Functional foods provide health benefit beyond basic nutrition. Functional foods fortified with plant ingredients are well known. Ayurveda (Indian System of Medicine) has found several ways in which the medicinal benefits of herbs can be conveyed via certain foods as carriers. Milk is one such carrier which has been effectively used to deliver phytochemicals for targeted health benefits. Indian Kudzu or
The use of botanicals in foodstuffs is well established. It includes use as vegetables, fruits, herbs, and botanical food supplements. While medicinal products are intended to prevent or treat a disease or modify the way in which the body functions, food supplements and nutraceuticals are intended to complement the diet with substances possessing health-maintenance or -promoting properties [
Indian Kudzu or
Recently, we have investigated the
Since the health benefits of the nutraceuticals or functional foods containing different botanicals are due to the presence of the phytoconstituents of the added botanicals, it is important to have a biological marker and also to be able to associate that biological marker with the quality of life. Puerarin is the major constituent of
Several methods have been reported for the identification and analysis of puerarin [
Puerarin was obtained from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade methanol and water were obtained from Merck (Darmstadt, Germany). Whatman (Florham Park, NJ) No. 1 filter paper was used for filtration of the samples. Other chemicals and solvents were purchased from Merck Chemicals, Mumbai, India.
Tubers of
The coarse air-dried, (40°–50°C), powdered tubers (500 g each) of
Different milk combinations with
Fortified milk samples and milk controls were left at room temperature for 24 hr, followed by storage at 2°C to 8°C for 15 days. Stability profile was checked daily for precipitation profile and pH by bringing the samples to room temperature. Aliquots of the fortified milk samples and milk controls were observed during this period and sampled after two weeks from each of the three batches, for subsequent analyses. After sampling, the pH values of all fortified milk sample and milk controls were measured. Following the overall stability check, the analytical profiles of the 1st day and 15th day samples were compared.
Fortified milk samples and milk controls were kept in freezer at lower temperature (−10°C) for 24 hours, frozen samples then freeze dried by FREEZONE 4.5 lyophilizer, and dried powder weighed to predict the yield from wet mass. Dry lyophilized powder was defatted three times with hexane (1 : 5 w/v) and finally extracted with methanol (1 : 3 w/v) by warming on water bath. Isolated fractions were then dried under reduced pressure and temperature. The dried ethanolic and hot water extracts (EE and HWE) of
Analyses were performed on a liquid chromatography system (Waters, Milford, MA, USA) with 515 pumps and equipped with an online degasser, a Waters Pump Control Module (PCM), an autosampler 717, a Waters 2996 photodiode array detector (PDA), and Waters Empower software. Separation was carried out using a Supelcosil LC-8-DB column (250 × 4.6 mm i.d.; 5
Validation studies were performed for determining linearity, limit of detection, limit of quantification, repeatability, and percentage recovery. Five concentration points were used to prepare the calibration curve. The calibration plot was prepared by plotting peak area against the amount of puerarin and the regression coefficient (
Stability profile with pH was checked daily for precipitation profile and pH by bringing the samples to room temperature. Aliquots of the fortified milk samples and milk controls were observed during this period. No precipitate formation was observed in the samples. No significant change in the pH was observed. The observations are tabulated in Table
Stability profile of milk samples.
Milk samples | Day 1 sample | Day 5 sample | Day 10 sample | Day 15 sample | ||||
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Precipitate | pH | Precipitate | pH | Precipitate | pH | Precipitate | pH | |
Milk + 0.5% EE (w/v) | Absent | 6.50 | Absent | 6.53 | Absent | 6.55 | Absent | 6.58 |
Milk + 1.0% EE (w/v) | Absent | 6.46 | Absent | 6.59 | Absent | 6.60 | Absent | 6.69 |
Milk + 0.4% HWE (w/v) | Absent | 6.42 | Absent | 6.50 | Absent | 6.59 | Absent | 6.63 |
Milk + 0.7% HWE (w/v) | Absent | 6.43 | Absent | 6.45 | Absent | 6.48 | Absent | 6.51 |
Milk + 1.0% HWE (w/v) | Absent | 6.42 | Absent | 6.50 | Absent | 6.55 | Absent | 6.59 |
Milk + 0.01% puerarin (w/v) | Absent | 6.52 | Absent | 6.60 | Absent | 6.64 | Absent | 6.68 |
Milk (control) | Absent | 6.61 | Absent | 6.65 | Absent | 6.68 | Absent | 6.71 |
EE: ethanolic extract of
HWE: hot water extract of
To determine the content of puerarin in fortified milk samples, the dried ethanolic and hot water extracts of
Puerarin content in Indian Kudzu fortified milk samples.
Milk sample | Day 1 sample | Day 15 sample |
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(mg/10 mL fortified milk) | (mg/10 mL fortified milk) | |
Milk + 0.5% EE (w/v) |
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Milk + 1.0% EE (w/v) |
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Milk + 0.4% HWE (w/v) |
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Milk + 0.7% HWE (w/v) |
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Milk + 1.0% HWE (w/v) |
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Milk + 0.01% puerarin (w/v) |
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EE: ethanolic extract of
HWE: hot water extract of
Calibration curve for standard Puerarin.
HPLC profiles of fortified milk samples (a) Puerarin standard; (b) methanolic extract of milk + 1.0% HWE of
UV spectrum for Puerarin.
Linearity, limit of detection, limit of quantification, repeatability, and percentage recovery were studied. The linear range for puerarin was 1.0–5.0
In the present investigation, different herb-milk combinations were prepared by homogenizing pasteurized toned milk with extracts (EE and HWE) of
Milk, being one of the most widely consumed foods in the world, is an ideal vehicle for fortification. However, it is necessary that the samples are stable and the concentration and nature of the herbs/extracts that have been added for fortification do not change on storage. It was observed that there was no precipitate formation and the pH also did not change during the study period. This indicated that the milk samples were physically stable under the experimental conditions mentioned above. Also, the biomarker used for chemical analysis was puerarin, which is the major constituent of Kudzu. HPLC analysis results showed that the puerarin content can directly be a measure of the amount of ethanolic or hot water extracts of
A modern lifestyle is fast paced and mostly hurried where most people battle with time poverty. As a result, it is often difficult to find the time and energy to eat correctly and supply your body with the correct type of nutrition. On top of that, an individual’s health and nutrition needs do also change throughout his or her life. It is for this reason that development of functional foods and nutraceuticals with special health-promoting benefits is the need of the day. Herbal extracts in all their forms possess arguably the greatest potential for innovative functional food products. There are many companies already capitalizing on growing consumer acceptance of food and beverages containing herbal extracts [
The authors do not have any conflict of interests in this paper.
The authors are grateful to the Director, CSIR-National Botanical Research Institute, Lucknow, for providing the research facilities to carry out this work. This work was supported by the National Agriculture Innovation Project (Component 4: Basic and Strategic Research Subproject Code C30029), Indian Council of Agricultural Research, New Delhi, India.