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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access Full Text Article                                                              Research Article

Preformulation Insights into Daidzein: An Isoflavone with Therapeutic Potential

Neha Mandle , Pratibha Sahu, Rajesh Choudhary , Swarnali Das Paul , Jaya Shree * 

Shri Shankaracharya College of Pharmaceutical Sciences, A Constituent College of Shri Shankaracharya Professional University, Junwani, Bhilai, Chhattisgarh, 490020, India

 

 

INTRODUCTION: 

Medicinal plants are frequently used to create plant-derived medications that are a good substitute for synthetic chemicals. Medicinal plants have proven to be extremely important.¹ Harnessing natural substances from the natural world is the origin of many traditional treatments. The soybean was first grown in northeastern China before 1100 BC², making it one of the earliest domesticated crops. The consumption of these plants, particularly soybeans, can significantly impact the health of both humans and animals. Daidzein is part of the soybean. Daidzein is categorized as an isoflavonoid, which is widely recognized for its potential health benefits. Isoflavones, as a prevalent class of phytoestrogens, are known for their phenolic nature and are often utilized in the prevention and treatment of chronic illnesses. Daidzein, also known as 4′,7-dihydroxy isoflavone, is a naturally occurring phytoestrogen that falls within the category of non-steroidal estrogens. It is a compound with an interesting chemical structure, which can be observed in Figure 1. This phytoestrogen is derived from various plants, including soybeans, alfalfa (Medicago sativa), and red clover (Trifolium pratense), all of which are rich sources of glucosides. Additionally, certain legumes such as Glycine Max also contain Daidzein. Despite its promising properties, it is important to note that Daidzein's pharmacological benefit rate is hindered by its poor solubility, requiring a higher dosage to achieve the intended effects.³ Among the main food crops, soybeans are a major source of isoflavones and a crucial part of the human diet.  Studies have demonstrated that DAID demonstrates a range of pharmacological actions, including antioxidant, anti-hemolytic, and anti-inflammatory properties, making it a potential isoflavone. ^4, 5,6  Additionally, daidzein may prevent the activation of NF-κB. This transcription factor is closely linked to inflammation by controlling the transcriptional activation of various target genes, including adhesion molecules, pro-inflammatory mediators such as iNOS and COX-2, and other cytokines and chemokines. TNF-α is one of the numerous triggers that can activate it.⁷  Murine lung treated with TNF-α the cell models used to clarify daidzein's fundamental anti-inflammatory action were MLE-12 epithelial cells. By directly binding to PARP-1, daidzein significantly decreased the level of TNF-α-induced protein poly-adenosine diphosphate-ribosylation (PARylation). This led to the suppression of pro-inflammatory gene transcription, including NF-κB, which further reduced the expression of the chemokine Cxcl2. 

Figure 1: Structure of Daidzein 

MATERIAL AND METHODS:

Daidzein (purity 98% by HPLC) was purchased from NAC Chemicals (India) Pvt. Ltd. All the other analytical grade chemicals were used for the proposed study.

Preparation of stock solution: For the preparation of a stock solution of 1000 μg/ml, 10 mg of precisely weighed daidzein was placed into the calibrated volumetric flask and dissolved in 10 ml of ethanol. Stock I solution is further diluted with an ethanol solvent to achieve the target concentration of 10 µg/ml (Stock II solution).

Determination of wavelength of maximum absorbance (λmax): Using ethanol as a blank, the Stock-II solution was scanned in full scan mode across the whole UV and visible spectrum, or 800 to 200 nm. Using the software, λmax was determined once the spectrum was obtained. ⁸.

Preparation of a Calibration Curve. The stock II solution was diluted to achieve six distinct calibration standards, which corresponded to 1, 2, 3, 4, 5, and 6 μg/ml of strength. Using a fixed wavelength measurement mode, the absorbance of each calibration standard was measured at a predetermined λmax of 255 nm. Plotting the calibration curve showed concentration vs absorbance. To achieve repeatable results, the aforementioned technique was carried out five times. ⁹.

FTIR studies: Analysis of Daidzein was performed by using the sample technique. Samples were placed in a KBr crystal, and spectra were scanned with an FTIR spectrophotometer.

Determination of Melting Point: Melting points were done by the Capillary tube method. Samples were filled in a capillary and sealed by flame. A capillary tube was placed in the melting point test apparatus. To achieve better results, this technique was carried out three times. 

Determination of the Partition coefficient: The partition coefficient was determined by an excess amount of the drug being dissolved and shaking it with two immiscible liquids, such as water and octanol. The drug's concentration in one of the layers is ascertained, and the value is calculated.

