Therapeutic Potential of Alkanna tinctoria Root in Ameliorating Obesity‐Associated Hepatic, Adipose, and Reproductive Dysfunctions in Diet‐Induced Obese Zebrafish

ABSTRACT

Obesity is a worldwide metabolic condition linked to various health problems such as non‐alcoholic fatty liver disease (NAFLD) and reproductive disorders. Anti‐obesity medications currently available on the market exhibit severe adverse effects, which leads to the exploration of safer natural alternatives. Alkanna tinctoria (L.) Tausch is well‐known for its excellent antioxidant, anti‐inflammatory, and antitumor activities; although, its anti‐obesity potential has yet to be comprehensively evaluated. The current study investigated the anti‐obesity effects of A. tinctoria root powder using diet‐induced obese (DIO) zebrafish as a model organism, through histopathological and Fourier Transform Infrared (FTIR) analysis. Zebrafish were assigned to three groups: control, DIO, and DIO + AT. Obesity was induced in the DIO and DIO + AT groups by overfeeding with brine shrimp, while the DIO + AT group additionally received A. tinctoria powder 30 min before each feeding. Overfeeding with brine shrimp significantly increased body weight gain, change in body mass index (BMI), gonadosomatic index (GSI), and hepatosomatic index (HSI), whereas supplementation with A. tinctoria root powder significantly reduced these parameters, along with hepatic lipid vacuolization area, mean vacuole diameter, adipocyte area, and adipocyte count in the DIO + AT group. Histopathological analysis further demonstrated marked attenuation of hepatic steatosis, restoration of normal liver architecture, and improved ovarian morphology characterized by enhanced follicular development and fewer cystic lesions in the DIO + AT zebrafish relative to the DIO zebrafish. ATR‐FTIR analysis revealed the reversal of obesity‐associated biochemical alterations, particularly in lipid, protein, carbohydrate, and phosphate‐associated spectral regions, suggesting improved metabolic balance in the DIO + AT zebrafish as compared to the DIO zebrafish. These findings demonstrate that A. tinctoria root powder effectively mitigated obesity‐associated metabolic, hepatic, adipose, and reproductive dysfunctions while restoring molecular homeostasis. Overall, our study highlights A. tinctoria root powder has promising potential as a natural anti‐obesity agent for the management of obesity and its associated metabolic and reproductive complications.

Significance Statement

Obesity has emerged as a global epidemic in recent times. Many drugs available on the market are associated with severe side effects, highlighting the need for safer alternatives. Alkanna tinctoria root is widely recognized for its antioxidant, anticancer, and anti‐inflammatory activities; nonetheless, its role in obesity management has yet to be fully explored. Therefore, the current investigation was conducted to assess the anti‐obesity effects of A. tinctoria root against obesity‐associated hepatic, adipose, and reproductive abnormalities. The findings show that A. tinctoria root powder reduces excess weight gain, improves hepatic steatosis and adipose tissue health, restores normal ovarian structure, and reverses obesity‐induced molecular alterations in DIO zebrafish. These results suggest that A. tinctoria root powder is a potential plant‐based therapeutic agent for obesity management and related metabolic and reproductive disorders.

Abbreviations

ATR‐FTIR

attenuated total reflectance‐fourier transform infrared spectroscopy

BMI

body mass index

DIO

diet‐induced obesity

DIO + AT

diet‐induced obesity + Alkanna tinctoria root

GSI

gonadosomatic index

HPLC

high‐performance liquid chromatography.

HSI

hepatosomatic index

NAFLD

non‐alcoholic fatty liver disease

PCOS

polycystic ovarian syndrome

1. Introduction

Obesity is a multifactorial and chronic disease that has attained pandemic proportions worldwide, resulting in severe health and economic consequences. According to the World Obesity Federation, approximately 800 million people worldwide are currently affected by obesity, including an estimated 39 million children younger than 5 years and 340 million individuals aged 5–19 years. In addition, at least another 1 billion people are reportedly at risk of becoming obese [1]. One of the contributing factors to obesity is the consumption of diets containing excessive levels of macronutrients. A fat‐rich diet adversely affects nutrient digestion, enhances fat deposition, causes persistent low‐grade inflammation and impaired insulin sensitivity, and may ultimately lead to obesity, cardiovascular disorders, and tumorigenesis [2, 3, 4, 5]. Increased carbohydrate consumption can also contribute to increased body fat, weight gain, and fatty liver disease [6, 7].

Although various pharmacological remedies have been developed to treat obesity, these medications are often associated with severe side effects such as drug addiction, restlessness, insomnia, and anxiety, which may limit their compliance and use by obese individuals [8]. In contrast, natural agents generally possess fewer or no side effects [9]. These natural agents contain a wide range of bioactive compounds, including polyphenols, flavonoids, and terpenoids, which have been shown to be effective in managing obesity and other health‐related problems [10]. Abiraamavalli and Namasivayam [11] demonstrated that a polyherbal metabolite consortium prepared from Terminalia chebula, Tinospora cordifolia, Smilax china, Coscinium fenestratum, and Acorus calamus exhibited enhanced antioxidant, anti‐inflammatory, and α‐amylase inhibitory activities, indicating its potential to mitigate obesity‐related metabolic disturbances. Similarly, microbial‐derived products have also shown promising hypocholesterolemic and anti‐obesity activities through the modulation of lipid metabolism. Kokila et al. [12] reported that Bacillus amyloliquefaciens strain KAVK1 inhibited pancreatic lipase activity, reduced lipid deposition in 3T3‐L1 adipocytes, promoted cholesterol degradation, and significantly improved body weight, serum lipid profiles, and obesity‐associated histopathological abnormalities associated with HFD‐induced obesity in rats.

