Cookies enriched with anethole and secoisolariciresinol diglucoside from flaxseed and fennel seeds improve hypercholesterolemia, lipid profile, and liver functions: A pilot study

Abstract Over millennia, flaxseeds and fennel seeds have captured greater attention owing to the broad spectrum of bioactive compounds and their respective therapeutic potential. They are well‐known therapeutic plants, frequently used in home treatments for a variety of medical conditions. The novelty of this pilot study is to assess the beneficial health effects of secoisolariciresinol diglucoside (SDG) and anethole‐based enriched cookies among hyperlipidemic patients. The result of sensory evaluation revealed that cookies with anethole and SDG (500 + 500 mg/day) were significantly acceptable in terms of color, texture, taste, and overall acceptability same as that of control. This study was performed among 34 patients with hyperlipidemia in a university‐affiliated hospital, Lahore, Pakistan. In this study, patients received dietary supplementation with anethole and SDG (500 + 500 mg/day) administered in cookies for 8 weeks. Patients were assigned into two groups, intervention (receiving anethole + SDG‐enriched cookies; n = 16) and placebo (n = 18), for 8 weeks. Both groups maintained the same diet and lifestyle. Pre‐ and postintervention weight, lipid profile, and liver enzyme levels were measured. Analysis of covariance and paired sample t‐test were used for comparing the two groups. After 8 weeks, a significant mean weight loss was observed in the intervention group (4.26%) as compared to the placebo group (0.3%). A significant reduction of TC (177.02 ± 5.14 mg/dL; p = .024), TG (150.19 ± 7.94 mg/dL; p = .032), and LDL (87.38 ± 3.58 mg/dL; p = .001) were compared to the control group and HDL level (57.09 ± 3.90 mg/dL; p = .035) were increased in the intervention group as compared to the placebo. Meanwhile, it had a minor improvement in AST (30.97 ± 2.95 U/L; p = .01), ALT (33.05 ± 1.52 U/L; p = .025), and ALP (112.15 ± 4.03 U/L; p = .03) among the intervention group. Thus, based on the results from the study, it can be said that anethole + SDG‐enriched bakery products could be developed as a functional dietary option for hyperlipidemia in developing countries like Pakistan.

One of the first domesticated crops flaxseed (Linum usitatissimum) was used to make flour for bread as early as 1000 BC (Parikh et al., 2019). Due to its potential health advantages, notably for cardiovascular protection, flaxseeds are currently being more employed in human diets. The main lignan ingredient in flaxseeds is secoisolariciresinol diglucoside (SDG), making it the richest natural source of plant lignans (Kezimana et al., 2018). The phytoestrogens (flax seed lignans and soy isoflavones) are often found in the human diet. A number of studies employing dietary flaxseeds have been carried out to date, and the results showed that SDG lignan may reduce plasma cholesterol concentrations (Noreen, Rehman, et al., 2023;Nwozo et al., 2023;Zhuang et al., 2021). The purpose of this study was to evaluate the effectiveness of SDG + anethole-enriched cookies for hyperlipidemic patients having high range of total cholesterol (>220 mg/dL), total triglyceride (>150 mg/dL), and low-density lipoprotein (>130 mg/dL).

| Materials
Total cholesterol (TC), low-density lipoprotein (LDL), very lowdensity lipoprotein (VLDL), high-density lipoprotein (HDL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were estimated using standard kits (Merck Specialties Pvt. Ltd.). Plastic bags were obtained from Reynolds company. SDG and anethole were obtained from flaxseeds and fennel seeds bought from the local market of Lahore, respectively, and kept at the Food Science and Technology Lab (Lab. No. 101) of the University Institute of Diet and Nutritional Sciences (UIDNS), University of Lahore, Pakistan. Wheat flour, eggs, butter, baking soda, brown sugar, skimmed dry milk, and salt were obtained from local market of Lahore, Pakistan.

