Quantification of the bioactive (-)-Pseudosemiglabrin at different growth stages of Tephrosia purpurea L. (Pers.) growing in Gizan, Saudi Arabia

1. Introduction

Tephrosia is a genus of about 400 species belonging to the family Fabaceae [1,2]. About 11 Tephrosia species were reported in the flora of Saudi Arabia [3-5]. The plants of Tephrosia purpurea are annual or perennial shrub can reach 40-80 cm [6]. The plants are more woody and closer to the ground with stiff coarse reddish hairs covering the trichomes [3-5].

T. purpurea used to treat many conditions in Unani medicine, Ayurveda and many pharmacological activities were reported for the plant, as summarized in Table 1 [7-11].

Phytochemically, this genus is characterized by the presence of the rare prenylated 5-deoxy flavonoids that can represent a biochemical marker [9]. The prenylated 5-deoxyflavonoids (-)-pseudosemiglabrin was the main component of T. apollinea and was reported to have in vitro and in vivo anti-inflammatory effects [12]. (-)-Pseudosemiglabrin exhibited significant dose-dependent antiproliferative effect against leukemia, prostate, and breast cancer cell lines without displaying any toxicity against normal human fibroblasts [13]. Recently we reported on the bronchodilation activity of T. purpurea total extract, fractions, and pure isolates. Our phytochemical investigation revealed that (-)-pseudosemiglabrin is the major active component in the plant. The physicochemical features of (-)-pseudosemiglabrin have been presented in Table 2 and Figure 1, as already described elsewhere [14]. The total extract and (-)-pseudosemiglabrin showed significant protective action in alleviating ischemia-reperfusion (IR)-induced damage in both the respiratory system and the pancreas. The mechanism of protection involves reducing the cytokine response, suppressing oxidative stress, modulating the HMGB1 and IL-22 pathway [15].

Table 2. The physicochemical properties of (-) Pseudosemiglabrin.

Property	Value
Name	[(12S,15S,16R)-14,14-dimethyl-6-oxo-4-phenyl-3,11,13-trioxatetracyclo[8.6.0.02,7.012,16]hexadeca-1(10),2(7),4,8-tetraen-15-yl] acetate
Formula	C23H20O6
Structure
Molecular weight	392.4 g/mol
Melting point	176.5 0C
Description and solubility	Colorless crystals isolated from (-)-Pseudosemiglabrin has been reported in T. purpurea and T. apollinea and freely soluble in methanol

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Figure 1.

UV spectrum of (-) Pseudosemiglabrin.

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It is well known that β₂-adrenergic receptor stimulants should be used with caution for the treatment of pulmonary and obstetrical disorders in epileptic patients receiving antiepileptic medications [16]. The connection between the use of selective β₂-adrenergic receptors stimulant and the development of seizures is controversial [17]. Our previous study revealed that (-)-pseudosemiglabrin expressed a comparable anti-epileptic efficacy to diazepam without sedation induction indicating that it can be a safer and creditable drug for the control of epilepsy besides its bronchodilator effect [18].

High-performance liquid chromatography (HPLC) is a versatile technique that detects compounds of various polarities and molecular masses. This technique is becoming increasingly popular as the main choice for fingerprinting and quality control studies [19]. As a result, HPLC has been described for quantifying and characterizing secondary metabolites in plant extracts, such as phenolic compounds, steroids, flavonoids, and alkaloids [20-23].

Although other flavonoids have been identified using HPLC and liquid chromatography-tandem mass spectrometry (LC-MS/MS), no one has quantified and validated (-)-pseudosemiglabrin. It is well known that the percentage of secondary metabolites varies based on environmental conditions and stages of plant growth [24]. Several studies were conducted to investigate the effect of the plant’s stages of growth on the secondary metabolites quantitatively and qualitatively [25,26]. From the early history of medicinal plants, the alkaloid morphine is one of the clearest examples of such variation [27]. The extracts components will be greatly affected by the polarity of the solvents used. Hence, in the present study, and due to the promising activities, we developed an HPLC method for the quantitative analysis of (-)-pseudosemiglabrin in T. purpurea extracts collected from Gizan, southern of Saudi Arabia. Moreover, we applied different methods of extraction using various solvents to optimize the extraction process of (-)-pseudosemiglabrin. Plants were collected in different stages of development to identify the stage with maximum contents of (-)-pseudosemiglabrin.