Alkanna tinctoria (L.) Tausch is a perennial plant species belonging to the Boraginaceae family, commonly referred to as dyer's alkanet or ratanjot. The plant possesses multiple pharmacological properties, including anthelmintic, antiseptic, and antipyretic activities and has traditionally been used to treat various ailments, such as eye disorders, bronchitis, and gastrointestinal disturbances [13]. In addition, several studies have reported the anticancer potential of bioactive compounds present in A. tinctoria against different types of cancer, including lung cancer, colorectal cancer, leukemia, and breast cancer [14, 15, 16]. Among the various parts of this plant, the root is considered the most useful and is widely used in cosmetic and medicinal products. The roots are particularly rich in naphthoquinone derivatives, mainly alkannin and shikonin, which are considered the major bioactive constituents of the plant [17].

Traditionally, it has been used to treat gastrointestinal ulcers, diarrhea, and cancer. In addition, it exhibits potent free radical scavenging activity, which contributes to its potential anti‐aging effects [18]. Studies have also demonstrated that the naphthoquinones present in A. tinctoria root (such as shikonin) inhibit the differentiation and lipid accumulation of 3T3‐L1 adipocytes along with glucose uptake enhancement [19]. Moreover, acetylshikonin, a phytoconstituent of the root of A. tinctoria, has been reported to exhibit anti‐obesity action via the reduction of lipid accumulation and enhancement of lipolysis in adipocytes [20]. However, further in vivo investigation is required to establish the anti‐obesity effects of A. tinctoria root powder.

Accordingly, the current study aimed to examine the anti‐obesity activity of A. tinctoria root powder through histopathological analysis and attenuated total reflectance Fourier transform infrared (ATR‐FTIR) spectral analysis. For the current investigation, adult zebrafish were chosen as a model organism because they possess all the necessary organs, including liver, ovaries, and white adipose tissue, similar to those in humans, which are required for regulating metabolic disorders [21]. Moreover, lipid metabolism mechanisms seen in zebrafish also show a remarkable similarity with that observed in humans concerning intestinal absorption mediated by liver‐made bile secretion [22], fat and cholesterol transmission through lipoproteins [23], β‐oxidation pathways [24], or storage of triacylglycerols in various adipose tissues like visceral, subcutaneous, and intramuscular adipocytes [22, 23, 24, 25].

In the present investigation, the effects of A. tinctoria root powder on obesity were examined using diet‐induced obese (DIO) zebrafish model emphasizing changes in the liver, adipose tissue, and ovaries. Moreover, serum and liver tissue samples were used to determine alterations in functional groups through FTIR analysis.

2. Materials and Methods

2.1. Model Organism

Adult zebrafish (Danio rerio), 3‐4 months old, weighing about 0.3 ± 0.5 grams were used for the experiment. The fish were procured from Marine Dreams, Mohali, Punjab and were acclimatized for at least 20 days in a 50‐liter tank containing dechlorinated water before starting the experiment. During this phase, environmental conditions were strictly maintained and water conductivity at 470–524 μS/cm was maintained. Other parameters, such as temperature (27 ± 2°C) and pH (7.2–7.6), were maintained throughout the study. A regulated light cycle of 12 h of light followed by 12 h of darkness was established to simulate natural day/night conditions. During the acclimation period, the zebrafish were fed a balanced diet of micro pellets and freshly hatched brine shrimp two times daily to enhance their well‐being. All handling and experimental procedures followed the ethical guidelines set by the institution and were sanctioned by the Institutional Animal Ethics Committee at Guru Nanak Dev University, Amritsar (Approval No. 226/CPCSEA/2023/36).

2.2. Experimental Design

The experimental design was adapted from our previously published study [26], with modifications for the present investigation. The feeding regimen for induction of diet‐induced obesity (DIO) was based on previously established protocols described by Oka et al. [27] and Kaur et al. [28]. The zebrafish were randomly assigned to one of three experimental groups. Each group included 60 zebrafish, consisting of an equal number of males and females (Figure 1). These groups were given different diets to examine the effects of A. tinctoria root powder on diet‐induced obesity (DIO). The control group (Group I) received a conventional commercial diet administered twice per day, with each fish receiving approximately 15 mg of feed per day. In Group II (DIO group), diet‐induced obesity was established by overfeeding the zebrafish with brine shrimp (Artemia) four times daily, along with a commercial diet provided twice daily, resulting in a total feed intake of approximately 60 mg per fish per day. Zebrafish in group III (DIO + A. tinctoria root powder; DIO + AT) were maintained under the same overfeeding conditions as the DIO group. Each fish received 60 mg of feed per day, divided into six meals. In addition, 6 mg of A. tinctoria root powder was given six times daily, 30 min prior to the regular feeding.

Figure 1.

Experimental design for evaluating the anti‐obesity potential of A. tinctoria root powder in adult zebrafish.