| Settings
The study was conducted at the following settings: Food Science

| Preparation of extracts
Prior to sample preparation, all glassware was silanized in a 5% dimethyldichlorosilane in heptane solution, followed by deactivation in methanol. Flaxseeds were ground in a coffee grinder and either used for analysis or further defatted by Soxhlet extraction in nhexane for 8 h (ISO 659:1998) and oven-dried to a consistent mass.
Magnetic stirrer was used for mixing of milled flaxseeds in n-hexane (1:5 w/v) at room temperature for 1 h to extract the oil, according to prior studies that utilized stirring with n-hexane. After extraction, gas chromatography mass spectrophotometer (GC-MS) was used for quantification (Hanaa et al., 2017).

| Preparation of cookies
Cookies were made using the straight dough technique by Riaz et al. (2020). In a Hobart Mixer (Model N-50; Hobart Corp.), the composite flours were mixed with other baking ingredients for 3 min as mentioned by Table 1, then eggs and water were added, and the process was continued until foaming occurred (around 5 min at a speed setting of 2). Before adding the flour and baking powder, the components were blended to make a homogenous mixture. The cookies were baked for 10-15 min at 175°C in a preheated baking oven using a coated aluminum pan (Kenmore Co.). The daily cookie serving (1 cookie equaling 25 g) was placed in separate plastic bags after chilling for 60 min.

| Physical analysis of cookies
The width, thickness, and spread factor for cookies were determined every fortnight up to 45 days according to the method described by American Association of Cereal Chemists. Approved Methods Committee (2000).

K E Y W O R D S
fennel seeds, flaxseed, functional cookies, hyperlipidemia

| Sensory evaluation
The sensory evaluation of the cookies was conducted using 10 trained panelists. Cookies were coded with two-digit numbers and positioned randomly. Cookies were distributed at random and coded with two-digit numbers. The sensory assessment sheet was given to the panelists who used a 9-point hedonic scale (9 = strongly liked; 1 = severely hated) as described previously (Meilgaard et al., 1999) to grade the color, appearance, flavor, texture, general acceptability, and affordability in accordance with their preferences. The panelists rinsed their taste receptors with mineral water and expectorant cups during the sensory examination (Adeboye et al., 2016;Sidel & Stone, 1993;Amagwula et al., 2022aAmagwula et al., , 2022b).

| Subjects
In this pilot trial, 45 patients signed up and seven people were eliminated because they did not meet the inclusion criteria ( Figure 1).
There were 38 patients in all who were randomized between the two groups. Of the 38 patients, three were removed from the intervention group due to travel, constipation, or the beginning of another medicine, while one patient left the placebo group to begin another medication. Sixteen participants in the therapeutic group and 18 participants in the placebo group eventually finished the trial and were evaluated after 8 months as mentioned in Figure 1. The patients took their existing medication as directed.
Participants who were diabetic, had serious illnesses or surgeries within the previous 3 months, or had secondary reasons for hyperlipidemia were not allowed to participate. Patients with gastrointestinal issues or documented sensitivities to fennel and flaxseed were also disqualified. All of the chosen participants were questioned in order to explain the experiment's purpose and to get their signed informed consent. The study comprised male and female participants aged 45-55 years. Patients having high range of total cholesterol (>220 mg/dL), total triglyceride (>150 mg/dL), and low-density lipoprotein (>130 mg/dL) were included in this study. At the beginning of the trial, within the first week, and at the end of the experiment, after 8 weeks, blood was obtained to determine the baseline values for plasma lipids (Riaz et al., 2020). All subjects from the placebo and interventional groups consumed 25 g of cookies daily which contained the combination of SDG and anethole (500 + 500 mg) for the period of 8 weeks as shown in Table 2. Methods used were authorized by the Research Ethical Committee of the University of Lahore (IRB-UOL-FASH/826/2021).