2. Materials and Methods

2.2. Plant material

The plants of T. purpurea (voucher specimen #MSA 10521) were described earlier [14]. For this study, plants were collected at different growth stages during a 6-month period in 2023 from Gizan, Saudi Arabia (Figure 2).

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Figure 2.

Diagrams of T. purpurea plant at different stages: pre-flowering stage (PFS), flowering stage (FLS), fruiting stage (FRS), and perennial herbs during the flowering stage (PHF).

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2.4. HPLC chromatographic conditions

The HPLC analysis of the plant samples and (-)-pseudosemiglabrin quantification were carried out using a 1260 Infinity II HPLC system (Agilent Technologies, Palo Alto, CA, USA), comprising of a binary solvent gradient flexible pump (G7111A), autosampler (G7129A), and UV detector (G7114A). Agilent HPLC column Poroshell 120 EC-C18 (4.6 mm x 150 mm, 4 µm) was used for chromatographic separation. All solvents were of HPLC grade. The mobile phase consisted of two solutions: A (water containing 0.05% formic acid) while B (methanol containing 0.05% formic acid). The gradient elution was set as follows: 2 min, 5% B; 5 min, 5-90% B; 7 min, 90-95% B; 4 min, 95% B; 1 min 95-5% B, 1 min 5% B, with a total run time of 20 min.

The flow rate was fixed at 1 mL/min, and the samples injection volume was10 µL. Prior to injection, all samples were prepared at a concentration of 1 mg/mL and, in some cases, further diluted and filtered. A purified standard of (-)-pseudosemiglabrin was used to identify and quantify the presence of this compound in all samples. The flavonoid was detected and quantified at a wavelength of 256 nm (Figure 1) (UV-Vis-NIR spectrophotometer, Agilent) and observed at a retention time of Rt = 11.7 min. (Figure 3). The quantification of the compound was processed using OpenLab CDS software and based on the integrations of the area of the corresponding UV absorption peaks.

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Figure 3.

Representative HPLC chromatograms of (a) (-)-pseudosemiglabrin, (b) Ethanol extract (ECE) and (c) Water extract (WE) for (-)-pseudosemiglabrin quantification showing the retention time of the compound at 11.67 min.

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3. Results and Discussion

Natural products are indispensable sources that contributed many drugs for diverse indications [28]. Considerable number of natural products are among the recently approved drugs [29]. (-)-Pseudosemiglabrin is emerging as an important secondary metabolite from Tephrosia species and may serve as a drug or lead compound for further development. One of the main problems in developing pharmaceutical products from natural compounds is the yield [30]. The main advantage of (-)-pseudosemiglabrin, besides its biological activity, is indeed tied to its abundance in certain species of Tephrosia plants, such as T. purpurea [14] and T. apollinea [12]. It is well known that the percentage of secondary metabolites varies based on environmental conditions and stages of plant growth [24]. In the current study, we developed and validated an HPLC method for the quantification of (-)-pseudosemiglabrin at the different stages of T. purpurea growth extracted with three different solvents by maceration and hot extraction using the Soxhlet apparatus.

Solvent selection based on their polarity and solubilization power. Ethanol is one of the most polar solvents and expected to extract all components in the plant. Ethanol extraction is expected to be efficient but not selective. Chloroform has strong solubilization power but less polar so extracts will be less contaminated with polar plant components such as sugars and salts. However, direct extraction of plant materials with chloroform will probably encounter tissue penetration problems. On the other hand, acetone is more polar than chloroform and its miscibility with water gives the advantage of better tissue penetration. However, the solubilization power of acetone is less than chloroform. Although it is not expected to get high yield from the WEs due to its high polarity, the extraction with water was conducted to mimic the common practice of using plants in traditional medicine. Extraction at room temperature is safer than hot extraction but takes longer time and more solvents. Soxhlet continuous hot extraction is very efficient, less time and solvents consuming but not suitable for volatile or thermolabile components [31]. The three solvents were used for cold and hot extractions aiming to optimize the extraction conditions for (-)-pseudosemiglabrin.