Apparent solubility studies: Solubility studies were performed with an excess amount of drug in different solvents such as water, ethanol, methanol, phosphate buffer, and n-hexane. Samples were filtered with a 0.45 μm membrane filter and analyzed by a UV spectrophotometer. ¹⁰

Screening of lipids based on solubility study: The screening process began with evaluating the solubility of the model drug, daidzein (DZ), in various solid lipids, oils. The solubility study was essential for selecting lipids that could effectively incorporate and retain the drug within the lipid matrix. Each lipid was weighed (10 ml) and transferred into individual glass test tubes. Daidzein was gradually added to the molten lipids in small increments under constant stirring until the point of saturation was visually observed—i.e., when no further drug could be solubilized and excess drug remained undissolved.

After reaching the saturation point, the samples were allowed to equilibrate, and any undissolved drug was separated. The remaining amount of drug that had successfully dissolved in each lipid was quantified by reweighing the undissolved drug. This method allowed for the calculation of the drug's solubility in each lipid, providing critical data for selecting the optimal lipid components that offer the highest solubilizing capacity and, consequently, the greatest potential for drug entrapment and loading in the novel formulations.¹¹

Data analysis: The R2 (correlation coefficient) number indicates how well the regression line fits the individual CT data points of the standard reactions. A value of 1.00 indicates a perfect match between the regression line and the data point. An R2 score above 0.998 is preferred.

RESULTS 

Determination of wavelength of maximum absorbance: Quantitative UV analysis requires the wavelength of maximum absorption to be determined. Usually, a solution with an absorbance value less than 1 is seen to be appropriate for determining the wavelength of maximum absorbance. Taking into account the necessary conditions and appropriateness, the UV-visible spectrophotometer's complete scan mode was utilized to determine the maximum wavelength of the daidzein solution. UV software was used to process the entire scan, and it was also used to identify the λmax. A wavelength determination of 255 nm was made for daidzein. 

Figure 2:  Maximum absorbance (λmax) of daidzein at 255nm

 

 

 

Preparation of standard curve: Any instrumental method of analysis, including the UV-visible spectrophotometer, requires a repeatable calibration curve and an equation expressing the relationship between concentration and response to quantify unknown materials. The method mentioned above is more commonly recognized and reproducible than the graphical method. Five distinct calibration standards were used to create the daidzein calibration curve to assess the usefulness of quantitative daidzein analysis. The absorbance of various calibration standards at 255 nm was measured using a UV-visible spectrophotometer operating in fixed-wavelength mode. Five repetitions of the calibration curve were made. 

FTIR: FTIR spectroscopy can be used to identify compounds by providing structural and purity information. Organic molecules vibrate when exposed to infrared radiation. Absorption results from these vibrations matching the radiant energy. Using the infrared spectrum aids in determining a drug's functional characteristics. Using an FTIR Spectrometer, major absorption bands in the 400–4000 cm-1 range were captured during FTIR analysis of medications and their formulations. The existence and absence of these bands, as well as the appearance of any new bands, were recorded within the infrared absorption spectrum. 

Figure 3: Standard curve of Daidzein in Ethanol

 

 

Figure 4: FTIR spectra of Daidzein

 

 

Melting Point: The melting point of daidzein was determined using a melting point apparatus to assess its purity and thermal stability. The compound exhibited a sharp and consistent melting point in the range of 325 ± 3 °C to 326 ± 3 °C, indicating its crystalline nature and high degree of purity. The minimal variation among the recorded values confirms the reproducibility of the results and suggests that the sample was free from impurities or degradation products.

Solubility study: Solubility was measured using different polar and nonpolar solvents such as Methanol, Ethanol, n-hexane, and water. Fig.5

Partition coefficient: The determined partition coefficient (log P) was 2.55, indicating that daidzein has a relatively lipophilic nature. This means that daidzein has a higher affinity for lipid or nonpolar (organic) phases compared to aqueous (polar) phases. In other words, it tends to dissolve more readily in fats, oils, and organic solvents than in water. Such a value suggests that daidzein can easily penetrate biological membranes, which are primarily composed of lipids, potentially influencing its absorption, distribution, and bioavailability in biological systems.

Figure 5: Solubility profile 

 

DISCUSSION: 

Daidzein, a naturally occurring isoflavonoid derived from plant sources such as soybean and alfalfa, possesses significant pharmacological potential owing to its antioxidant, anti-inflammatory, and estrogenic properties. However, before developing an effective formulation, it is essential to identify and understand its physicochemical characteristics through preformulation studies.