| Plasma lipids
Blood samples were collected for baseline and follow-up testing in ethylenediaminetetraacetic acid (EDTA)-treated tubes to assess the following hematological parameters: an enzymatic colorimetric method was used to quantify TC, triglyceride (TG), and HDL levels (Burstein et al., 1970). The LDL-c concentration was measured using the formula of Friedewald et al. (1972). To calculate VLDL-c, the following formula was used:

| Statistical analysis
The data were statistically processed by SPSS version 25. The mean ± SD values of numerical data like age, lipid profile, liver enzymes were presented. The quantitative variables between the two groups were compared using t test after fulfilling the parametric assumptions (Steel & Torrie, 2012).

| Physical parameters of cookies
There is no difference in width among control and fortified cookies as same standard fat was used in both groups (Table 3). Highest mean value for cookie width was observed in C 1 (51.30 mm) as compared to C 0 (51.12 mm). The mean values for cookies thickness revealed a significant difference between enriched and control cookies. The highest thickness value (7.14 mm) was noted in control cookies as compared to cookies enriched with anethole + SDG. Enriched cookies got minimum thickness (6.69 mm). Anethole and SDG caused the cookies to dry more quickly than previously thought because of the greater content of linolenic acid, which resulted in a reduction in cookie thickness (Masih et al., 2014). The flour's particle size index, moistness, and the type of fat used are the three main components that determine the spread factor of cookies while baking (Man et al., 2021). Spread ratio, which in turn is impacted by the relative water availability of various components and the capacity of wheat or any other component to absorb moisture during dough production, also has an impact on cookie diameter and thickness (Zhang et al., 2022).

| Sensory attributes of wheat flour and enriched cookies (anethole + SDG extract)
As indicated in Table 4, the sensory analysis of the fortified cookies revealed significant (p < .05) differences between control and fortified cookies in terms of texture, mouth feel, flavor, color, and general acceptance. A different pattern, reported by Sukhcharn (2008), emerged from the sensory assessment findings of the study. These varying score patterns might result from the panelists' varying rates of preference and acceptable values and the caliber of the generated completed cookies. One of the essential aspects of product quality is texture, which is the sensory expression of a cookie's structural makeup and how it responds to an applied force (Sruthi et al., 2021).
Texture analysis measures the characteristics that affect how a cookie feels in the mouth (initial bite). The texture of the cookies in the control treatment (C 0 ), which received the highest texture score, and those that included anethole + SDG extract (C 1 ) significantly differed (p < .05), similar result was found by Khouryieh and Aramouni (2012).
Taste is the key determinant of any product's acceptance and has the greatest influence on the product's commercial success (Mahmud et al., 2020). were used in their production, but also tells you how they were made (Marand et al., 2020). The average quality score for the cookies' color is shown in Table 2. The data clearly show that cookies made from C 0 scored lower, whereas cookies made from C 1 with SDG and anethole scored higher. When exposed to sensory examination, judges approved the color of the cookies made with SDG and anethole. The level of SDG and anethole supplementation in the wheat flour was raised, which may have contributed to the dark brown color of the F I G U R E 1 Consort form.

Samples
cookies, but it was okay. This resulted in a darker shade of brown in the cookies, but it was still acceptable. The mean scores were trending downward as the cookies' color shifted from light brown to dark brown. A possible cause of the deeper hue is the Maillard reaction between decreasing sugars and protein (Jiamjariyatam et al., 2022).
A significant factor in the organoleptic estimate is the overall acceptability, which has many ramifications. According to similar findings published by Rathi and Mogra (2012), flaxseed-based cookies were satisfactory up to a 70% supplementation level based on sensory qualities.  Note: The values are means of 18 replicates ± SD in placebo group and 16 replicates ± SD in interventional group. Significant value ≤.05.

| Clinical trial
TA B L E 5 (a) Anthropometric measurement and (b) liver enzymes of hyperlipidemic patients fed control and anethole + SDG-enriched cookies at baseline and after 8 weeks.
by Yilmaz and Altuntaş (2020), who reported that hepatoprotective effects could be found in herbal remedies and the essential oils of fennel and flaxseed. In the current investigation, it was shown that individuals receiving conventional treatment saw no improvement in their liver enzymes; rather, there is marginal degradation in liver enzymes, but they are negligible (Yilmaz & Altuntaş, 2020). Those who are taking fortified cookies with anethole and SDG showed improvement in liver enzymes. Similar study by Elghazaly et al. (2019) showed that herbal treatment improved liver enzymes among hyperlipidemic rats.

