Radiation processing: An effective quality control tool for hygienization and extending shelf life of a herbal formulation, Amritamehari churnam

Radiation processing: An effective quality control tool for hygienization and extending shelf life of a herbal formulation, Amritamehari churnam

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Journal of Radiation Research and Applied Sciences journal homepage: http://www.elsevier.com/locate/jrras

Radiation processing: An effective quality control tool for hygienization and extending shelf life of a herbal formulation, Amritamehari churnam Q6 Q1

Suchandra Chatterjee a,*, Vivekanand Kumar a, Swati Khole b, Bhaskar Sanyal a, T.S. Murali c, Prasad S. Variyar a a

Food Technology Division, Bhabha Atomic Research Centre, Mumbai 400085, India Department of Zoology, University of Pune, Pune, India c Arya Vaidya Sala, Kottakal, Kerala 676503, India b

article info

abstract

Article history:

Amritamehari churnam (AC) is an antidiabetic polyherbal formulation constituting of four

Received 9 February 2015

herbal medicinal plants namely Phyllanthus emblica, Salacia reticulata, Tinospora cordifolia and

Received in revised form

Curcuma longa. The feasibility of using gamma irradiation at doses between 2.5 and 10 kGy

31 August 2015

to reduce microbial load and enhance shelf life of this formulation was investigated. The

Accepted 27 September 2015

irradiated and non-irradiated products were stored at room temperature (25e32  C and 50

Available online xxx

e85% R.H., 1.5 years). Acceptability of the irradiated product was assessed based on sensory, microbial, physical and chemical attributes as well as their antioxidant status. A dose

Keywords:

7.5 kGy was sufficient to maintain microbial quality within acceptable limit up to 18

Herbal formulation

months of storage. No significant differences in sensory properties were observed between

Radiation processing

the non-irradiated and irradiated sample. The applied dose did not cause any significant

Shelf life extension

qualitative and quantitative changes in the chemical constituents, antioxidant activities as

Quality control

well as physical properties when measured by EPR spectroscopy. Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.

Introduction

Herbs are prone to contamination by microorganisms and insect pests during processing and storage. This shortens their shelf life as well as in rare cases cause major illness particularly if the herbs are in combination with food poisoning organisms such as Salmonella and Staphylococcus aureus. Adoption of Good Manufacturing Practices (GMP) can reduce the contamination during processing and storage to a

great extent. Conventional decontamination methods using heat and chemicals can reduce contaminant substantially. However these methods also result in changes in flavor as a result of loss/degradation of labile volatile aroma components with a consequent reduction in the quality of the herb. Food irradiation is a relatively new environmental-friendly technology that can aid in enhancing food safety, quality and trade (Diehl, 1995). Available reports have clearly demonstrated that exposure of food commodities to ionizing radiation (gamma/ electron beam) is an effective method for disinfestation,

* Corresponding author. Tel.: þ91 22 25592531; fax: þ91 22 25505151. Q2 E-mail addresses: [email protected], [email protected] (S. Chatterjee), [email protected] (P.S. Variyar). Peer review under responsibility of The Egyptian Society of Radiation Sciences and Applications. http://dx.doi.org/10.1016/j.jrras.2015.09.007 1687-8507/Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Chatterjee, S., et al., Radiation processing: An effective quality control tool for hygienization and extending shelf life of a herbal formulation, Amritamehari churnam, Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.09.007