The experiment was carried out for a duration of 30 days. Body weight and length were recorded on days 1, 14, and 30 of the experimentation. The body mass index (BMI) was then calculated by applying the following equation:

$$\text{BMI}=\frac{\text{Body}\text{weight}(\text{mg})}{\text{Body}{\text{length}}^2\left({\text{cm}}^2\right)}$$ (1)

After the experimental period, the fish were sacrificed for the evaluation of histopathological alterations in the liver, adipose tissue, and ovaries, as well as the overall metabolic state.

2.3. Hepatosomatic Index (HSI) and Gonadosomatic Index (GSI)

At the end of the 30‐day experimental period, fish from all three experimental groups were anesthetized by placing them on ice. The liver and gonads (ovaries and testes) were excised and weighed to calculate the HSI and GSI, respectively [29, 30]. The following formulas were used to calculate the HSI and GSI:

$$\text{HSI}=\frac{(\text{Liver}\text{weight},\mathrm{g})}{(\text{Body}\text{weight},\mathrm{g})}\times 100$$ (2)

$$\text{GSI}=\frac{(\text{Ovary}\text{or}\text{Testis} \text{weight},\mathrm{g})}{(\text{Body}\text{weight},\mathrm{g})}\times 100$$ (3)

2.4. Histopathological Examination

After completion of the experiment, the liver, adipose, and ovarian tissues were dissected out and washed with phosphate buffer saline (PBS) for histopathological examination. The collected tissues were fixed in 10% formalin for 24 h, followed by dehydration through a series of graded ethanol concentrations and clearing with xylene. Thereafter, the tissues were embedded in melted paraffin wax to facilitate sectioning with the microtome. Thin sections (5 μm thick) were prepared, and slides were stained with hematoxylin and eosin (H&E) following the procedure described by Cheng et al [31]. Following H&E staining, the tissue sections were mounted using DPX and covered with coverslips. The slides were air‐dried and were then examined using a light microscope at 10× and 40× magnifications.

Fat vacuolization, adipocyte number and size, and the proportions of pre‐vitellogenic, early‐vitellogenic, and mature‐vitellogenic oocytes were quantified using ImageJ software (NIH, Bethesda, MD, USA). For the assessment of fat vacuolization, histological images of liver tissue were first converted to 32‐bit grayscale and subjected to thresholding. The ImageJ “Analyze Particles” tool was subsequently used to determine the area occupied by vacuoles. A circularity filter was applied to distinguish fat vacuoles from hepatic sinusoids. The mean vacuolar diameter was measured using ten horizontal transects placed across each image. All vacuoles intersected by the transects were measured automatically, and the resulting measurements were averaged for each experimental group [32].

For the assessment of adipose tissue, selected tissue areas containing adipocytes were identified from the histological sections. Adipocyte size was measured manually, with the cells classified based on their size and circularity. The total number of adipocytes in each selected region was also manually counted using the multipoint tool in ImageJ software [33, 34].

Ovarian follicles were classified into three developmental stages: pre‐vitellogenic, early‐vitellogenic, and mature‐vitellogenic, based on the morphological criteria established in previous studies [35, 36, 37]. Oocytes measuring up to 250 µm in diameter were categorized as pre‐vitellogenic. Follicles measuring > 250 to 500 µm in diameter were classified as early‐vitellogenic, whereas those exceeding 500 µm were categorized as mature‐vitellogenic based on the presence of yolk‐filled alveoli throughout the ooplasm.

2.5. Attenuated Total Reflectance‐Fourier Transform Infrared (ATR‐FTIR) Spectroscopy

The ATR‐FTIR analysis was done using a Shimadzu IR Tracer‐100 interferometer (Shimadzu corporation, Kyoto, Japan) with a spectral range of 4000‐500 cm⁻¹. Each sample was scanned 20 times at a resolution of 4 cm⁻¹, resulting in an average acquisition time of about 30 s per sample. Sample preparation entailed the freeze‐drying of 30 μL aliquots of serum and liver tissue samples to freeze out any remaining moisture in the sample prior to carrying out the analysis. Samples were analyzed using a diamond ATR accessory. After each scan, the ATR crystal was carefully cleaned with ethanol of high‐performance liquid chromatography (HPLC)‐grade to prevent any noise or interference from the ambient conditions; background spectra were recorded for correction. The Savitzky–Golay smoothing method (second‐order polynomial, 15 data points) was applied in the spectral range to reduce noise and smooth abrupt changes in the spectral bands, thus ensuring the spectra were clearer and more reliable.

2.6. Statistical Analysis

Statistical analysis was carried out using SPSS software (version 21). All results were expressed as mean ± standard error (SE). One‐way ANOVA was performed on the data to determine the statistical significance of the variations between groups at different times. Post hoc Tukey's test was applied to see the difference between different groups in different time durations. The p < 0.05 values were regarded as statistically significant. Pearson's correlation analysis was conducted using Past 4.03 software to assess the relationship among the measured parameters (p < 0.05).

3. Results

3.1. Body Weight Gain and Change in BMI

Obesity was effectively established in zebrafish by feeding them an energy‐rich diet consisting of brine shrimp. Body weight, total body length, and BMI measurements were taken on day 1, day 14, and day 30 of the experimental period. A significant increase in body weight gain and change in BMI was observed in the DIO zebrafish compared with the control group. Conversely, treatment with A. tinctoria root powder led to a marked decrease in both the parameters (p < 0.01) compared with the DIO group (Figure 2A,B).

Figure 2.

