revealed that the CA sample with the highest level of lupeol also exhibited the highest inhibition effect against protein denaturation, with an IC₅₀ value of 5.10 ± 0.46 mg/mL. This result indicates that the potential of the plant extracts to counter inflammation was associated with the amount of the active compound lupeol existing in the plants. However, a comparison of CA and MS samples revealed that the quantified lupeol in their extracts differed substantially. The content of lupeol in MS extract (0.06 ± 0.01%w/w) was found to be about 100 times lower than in CA extract, but they both had similar anti-inflammatory activity. This shows that the activity of the MS extract was affected not only by the lupeol compound but also by the other active ingredients. The presence of indole glycosides, cappariloside A and B, a flavonoid chrysoeriol, and a phenylpropanoid cinnamic acid, which were isolated from M. siamensis in a previous report (Nobsathian et al. 2018), might well be attributed to the anti-inflammatory ability of MS extract in our observation. These phytoconstituents have already been demonstrated to be active molecules with anti-inflammatory properties (Li et al. 2018; Pontiki and Hadjipavlou-Litina 2019; Yoon and Park 2021). Likewise, the intriguing action was also found in the MR sample, whereas its extract had the smallest amount of lupeol (0.04 ± 0.01%w/w), which was comparable to MS and much lower than CA samples. Based on this result, it seems likely that the other active constituents of the MR extract might work synergistically with the lupeol compound to respond to the activity. Previous reports on phytochemical studies of M. repandus stem extracts revealed that the plant contains an isocoumarin bergenin as one of its active constituents (Rivière et al. 2010; Sriset et al. 2021). Bergenin was examined in various anti-inflammatory models and was proven to be an efficient anti-inflammatory compound (Oliveira et al. 2019). The compound has been thoroughly studied in terms of pharmacology and has emerged as a promising candidate for therapeutic uses (Mehta et al. 2022). Due to the anti-inflammatory agents that were identified as chemical constituents of M. siamensis and M. repandus, as well as the abundant lupeol compound found in C. adansonii, there could be a reason to support the anti-inflammatory activity of the mixture of these three plants, which was the most potent in our observation. The existence of these active phytoconstituents in the mixture might even increase its activity to a level comparable to that of diclofenac, a nonsteroidal anti-inflammatory drug used as a positive control in this study. Our finding suggests that a variety of chemical compounds might be responsible for the anti-inflammatory effect of the mixture formula. Therefore, an extensive study on the phytochemical analysis of other promising anti-inflammatory agents, as well as the use of multi-chemical markers for performing HPLC analysis in the mixture, should be considered in order to develop this combination into an effective pharmaceutical herbal compound for the treatment of inflammation.