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decontamination, overcoming quarantine barriers in international trade and for improving nutritional attributes and shelf life (Hong et al., 2008). Due to their low moisture content irradiation of herbs at higher doses, up to 10 kGy, results in considerable improvement in the microbial quality. Effect of radiation processing on the chemical and microbial quality of herbs has been extensively investigated (Alothman, Bhat, & Karim, 2009). The quality parameters of herbs and spices do not change appreciably after radiation processing. An enhancement in aroma and flavoring properties as well as extractability of active principles has been noted in several cases. This results in positive impact on the quality of the final herbal preparation. Very few reports, however, exists on the effect of radiation processing on the quality of herbal formulations. Diabetes mellitus is a very common disease that has been implicated to be the main cause of death in human population throughout the world including India. Several antidiabetic drugs and herbal remedies are used for treatment of diabetes. One such promising antidiabetic herbal drug widely used in clinical practice in Ayurveda is herbal formulationAmritamehari churnam (AC). The product is a mixture of Phyllanthus emblica, Salacia reticulata, Tinospora cordifolia and Curcuma longa. The formulation is prone to microbial contamination on storage and thus has a low shelf life. Radiation processing as a method for extension of shelf life of this product was thus attempted.

2.2.

Testing was carried out by an experienced sensory panel (15 members) using a 9 point scale with 1, dislike extremely or not characteristic of the product and 9, like extremely or very characteristic of the product. Values less than 4 were considered to be unacceptable to the consumers. Parameters evaluated were color, aroma, texture and overall acceptability. The sensory evaluation was done at every three months of storage intervals.

2.3.

Materials and methods

2.1.

Materials

AC (4 Kg) samples (mesh size 40) procured from Kottakal Ayurvaidyashala, India, was divided into two sets (1 Kg each). One set was kept as non-irradiated sample. The other set of samples, packed in high density polyethylene bags of 0.12 mm thickness were irradiated in a gamma chamber with cobalt-60 source (GC 5000, BRIT, DAE, Mumbai) to doses of 2.5, 5, 7.5 and 10 kGy at the dose rate of 6.01 kGy/h. Gamma Chamber was calibrated using Fricke dosimeter. Irradiated and corresponding non-irradiated samples were stored under ambient conditions (25e32  C, 50e85% R.H.) for 2 years. Samples were taken at predetermined storage intervals (every three months) were analyzed in triplicate for the analysis of chemical constituents and antioxidant activities. For EPR studies, powdered samples were grouped into three lots. One served as control, second lot was irradiated at 2.50e10.0 kGy. The third lot was subjected to thermal treatment at 100  C for 1 h using a laboratory oven to study the induced radicals by thermolysis (Sanyal, Chawla, & Sharma, 2009). During the time kinetics study of the paramagnetic species for a period of 90 d, irradiated, thermally treated and non-irradiated samples were stored inside EPR quartz tube under normal laboratory conditions at ambient temperature (25e32  C). Three replicates of each sample were evaluated. All chemicals were purchased from SigmaeAldrich Chemicals, USA. Solvents (s.d Fine Chemicals Ltd., India) were redistilled before use.

Microbiological analysis

Standard methods were used to enumerate microorganisms present at each sampling time and treatment for 2 years of storage. The mesophilic bacteria, yeast and mold counts were carried out using Plate Count Agar and the pour plate method. The sample (5 g) was homogenized in 45 ml of sterile physiological saline. Appropriate dilutions were pour plated using plate count agar. The colonies were counted after 24 h of incubation at 37  C. Total yeast and mold count was performed by pour plate method using potato dextrose agar supplemented with 10% tartaric acid to maintain pH of media to 3.5. Plates were incubated at 27  C for 48 h. Microbial counts were expressed as cfu g1 of herbal products. Each analysis was performed in triplicate (Khurana, Sharma, & Bhaduria, 2011).

2.4.

2.

Sensory analysis8

Extraction

All samples (50 g each) were defatted with hexane and subsequently extracted with methanol (4  250 ml), 80% aq. methanol (4  250 ml) and finally with distilled water (4  200 ml) in an omnimixer. Three replicates were prepared. The respective extracts were dried in vacuum and made to 10% (w/v) solution of the respective solvents. These extracts were used for chemical analyses as well as antioxidant activity measurements.

2.5.