(A) Δ Weight (B) Δ BMI (C) Hepatosomatic index (HSI) and (D) Gonadosomatic index (GSI) of adult zebrafish ovaries and testes from the Control, Diet‐Induced Obese (DIO), and DIO treated with A. tinctoria root powder (DIO + AT) groups. Statistical significance was assessed using one‐way ANOVA (p < 0.05). Different lowercase letters (a, b) denote significant differences across the experimental groups within same time interval. Data are presented as mean ± SE.

3.2. HSI and GSI

A noticeable increase in HSI was observed in the DIO group compared with the control group (p < 0.05), whereas the DIO + AT group showed no statistically significant difference in comparison to the control group (Figure 2C). Similarly, both male and female zebrafish exhibited a significant increase in GSI in the DIO group compared with the control and DIO + AT groups (p < 0.01) (Figure 2D).

3.3. Histopathological Analysis of Liver, Adipose, and Ovarian Tissues

As shown in Figure 3, DIO zebrafish exhibited marked accumulation of visceral adipose tissue around the liver. This accumulation was considerably higher than that observed in the control and DIO + AT groups, as visualized using an Olympus SZX7 stereomicroscope. To further understand the hepatoprotective properties of A. tinctoria root powder, histopathological analysis was performed on liver sections from the control, DIO, and DIO + AT groups (Figure 4A–C). The DIO group exhibited evident hepatic damage, characterized by the hepatocytes containing excessive lipid content (reflected in microvesicular as well as macrovesicular steatosis), as well as vacuolization. However, the liver sections of the DIO + AT group showed only minimal histopathological alterations, indicating the potent hepatoprotective effect of A. tinctoria root powder. Histopathological analysis also showed that the DIO group exhibited a significantly greater area of fat vacuolization and a larger mean vacuole diameter compared with the control group. In contrast, administration of A. tinctoria root powder resulted in significant reductions in both parameters (p < 0.01) (Figure 6A,B).

Figure 3.

Gross liver anatomy (outlined with yellow dotted lines) of zebrafish from the (A) control group, (B) DIO group, and (C) DIO + AT group, captured at 2.6× magnification. The blue arrow highlights prominent adipose deposits in the visceral region of the DIO group.

Figure 4.

Histological liver sections stained with hematoxylin and eosin (H&E) from (A) control, (B) DIO, and (C) DIO + AT groups, examined at 40× magnification. The control and DIO + AT groups displayed normal hepatocyte morphology (yellow arrows) with well‐organized hepatic plates (blue arrows). In contrast, the DIO group exhibited prominent hepatocellular steatosis (dotted black arrows) and vacuolation (bold black arrows).

Figure 6.

Fat vacuolization area (%) (A) and mean diameter fat vacuoles (µm) (B), adipocyte number (C) and visceral adipocyte size (µm²) (D) in Control, Diet‐Induced Obesity (DIO), and DIO treated with A. tinctoria root powder (DIO + AT) groups. Statistical significance was assessed using one‐way ANOVA (p < 0.05). Different lowercase letters (a, b) denote significant differences across the experimental groups within same time interval. Data are presented as mean ± SE.

The H&E staining of cross‐sections from zebrafish showed that the DIO group had a significantly greater number of visceral adipocytes compared to both the control and DIO + AT groups (Figures 5A–C and 6C) (p < 0.01). Stereological analysis revealed that the adipocytes in the DIO group were significantly larger than those observed in the control group (p < 0.01). Conversely, no significant difference in adipocyte size was observed between the DIO + AT and control groups (Figures 5D–F and 6D).

Figure 5.

(A–C) Histological visceral adipose tissue sections stained with hematoxylin and eosin (H&E) from control, DIO, and DIO + AT zebrafish, respectively, captured at 40× magnification. (D–F) Enlarged views highlighting the adipocyte morphology within the visceral adipose tissue of the corresponding groups. Marked adipocyte enlargement was observed in DIO zebrafish, while A. tinctoria root powder supplementation decreased adipocyte size and improved tissue morphology relative to the DIO group.

Histopathological examination of the ovarian sections revealed that the DIO group primarily contained pre‐vitellogenic oocytes with a significant reduction in the numbers of early‐ and mature‐vitellogenic oocytes compared with the control group, which exhibited higher numbers of early‐ and mature‐vitellogenic oocytes (Figure 7B) (p < 0.05). Administration of A. tinctoria root powder in the DIO + AT group resulted in a recovery of early‐ and mature‐vitellogenic oocyte numbers, bringing them to levels similar to those observed in the control group (Figures 7C and 8). Furthermore, ovarian tissues from the DIO group developed cystic lesions that were characterized by disorganized follicular membranes, hypertrophy of the theca layer, and invagination of the zona pellucida. These pathological features were absent in both the control and DIO + AT zebrafish (Figure 7D–F). The percentage distribution of oocyte developmental stages among the three groups is presented in Figure 8.

Figure 7.

(A–C) Histological sections of ovaries stained with hematoxylin and eosin (H&E) from control, DIO, and DIO + AT groups. Ovaries from the control and DIO + AT groups primarily contained early‐ and mature‐vitellogenic oocytes (indicated by brown and yellow arrows), while those from the DIO group predominantly exhibited pre‐vitellogenic oocytes (red arrow). (D–F) Ovarian sections of the DIO group displayed prominent cystic lesions, such as invagination of the zona pellucida (blue arrow), hypertrophy of the thecal layer (pink arrow), and disorganized follicular membrane (green arrow). Images captured at 10× magnification.

Figure 8.