| Plasma lipid
The study's findings demonstrated that all patients who took part in the study had changes in their plasma levels of triglycerides, LDL and HDL cholesterol, and total cholesterol between the study's baseline and after 8 weeks of dietary therapy. The interventional group considerably reduced plasma total cholesterol (177.02 ± 5.14 mg/dL; p = .024), TG (150.19 ± 7.94 mg/dL; p = .032), and LDL (87.38 ± 3.58 mg/dL; p = .001) than the control group, as indicated in Table 6. As compared to the control group, significant improvement in HDL (57.09 ± 3.90 mg/dL; p = .035) was observed in interventional group. The results of this study are consistent with the findings from the previous study by Naik et al. (2018). There was no evidence of a beneficial impact of flaxseeds on lipid profiles in the study by Wu et al. (2010) on the relationship between lifestyle choices and flaxseed supplementation and metabolic syndrome.
Due to higher exogenous cholesterol absorption and subsequent buildup, hyperlipidemic rats' plasma and hepatic cholesterol levels rose in addition to decreased cholesterol catabolism, as demonstrated by a reduction in bile acid formation and turnover of bile acids (Barakat & Mahmoud, 2011). In this current investigation, consumption of cookies enriched with anethole + SDG restored the lipid profile to normal ranges. Studies have suggested similar effects (Noreen, Rehman, et al., 2023;). An earlier which study found that rats on a high-cholesterol diet with flaxseed oil supplementation had their blood lipid profiles improved was used to support this conclusion (Adolphe et al., 2010;Farook et al., 1991).
They explained that ALA, which encourages cholesterol release into bile, depletes the intrahepatic pool of cholesterol and, as a result, increases cholesterol synthesis and metabolism. Furthermore, an ALA-rich diet helps in reduction of hepatic lipid deposition both by stimulating β oxidation and by inhibiting fatty acid synthesis (Hussein et al., 2014). An earlier study by Zhang et al. (2022) also supported this study analyses as it showed significant reduction in TC (22%) and LDL-c (24%) among hypercholesterolemic patients after 8 weeks of consuming 600 mg/day SDG derived from flaxseeds (Torkan et al., 2015). The same outcomes were obtained in a second research in which volunteers received a single 500 mg dosage of trans-anethole for 7 weeks (12 mg/kg body weight) in order to manage hyperlipidemia in patients (Mehra et al., 2021). Therefore, the combination of anethole + SDG is proposed as a new approach for the therapeutic therapy of patients with hyperlipidemia.

| CON CLUS ION
Anethole and SDG, active compounds of fennel seeds and flaxseeds, are extremely beneficial for positive health and help sustaining essential physiological functioning of the human body, and same has been validated by this study effort. Our study endorsed this fact by examining the lipid profiles of hyperlipidemic patients over the course of 8 weeks while they were fed anethole and SDG extractbased cookies, which were successful in reducing TC, TG, and LDL concentrations in the interventional group. Due to this assurance, the ready-to-serve anethole + SDG-enriched cookies, which are better referred to as "functional cookies," may be made available to satisfy customer demand for a nutritious snack. In future, more studies can be performed on anethole + SDG based products for human welfare. Note: The values are means of 18 replicates ± SD in placebo group and 16 replicates ± SD in interventional group. Significant value ≤.05.

TA B L E 6
Effect of anethole + SDGenriched cookies among hyperlipidemic patients.

ACK N OWLED G M ENTS
The authors are thankful to the University of Lahore, Lahore and Kampala International University, Kampala, for providing the facilities used for this study.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used for this study are available on request through the corresponding author, though all the relevant data have been provided here.

E TH I C A L A PPROVA L
The methods used were authorized by the Research Ethical Committee of the University of Lahore (IRB-UOL-FASH/826/2021).