Thin layer chromatography (TLC) densitometry

TLC densitometry was carried out using toluene:ethyl formate:formic acid (5:4:1) as solvent system on silicagel G plates. Separated bands were visualized by spraying 50% sulfuric acid and heating at 110  C and identified using standards. Quantitative estimation was performed on a dual wavelength flying spot scanning densitometer, CS-9301PC, Shimadzu, (Kyoto, Japan). Density of the spots of interest was determined in the reflectance mode (529 nm) and concentrations of the samples were obtained from the standard curve (correlation coefficient 0.99) prepared using gallic acid.

2.6. High performance liquid chromatography (HPLC) analysis Identification and quantification of the major active constituents by HPLC analysis was carried out on a HPLC system (Jasco Corporation, Tokyo, Japan) equipped with a C-18 reverse phase stainless steel column (30 cm  0.46 cm) and a UV detector. Extracts were eluted with three different solvent systems. [1] Gallic acid and ellagic acid were separated using

Please cite this article in press as: Chatterjee, S., et al., Radiation processing: An effective quality control tool for hygienization and extending shelf life of a herbal formulation, Amritamehari churnam, Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.09.007

Q3

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Solvent A (1.5% H3PO4) and solvent B (CH3COOH/CH3CN/ H3PO4/H20 (20:24:1.5:54.5)) starting with 80% A, linearly decreasing to 33% A after 30 min, 10% after 33 min, and 0% after 39.3 min (269 nm) and estimated using standard curve prepared by gallic acid. [2] Solvent A (H20) and solvent B (CH3CN), with a gradient starting with 100% A, linearly decreasing to 0% A after 25 min, for separation of curcuminoids at 420 nm and using curcumin for preparing standard curve (correlation coefficient 0.99). [3] An isocratic elution using CH3CN:H20 (16:84) at 254 NM to estimate mangiferin using the same compound for the preparation of the standard curve (correlation coefficient 0.99).

2.7.

Estimation of total phenolics content (TPC)

Sample (40 ml, 1 mg/ml) was mixed with 200 ml Folin Ciocalteus reagent and 1160 ml of distilled water. After 3 min incubation 600 ml 20% sodium carbonate solution was added to the mixture and kept in dark for 2 h at room temperature and absorbance was measured at 756 nm. Gallic acid was used as a standard and the total phenolic content was expressed as mg of gallic acid equivalents (GAE) per mg of extract (Gao, Ohlander, Jeppsson, Bjork, & Trajkovski, 2000).

2.8.

DPPH radical scavenging assay

Test compound (25 ml, 1 mg/ml) was added to 1 ml of freshly prepared DPPH solution (0.025 gm/L in methanol). The absorbance was measured at 517 nm after 20 min. The calibration curve was plotted with %DPPH scavenged versus concentration of the standard antioxidant (Ascorbic acid). The results were expressed as Ascorbic acid Equivalent Antioxidant capacity (AEAC) (Aquino et al., 2001).

2.9.

Ferric reducing antioxidant power (FRAP) assay

The stock solutions of 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl, 20 mM FeCl3.6H2O and 0.3 M acetate buffer (pH 3.6) were prepared. A conglomerate of 2.5 ml TPTZ, 2.5 ml FeCl3.6H2O and 25 ml acetate buffer warmed at 37  C and 900 ml of FRAP reagent was mixed with 90 ml of distilled water and 30 ml of test compound/methanol/DW/standard solution. The reaction mixture was incubated for 30 min at 37  C and absorbance was recorded at 593 nm. A standard curve was prepared using different concentrations of trolox. The results were expressed in lM trolox per 100 g dry weight (Benzie & Strain, 1996).

2.10.

Hydroxyl (OH) radical scavenging assay

The reaction mixture contained 100 ml of 28 mM deoxyribose, 100 ml of 250 mM FeCl3, 100 ml of 28 mM H2O2, 100 ml of 1 EDTA, 100 ml of sample and 100 ml of ascorbic acid. The final volume was made up to 1 ml by using 20 mM KH2PO4eKOH buffer (pH 7.4). It was incubated at 37  C for 10 min. Then 1 ml of 2.8% TCA and 1 ml of TBA reagent were added. The mixture was boiled for 20 min and absorbance was read at 532 nm (Halliwell, Gutteridge, & Aruoma, 1987).