Percentage distribution of oocyte developmental stages in zebrafish from the control, diet‐Induced Obese (DIO), and DIO treated with A. tinctoria root powder (DIO + AT) groups: (A) pre‐vitellogenic, (B) early‐vitellogenic, and (C) mature‐vitellogenic oocytes. Statistical significance was assessed using one‐way ANOVA (p < 0.05). Different lowercase letters (a, b) denote significant differences across the experimental groups within the same time interval. Data are presented as mean ± SE.

3.4. Pearson Correlation Analysis

Figure 9 displays the correlation matrix that evaluates the relationships among parameters associated with obesity. Negative correlations are indicated by red, while positive correlations are shown in blue, with the color intensity reflecting the strength of the correlation coefficient. Statistically significant correlations (p < 0.05) are highlighted with boxed circles, while non‐significant correlations are represented by unboxed circles. The analysis revealed strong positive correlations between body weight gain, change in BMI, HSI, GSI (ovary and testis), fat vacuolization area percentage, mean vacuolar diameter, adipocyte area, adipocyte number, and percentage of pre‐vitellogenic oocytes. Conversely, the proportions of early‐vitellogenic and mature‐vitellogenic oocytes showed significant negative correlations with the previously mentioned obesity‐associated parameters.

Figure 9.

The effects of A. tinctoria root powder on obesity‐associated parameters in diet‐induced obese zebrafish were assessed using Pearson's correlation. Δ Weight (body weight gain), Δ BMI (change in BMI), HSI (Hepatosomatic index), GSI (Gonadosomatic index: ovary and testis), Fat Vac.% (Fat Vacuolization area %), V. D. (mean vacuolar diameter), Adipo. area (Adipocyte area), Adipo. no. (Adipocyte number), pre‐vit. (Pre‐vitellogenic oocytes), early‐vit. (Early‐early‐vitellogenic oocytes), mature‐vit. (Mature‐vitellogenic oocytes).

3.5. FTIR Spectral Analysis

The FTIR spectroscopy was employed to characterize the chemical profiles of blood serum and liver tissue samples from the three groups, enabling a comparative assessment of their molecular composition. Figure 10 presents the mean FTIR spectra of dried blood serum samples obtained from the control, DIO, and DIO + AT groups over the wavenumber range of 4000–500 cm⁻¹.

Figure 10.

FTIR spectra of dried serum samples from control, diet‐induced obese (DIO), and DIO + AT groups, depicted by black, red, and blue curves, respectively.

The serum FTIR spectra were analyzed based on the characteristic regions corresponding to the major biochemical components present in the samples. The bands observed between 3000 and 2800 cm⁻¹ were mainly related to lipids, while those in the 1700–1500 cm⁻¹ range were largely associated with proteins. The bands appearing between 1300 and 950 cm⁻¹ were mainly attributed to carbohydrates and nucleic acids. The major bands observed in the serum samples from the three experimental groups, together with their corresponding assignments, are given in Table 1.

Table 1.

Peak positions and differences in peak positions in FTIR spectra of blood serum samples collected from the DIO, control, and DIO + AT groups, along with the description of peaks analyzed in FTIR spectra. Measuring range 4000–500 cm⁻¹.

Wavenumber in FTIR DIO spectrum (cm−1)	Wavenumber in FTIR Control spectrum (cm−1)	Wavenumber in FTIR DIO + AT spectrum (cm−1)	Differences in peak positions DIO‐Control (cm−1)	Differences in peak positions DIO‐DIO + AT (cm−1)	Peak description
2854	2852	2852	2	2	νs CH2 Symmetric stretching vibration of CH2 of Acyl chains (lipids)
1533	1540	1533	−7	0	Amide II
1217	1224	1224	−7	−7	Collagen Asymmetric stretching of phosphate groups of phosphodiester linkages in DNA and RNA, Asymmetric PO2 ‐ stretching in RNA and DNA, Symmetric stretching of phosphate groups in phospholipids
1149	1163	1163	−14	−14	CH'9, CH7, CH'7 deformations
1039	1053	1058	−14	−19	νC‐O & δC‐O of carbohydrates

The FTIR analysis of serum samples from the control, DIO, and DIO + AT groups showed clear differences in the intensity and position of characteristic absorption peaks corresponding to major biomolecules. The symmetric stretching vibrations of CH₂ groups from the lipid acyl chains were detected around 2852 cm⁻¹, while the amide II band indicative of protein structures appeared close to 1540 cm⁻¹. A notable band near 1224 cm⁻¹ was attributed to the asymmetric stretching of phosphate (P = O) groups associated with nucleic acids and phospholipids. The band detected at 1163 cm⁻¹ is linked to C–O–P stretching vibrations of phosphate esters and is likely originating from DNA, RNA, and phospholipids. The absorption band at 1053 cm⁻¹ is related to stretching (νC–O) and bending (δC–O) vibrations. These are typical features of carbohydrate structures and glycosidic linkages. There was a noticeable difference in both absorbance intensity and wavenumber position between the groups. The DIO group showed higher absorbance in the ${\text{PO}}_2^{-}$ and lipid‐associated regions than the control and DIO + AT groups. This may be associated with increased lipid accumulation and changes in the structure of nucleic acids. In addition, distinct shifts in the positions of several peaks were observed in the DIO group. The phosphonate (P = O) and amide II bands shifted toward lower wavenumbers, while the CH₃ stretching bands shifted toward higher wavenumbers compared with the control and DIO + AT groups.