In the present study, daidzein was successfully identified and characterized using both UV–Visible spectrophotometry and Fourier Transform Infrared (FTIR) spectroscopy. The UV–Visible analysis demonstrated a distinct maximum absorbance (λ_max) at 255 nm when measured in ethanol. This absorption peak corresponds closely with previously reported literature values for daidzein, thereby supporting the accuracy of the identification and confirming the compound’s purity. Furthermore, the method exhibited excellent linearity across the tested concentration range, as evidenced by a high regression coefficient (R² = 0.998). This strong linear relationship indicates that the UV–Visible spectrophotometric technique used in this study is highly reliable, accurate, and reproducible for quantitative analysis of daidzein. Collectively, these findings validate the analytical methods employed and reinforce their suitability for routine evaluation of daidzein in research and quality. 

FTIR spectral analysis further confirmed the structural integrity of daidzein by exhibiting characteristic absorption bands corresponding to its functional groups, such as hydroxyl (–OH), carbonyl (C=O), and aromatic C=C stretching vibrations. These peaks validated the chemical identity and stability of the compound under the experimental conditions.

The partition coefficient value obtained for daidzein indicated a predominantly lipophilic character, reflecting its stronger affinity for lipid phases than for aqueous environments. This inherent lipophilicity suggests that the compound may readily permeate biological membranes, which is advantageous for certain pharmacokinetic processes. However, it also implies limited solubility in water, a factor that can significantly restrict its dissolution rate and, consequently, its overall bioavailability upon oral administration. In addition to partitioning behavior, melting point analysis further corroborated the compound’s physicochemical characteristics. The sharp, well-defined melting point observed in the present study is indicative of a highly crystalline structure and supports the conclusion that the isolated daidzein sample was highly pure. Collectively, these findings provide essential insights into the compound’s structural integrity and its potential performance in biological systems.

Solubility studies revealed that daidzein exhibits very low aqueous solubility, which aligns with its classification under the Biopharmaceutical Classification System (BCS) as a Class IV compound—defined by both poor solubility and limited permeability. These physicochemical limitations pose significant challenges for oral delivery, as inadequate dissolution in gastrointestinal fluids often results in low and inconsistent absorption. To address these formulation barriers, a comprehensive lipid screening study was conducted to identify suitable lipid carriers that enhance the solubility and subsequent bioavailability of daidzein. Among the various lipids evaluated, tocopherol α (Vitamin E) demonstrated complete solubility for the compound, signifying a strong affinity between daidzein and this lipid medium. This pronounced solubilization capacity highlights tocopherol α as a promising candidate for use as the lipid phase in advanced drug delivery platforms. Its compatibility with daidzein makes it particularly suitable for the development of innovative systems such as nanostructured lipid carriers (NLCs) or nanoemulsions, which rely on effective solubilization and stabilization within a lipid matrix to improve drug loading, enhance permeability, and ultimately increase bioavailability. These findings lay a strong foundation for subsequent formulation development and optimization.

Overall, the findings from the identification and preformulation studies provide a comprehensive understanding of daidzein’s physicochemical profile. The insights gained from lipid screening and solubility evaluation will be valuable for designing advanced formulations to enhance the solubility, permeability, and bioavailability of daidzein for therapeutic applications.

CONCLUSION: 

The study successfully identified Daidzein via UV and FTIR spectroscopy, confirming its characteristic λmax at 255 nm and high linearity (R² = 0.998). Preliminary physicochemical evaluations, including melting point, partition coefficient, and solubility screening, established Daidzein’s classification as a BCS Class IV compound with poor solubility and bioavailability. The complete solubility of Daidzein in α-tocopherol highlights its potential as a suitable lipid for novel formulation development aimed at improving Daidzein’s solubility and therapeutic efficacy.

Acknowledgement: The authors express heartfelt gratitude to the college administration for its cooperation.

Conflict of Interest: There is no conflict of interest. 

Authors’ Contribution: 

Conceptualization: Neha Mandle, Jaya Shree; Methodology: Neha Mandle, Jaya Shree, Rajesh Choudhary, Swarnali Das Paul; Investigation: Neha Mandle, Jaya Shree, Rajesh Choudhary, Swarnali Das Paul; Supervision: Rajesh Choudhary, Jaya Shree; Writing – Original draft: Neha Mandle; Writing – Review & Editing: Neha Mandle; Jaya Shree; Rajesh Choudhary, Swarnali Das Paul.

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