The mobile phase composition was optimized to obtain the best separation of (-)-pseudosemiglabrin from other plant matrix components in the shortest time. During the method development process, different mobile phases and compositions were tried to achieve optimum separation and water+0.05% formic acid and methanol+0.05% formic acid was chosen as the eluent in gradient mode. The UV spectrum of (-)-pseudosemiglabrin was checked and 256 nm maximum absorbance was selected for detection (Figure 1).

The designed HPLC method was developed and validated in terms of specificity, linearity, accuracy, precision, robustness, limit of detection (LOD), and limit of quantification (LOQ) complying with the guidelines of the International Conference on Harmonization (ICH) [32].

The established method selectivity was confirmed as there was no interference from the other components of the T. purpurea plant matrix at the analyte retention time. The chromatograms of standard (-)-pseudosemiglabrin and (-)-pseudosemiglabrin present in T. purpurea ECE, and T. purpurea WE have been depicted in Figures 3(a-c). The retention time of standard (-)-pseudosemiglabrin was found to be 11.65 min and the (-)-pseudosemiglabrin present in T. purpurea ECE, and T. purpurea WE were found to be 11.68 min. This showed that the method was specific in identifying the peak of (-)-pseudosemiglabrin with no interference. Moreover, solvent blanks contained no peaks of the target analytes indicating that no carry-over effect was detected.

Results of (-)-pseudosemiglabrin HPLC analysis showed a linear correlation between peak area and concentration with a good correlation coefficient (R² = 0.9995) in the range of 15.63 to 200 ppm, which was determined by constructing a calibration curve at seven concentrations levels (31.25 50 62.60, 100, 125, 150, and 200 ppm) (Figure 4). Table 3 summarizes the regression data of the proposed HPLC method.

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Figure 4.

Calibration curve of (-)-Pseudosemiglabrin.

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LOD and LOQ were determined as 9.32 ppm and 28.24 ppm, respectively. These values were calculated using the slope of the calibration (S) curve, SD of the blank sample, and applying the following formulae

$\begin{array}{c}\text{LOD}=\text{3}.\text{3}\times \left(\text{SD}/\text{S}\right)\\ \text{LOQ}=\text{1}0\times \left(\text{SD}/\text{S}\right)\end{array}$

The standard deviation of the response was determined using the y-intercepts of the regression lines’ standard deviation.

Accuracy was determined concerning the % recovery by the assay of the known added amounts of (-)-pseudosemiglabrin reference standard. Samples of (-)-pseudosemiglabrin (100 ppm) previously analyzed were spiked with additional amounts of the analytes at 0, 50, 100, and 150% of the target concentration and five replicates of each concentration. Consequently, the mixtures were resubmitted for analysis. Indication for accuracy was deduced from the percentage of recovery and relative standard deviation (RSD, %) for each concentration level (Table 4).

Precision was determined as repeatability and intermediate precision, which measure intraday and interday variation, respectively. For this validation, three different quality control levels (50, 100, and 150 ppm) covering the specified range were analyzed (n=5). The results of the precision evaluation have been summarized in Table 5.

Robustness was assessed to determine how small and intentional modifications in the analytic conditions affected the newly designed method. Minor changes to method parameters such as the duration of mobile-phase saturation, mobile-phase composition, and the mobile-phase volume during the analysis of (-)-pseudosemiglabrin were implemented. Moreover, the compound was quantified at 256 nm, whereas no effect was observed for the quantification at 260 nm. The method performance remained unaffected by these changes, which is an indication of the method’s suitability and reliability during normal use. Other parameters have been summarized in Table 6.