The FTIR spectra of liver tissues from the control, DIO, and DIO + AT groups were recorded in the range of 4000‐500 cm⁻¹ and showed characteristic absorption bands corresponding to the major biochemical constituents such as lipids, proteins, phospholipids, and nucleic acids (Figure 11). The main IR bands identified in the liver tissue samples of the three experimental groups, along with their corresponding assignments, are presented in Table 2. Noticeable differences in peak positions and absorbance intensities were observed between the FTIR spectra of the DIO and DIO + AT groups, indicating changes in the biochemical composition of the liver associated with obesity in DIO zebrafish and the possible protective effect of A. tinctoria root powder.

Figure 11.

FTIR spectra of dried liver tissue samples obtained from the control, DIO, and DIO + AT groups, shown in black, red, and blue curves, respectively, with annotated peaks.

Table 2.

Peaks position and differences in FTIR spectra of liver tissue collected from the DIO, control, and DIO + AT groups with the description of peaks analyzed in FTIR spectra. Measuring range 4000–500 cm⁻¹.

Wavenumber in FTIR DIO spectrum (cm−1)	Wavenumber in FTIR Control spectrum (cm−1)	Wavenumber in FTIR DIO + AT spectrum (cm−1)	Differences in peak positions DIO‐Control (cm−1)	Differences in peak positions DIO‐DIO + AT (cm−1)	Peak description
2862	2853	2853	9	9	νs CH2 of lipids, symmetric CH2 stretching mode of the methylene chains in membrane lipids
1735	1740	1740	−5	−5	C = O, C = O stretching (lipids), Ester C = O stretching vibration (phospholipids)
1633	1639	1639	−6	−6	Amide I
1094	1085	1094	9	0	PO2 ‐ symmetric (phosphate II)
952	960	960	−8	−8	Symmetric stretching vibration of ν1PO4

A prominent absorption band near 2853 cm⁻¹ attributed to the symmetric CH₂ stretch of methylene chains in membrane lipids. The DIO group exhibited a + 9 cm⁻¹ shift relative to the control group, while the administration A. tinctoria root powder demonstrated restorative effect. In the carbonyl region, the strong band at 1740 cm⁻¹ for the C = O stretch of ester groups of phospholipid molecules showed a − 5 cm⁻¹ shift for the DIO group in comparison to the control and DIO + AT groups. Protein‐related absorptions were observed in the amide I band (1639 cm⁻¹) corresponding to C = O stretching and N‐H bending of amides and is a probe for the secondary structure of proteins. In the DIO group, this peak demonstrated shift of –6 cm⁻¹ towards lower wavenumber whereas DIO + AT group spectra showed no significant difference in wavenumber when compared to the control group. The regions related to phosphate also exhibited significant changes. The symmetric stretching of phosphate (P = O) was noted near 960 cm⁻¹. These phosphate vibrations are primarily related to phospholipids and nucleic acids. The DIO group showed a downward shift of −8 cm⁻¹, suggesting potential disruptions in nucleic acid and phospholipid structure within hepatocytes whereas supplementation of A. tinctoria root powder in DIO + AT group notable showed restorative effects.

4. Discussion

In the present investigation, the anti‐obesity and non‐alcoholic fatty liver disease (NAFLD) ameliorating potential of A. tinctoria root powder were investigated through histopathological assessments of the liver, adipose, and ovarian tissues, as well as FTIR spectroscopic analysis. The results indicate that supplementation with A. tinctoria root powder for 30 days resulted in notable reductions in body weight gain and HSI. In addition, GSI was significantly decreased in both sexes of zebrafish following A. tinctoria root powder supplementation, whereas food intake remained unchanged. In addition, a significant decrease in the change in BMI was recorded in the DIO + AT group compared with the DIO group. These results indicate that the phytochemical present in the root powder of A. tinctoria, particularly shikonin, alkannin, and their derivatives may have played a key role in the regulation of lipid metabolism and body weight.

Our observations are consistent with those reported by Gwon et al. [38], who found that treatment with shikonin (a phytoconstituent in A. tinctoria root) resulted in a marked reduction in body weight in HFD‐induced obese mice. This activity was attributed to the potential of shikonin to suppress the expression of lipogenesis‐related genes, including sterol regulatory element‐binding protein 1 (SREBP‐1), fatty acid synthase (FAS), glycerol 3‐phosphate acyltransferase (GPAT), acetyl‐CoA carboxylase alpha (ACCα), and stearoyl‐CoA desaturase 1 (SCD‐1). These genes are involved in fatty acid synthesis and lipid accumulation and therefore play an important role in lipid storage in adipose and liver tissues [38]. The decrease in body weight gain noted in the DIO + AT group may be associated with the inhibitory effects of shikonin on lipogenesis, which could reduce lipid deposition and support better lipid utilization.

In the present study, administration of A. tinctoria root powder also resulted in marked reduction in HSI in the DIO + AT group compared with the DIO group. The HSI is commonly used as an indicator of hepatic health [39]. The DIO group exhibited a higher HSI, indicating alterations in liver function associated with obesity in zebrafish. However, the supplementation with A. tinctoria root powder in the DIO + AT group notably lowered the HSI value to levels comparable to those of the control group, highlighting the liver‐protective properties of A. tinctoria root powder. The present findings are in agreement with those reported by Arika et al. [40], who observed a significant decrease in HSI among rats fed on a fat‐rich diet and administered a dichloromethane extract prepared from Gnidia glauca leaves. Similarly, Zhao et al. [41] observed a significant reduction in liver weight in the Zhuyu Pill‐treated groups compared with the high‐fat, high‐fructose (HFHF) diet group, further supporting our findings.

Elevated plasma free fatty acids (FFA) can promote lipid accumulation in hepatocytes [42]. The enzyme 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMGCR) plays a pivotal role in cholesterol biosynthesis and is also involved in the regulation of fatty acid metabolism [43]. Moreover, the expression of enzymes involved in lipogenesis, such as ACC and FAS is regulated by the transcription factor SREBP‐1 thereby promoting the fatty acid synthesis, triglycerides, and cholesterol in hepatocytes. Activation of these cellular pathways can lead to increased hepatic lipid accumulation through the influx of excess FFAs and enhanced de novo lipogenesis [43].

In the present study, the histopathological showed a clear accumulation of lipid droplets in the liver of the DIO zebrafish, indicating increased hepatic steatosis due to dietary‐induced obesity. In contrast, the DIO + AT group displayed a notable decrease in hepatic lipid accumulation, which highlights the protective effect of A. tinctoria root powder in reducing lipid deposition and maintaining hepatic lipid homeostasis. These results are consistent with those of Su et al. [44], who reported that administration of acetylshikonin (a phytochemical present in A. tinctoria root) reduced the expression of the lipogenic regulators SREBP‐1, FAS, and HMGCR in db/db mice, thereby attenuating hepatic lipid accumulation. Similar results were reported by Yang et al. [45], who observed that shikonin treatment markedly improved the liver histopathology of HFD‐induced obese rats. The treatment reduced hepatic changes such as disorganization of the hepatic lobules, fatty degeneration of hepatocytes, and excessive accumulation of lipid droplets. The reduced hepatic steatosis observed in the DIO + AT group implies that A. tinctoria root powder and its bioactive compounds, such as acetylshikonin and shikonin, may exert hepatoprotective and anti‐obesity effects by inhibiting crucial regulators involved in lipogenesis and cholesterol production.

Adipose tissue acts as an essential endocrine organ, as it is involved in storing excess energy and in regulating the energy balance through the release of various adipokines. As a result of overnutrition, particularly if the fat and carbohydrate components in the diet exceed the metabolic needs of the body, the primary function of adipose tissue is to store surplus energy as neutral lipids within the adipocytes [46, 47]. This causes the hypertrophy and hyperplasia of adipocytes, and subsequently results in their proliferation as well as the pathophysiology associated with obesity [26, 47, 48]. This is further associated with the enlargement of the adipose cell volume resulting from lipid droplet accumulation within the adipose cells [46].

Peroxisome proliferator‐activated receptor gamma (PPAR‐γ) is considered a principal transcription factor regulating the early steps of the adipogenesis process and, consequently, its development. Activation of PPAR‐γ induces the expression of genes involved in the differentiation of preadipocytes into white adipocytes and promotes lipid accumulation, thereby contributing to adipocyte maturation [49]. In the present study, the DIO group exhibited significant increases in the adipocyte size and number, suggesting enhanced adipogenesis along with increased lipid accumulation as a consequence of dietary‐induced obesity. In contrast, treatment with A. tinctoria root powder significantly attenuated these changes, resulting in restoration of adipocyte morphology and suggesting a potential inhibitory effect of A. tinctoria root powder on adipocyte hypertrophy. These results align with the findings of Arampatzis et al. [50] who reported that alkannin–shikonin derivatives reduced the expression of PPAR‐γ in 3T3‐L1 preadipocyte cells. Likewise, Su et al. [44] demonstrated that acetylshikonin reduced adipocyte size and lipid accumulation in HFD‐induced obese rats by suppressing the adipogenic transcription factors PPAR‐γ and CCAAT/enhancer‐binding protein alpha (C/EBPα) while promoting lipolysis through increased phosphorylation of protein kinase A (PKA) and hormone‐sensitive lipase (HSL). These findings suggest that the anti‐adipogenic effects of A. tinctoria root powder may be mediated by its phytoconstituents, particularly acetylshikonin, which may help limit adipocyte differentiation and promote the mobilization of stored lipids.

Polycystic ovary syndrome (PCOS) is an endocrine‐reproductive disorder prevalent among women. It is influenced by metabolic factors and lifestyle‐related conditions such as obesity, insulin resistance, and a sedentary lifestyle [51]. Women diagnosed with PCOS have been shown to have enlarged ovaries along with follicular development disorders, and PCOS‐like reproductive abnormalities can also be effectively modeled in zebrafish. Similar to women, PCOS zebrafish have been shown to have enlarged ovaries along with cellular and molecular abnormalities [52].

Moreover, in the present study, the DIO zebrafish displayed a significant increase in GSI (%) and ovarian cystic alterations, which were characterized by disrupted follicular membrane, hypertrophy of the theca layer, and invagination of the zona pellucida. These pathological alterations show similarities to ovarian abnormalities associated with PCOS. Nevertheless, treatment with A. tinctoria root powder markedly reduced GSI (%) and attenuated the observed ovarian histopathological lesions, resulting in a morphology more comparable to that of the control group. These findings indicate that A. tinctoria may have potential in mitigating obesity‐associated ovarian dysfunction and PCOS‐like reproductive abnormalities, possibly through its effects on ovarian development and lipid metabolism. Within the DIO group, the majority of follicles remained arrested at the previtellogenic stage, demonstrating a significant decrease in early‐ and mature‐vitellogenic oocytes. On the contrary, A. tinctoria dietary supplementation stimulated oocyte maturation and promoted a higher prevalence of early‐ and mature‐vitellogenic oocytes, signifying an improved follicular development and reproduction.