The developed method was applied to quantify the amounts of (-)-pseudosemiglabrin in the different extracts at the four growth stages (Tables 7, 8). Both efficiency of the three selected solvents in the extraction process presented by the yield of the obtained extracts as well as the selectivity indicated by the percentage of (-)-pseudosemiglabrin in the extracts were compared among the different growth stages, used solvents and method of extraction. Selectivity index (SI) (Table 9) was calculated for each extract using the following formula:

$\text{SI}=\text{Weight\ of\ }(-)-\text{pseudosemiglabrin}/\text{Weight\ of\ the\ extract}$

The results of (-)-pseudosemiglabrin quantification have been presented in Table 7 as milligram of the obtained extracts/mg of (-)-pseudosemiglabrin, while Table 8 represents the percentages of (-)-pseudosemiglabrin relative to the dry plant materials used as well as relative to the weight of the obtained extracts. The obtained results indicated that the FLS gives the best extractive values with all solvents used. Plants in the PFS come in second place while values obtained from FRS and PHF were close. These values can be explained as the plant activity reaches the peak during the FLS to ensure the production of fruits and seeds necessary for reproduction and propagation of the species. Flavonoid biosynthesis was found to involve structural genes when expressed they promote flavonoids accumulation. Flavonoids in turn are crucial for the successful flower development [33,34]. Flavonoids also act as sunscreen for the vegetative tissue from UV radiation, signaling molecules for the nitrogen-fixing rhizobia as well as providing visual attractions to pollinators and seed distributors [33]. Flavonoids in flowers produce UV-absorbing patterns visible and attractive to insect pollinators [35].

After this stage, the plant’s activity decreased gradually. Another factor is the high amount of fibrous tissues, fixed oils, and fats produced with the fruits that are considerably larger than the delicate flowers tissue. PHF similarly accumulated more fibrous tissue than the plants grow for the first season. The quantification results indicated that (-)-pseudosemiglabrin maximum concentration was detected in the ECE of the FLS with 186±5.7 mg/10 g dried plants (1.86%). Correlating the yield of (-)-pseudosemiglabrin to the dry extract weight, the ECE extract contained 10.5% (-)-pseudosemiglabrin. Almost the same pattern was observed with other solvents (Table 8). Apparently, extraction with CHE and AHE gave better yield than the CCE and ACE, respectively. Also, chloroform was more effective in extraction than acetone both at room temperature and hot extraction. Another important point is the % of (-)-pseudosemiglabrin in the obtained extracts that reflects the selectivity (Table 9). Extraction with 95% ethanol gave a better yield but was less selective than chloroform and acetone as indicated by the lowest SI 0.080- 0.105 (Table 9) and % of (-)-pseudosemiglabrin (Tables 7 & 8). The obtained values put the chloroform in the first place in giving relatively pure extracts as indicated by the SI (Table 9). However, the use of chloroform either by maceration or continuous hot extraction was not as effective as ethanol in extracting (-)-pseudosemiglabrin.

4. Conclusions

In conclusion, an HPLC method was developed successfully and validated according to the ICH guidelines for the quantification of (-)-pseudosemiglabrin in T. purpurea. The method was applied to quantify (-)-pseudosemiglabrin at different stages of plant growth. Also, the application of three solvents; ethanol, chloroform, and acetone for the extraction of the plant materials was conducted. Extraction was done at room temperature for 24 h and for chloroform and acetone by hot extraction using Soxhlet. Ethanol 95% was the most effective solvent in the extraction of (-)-pseudosemiglabrin. Chloroform was more effective than acetone and gave more pure extracts than 95% ethanol. The data indicated that the FLS produces the highest yield of (-)-pseudosemiglabrin. The proposed HPLC method provides a valuable tool for future studies and contributes to the understanding of the therapeutic potential of T. purpurea as a source of (-)-pseudosemiglabrin. The method can serve as a tool for the quality control and standardization of herbal products containing T. purpurea. Future breeding studies for obtaining high yield of (-)-pseudosemiglabrin can rely on the developed method. It can also be applied for pharmacokinetic studies of this promising molecule.