These results align with the studies reported by Ramamurthy et al. [53], which demonstrated that an isatin‐linked pyrazole K1 compound significantly reduced GSI (%) and increased the proportions of early‐ and mature‐vitellogenic stages of oocytes in triclosan‐induced PCOS model of zebrafish. Reproductive abnormalities have been associated with dysregulation of the janus kinase/signal transducer and activator of transcription (JAK/STAT) and phosphoinositide 3‐kinase/protein kinase B (PI3K/AKT) signaling pathways, which play important role in maintaining hormonal homeostasis and regulating ovarian and oocyte development [54, 55]. The improvements observed in the A. tinctoria treated group suggest that its phytoconstituents may have may have contributed to the restoration of ovarian homeostasis and reproductive function, potentially through modulation of these signaling pathways.

The use of FTIR spectroscopy provides valuable insights into biomolecular changes occurring in biological samples. The present investigation assessed the restorative potential of A. tinctoria root powder in DIO zebrafish using FTIR analysis of serum and liver samples. In the FTIR spectra of serum samples from the DIO group, noticeable shifts in the intensity of peaks corresponding to 1053 cm⁻¹, 1163 cm⁻¹, 1224 cm⁻¹, and 2852 cm⁻¹ were observed compared with those in the control and DIO + AT groups. Similarly, in the FTIR spectra of liver samples from the DIO group, significant shits in the intensity of peaks at 960 cm⁻¹, 1639 cm⁻¹, 1740 cm⁻¹, and 2853 cm⁻¹ were observed relative to the control and DIO + AT groups. These observations are consistent with the findings of Guleken et al. [56], who reported significant peak shifts in the serum FTIR spectra of obese patients. Their results showed that the most prominent structural change occurred in the vibrational regions corresponding to phospholipids, lipids, and amide I, reflecting obesity‐associated biochemical changes. Likewise, the present study demonstrated comparable changes in the FTIR spectral profiles, supporting the notion that obesity induces distinct molecular changes in lipid and protein structures, whereas supplementation with A. tinctoria root powder attenuated these changes. These findings indicate that A. tinctoria root powder supplementation reduced obesity‐associated changes in lipid‐, protein‐, and phosphate‐containing biomolecules in the serum and liver of zebrafish.

A limitation of the present study is the lack of a sex‐specific assessment of the anti‐obesity effect of A. tinctoria root powder. Although equal numbers of male and female zebrafish were included in each experimental group, the data were analyzed collectively rather than separately according to sex. Thus, the present study cannot determine whether sex contributes to differences in the magnitude of the observed anti‐obesity effect of A. tinctoria root powder. Future investigations employing sex‐stratified experimental designs and appropriate statistical analyses are warranted to clarify the potential role of sex in modulating the anti‐obesity response to A. tinctoria root powder.

The present study showed that A. tinctoria was well tolerated during the experimental period, with no treatment‐related mortality, behavioral abnormalities, or visible morphological alterations. However, further assessment of its safety is needed before considering its therapeutic application. In this regard, Aswinanand et al. [57] evaluated the safety of a thiazol‐sulfonyl derivative using both in vitro HepG2 cells and in vivo zebrafish models. Similarly, Priyanka and Namasivayam [58] assessed the hepatocompatibility of phycocyanin obtained from Spirulina maxima in zebrafish through biochemical, oxidative stress, and histopathological analyses. These studies highlight the need to assess both therapeutic efficacy and safety when evaluating potential herbal products. Therefore, future studies on A. tinctoria should include comprehensive toxicological assessments, such as standardized cytotoxicity assays, OECD‐guided acute and subchronic toxicity studies, as well as histopathological, hematological, and biochemical analyses to provide a clearer understanding of its long‐term safety.

5. Conclusions

The present study showed that supplementation of A. tinctoria root powder led to a notable decrease in body weight gain, change in BMI, HSI, GSI, and improved hepatic steatosis, as well as prevented the alterations in adipose and ovarian tissue in the DIO + AT zebrafish. The ATR‐FTIR spectral analysis of serum and liver tissues demonstrated that the biochemical perturbations caused by obesity in the lipids, proteins, and phosphates spectral regions were attenuated by the supplementation of A. tinctoria root powder. These findings underscore the potential of A. tinctoria root powder as a potential plant‐based natural therapeutic agent for obesity and its associated metabolic and reproductive complications. Nevertheless, the underlying molecular mechanistic pathways were not investigated in the current investigation. Future studies should aim to elucidate the molecular pathways involved, validate these findings in mammalian models and perform comprehensive toxicological evaluations to further establish the long‐term safety profile of A. tinctoria and support its clinical translation.

Author Contributions

Samriti Sharma: conceptualization, methodology, investigation, formal analysis, data curation, writing – original draft, visualization, validation. Deepika Choudhary: methodology, investigation, formal analysis, data curation. Anupam Kaur: conceptualization, supervision. Pooja Chadha: conceptualization, visualization, validation, supervision, writing – review and editing.

Funding

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

Data will be made available on request.