2.11.

3

Superoxide (SO) radical scavenging assay

This activity was measured by the reduction of nitro blue tetrazolium (NBT). The 1 ml reaction mixture contained phosphate buffer saline (1X PBS, pH 7.4), 0.98 M nonenzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS/NADH), NBT (0.62 M), PMS (0.33 M) and various concentrations of sample solution. After incubation for 5 min at ambient temperature at 540 nm absorbance was measured against an appropriate (Nagakawa & Yokozawa, 2002) blank.

2.12. Thiobarbituric acid reactive substances (TBARS) assay for lipid peroxidation activity For isolation of mitochondrial fraction three month old female Wistar rats were used. Rat liver was excised, homogenized in 0.25 M sucrose containing 1 mM EDTA and centrifuged at 3000  g for 10 min. The supernatant was centrifuged at 10,000  g for 10 min to sediment mitochondria, pellet was washed with 50 mM Phosphate buffer, pH 7.4, to remove sucrose. The pellet was finally suspended in 50 mM phosphate buffer and protein content was estimated. The incubation mixture (0.5 ml) contained 0.35 ml of buffering medium (1 mM KH2PO4 in 0.15 M TriseHCl buffer, pH 7.4), 50 ml of FeSO4 (final concentration 50 mM) in 0.1 N HCl, 50 ml of membrane fraction containing 0.25 mg protein and 50 ml of ascorbic acid to give a final concentration of 0.4 mM. The reaction was started by the addition of ascorbic acid. Incubation was carried out at 37  C in a shaker water bath. Then, TBA reagent containing 20% TCA, 0.5% TBA, 2.5 N HCl and 6 mM EDTA was cooled and centrifuged at 2000 rpm for 10 min and the precipitate obtained was removed. The absorbance of the supernatant was determined at 532 nm against a blank that contained all the reagents except the biological sample. Concentration of TBARS was then calculated with the help of standard graph using 1,1,3,3tetraethoxypropane, as malonaldehyde equivalents (Devasagayam, Pushpendran, & Eapen, 1983).

2.13.

EPR spectroscopy

The samples were transferred to 2-mm quartz capillary tubes and packed with gentle tapping to a length of 25.4 mm (active length) and the weight of the sample was determined in the range of 52 ± 4 mg. The results for the signal intensity of samples were normalized to the packing weight. EPR spectroscopy was carried out using a Bruker EMX spectrometer (Bruker, Gemany). All the spectra were recorded after 3 accumulated scans at ambient temperature (26 ± 2  C). Operating conditions of the EPR spectrometer were center field 336 mT, scan range 30 mT, microwave power 0.633 mW, microwave frequency 9.36 GHz, modulation frequency 100 kHz, receiver gain 4  104, and time constant 40.96 msec. The position of the radiationeinduced EPR signal was compared with that of the standard 2, 2, diphenyl-1-picrylhydrazyl (DPPH) with g ¼ 2.0032.

2.14.

Statistical analysis

Data presented for antioxidant activity assays are means of values obtained from three independent samples each

Please cite this article in press as: Chatterjee, S., et al., Radiation processing: An effective quality control tool for hygienization and extending shelf life of a herbal formulation, Amritamehari churnam, Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.09.007

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analyzed in triplicate. Thus a total of nine replications was carried out for each complete set of analysis and the values reported as means ± SD, n ¼ 9. One-way analysis of variance (ANOVA) followed by Tuckey's post test was used to assess significant differences (p < 0.05) between the samples. All the statistical analyses were accomplished using the computer software Graph Pad Prism 3.02 for Windows (Graph Pad Software, USA). Principal component analysis (PCA) was performed with the amount of gallic acid, ellagic acid, mangiferin, curcuminoids in mg/g in herbal drug as analyzed using HPLC. DPPH radical, hydroxyl radical, superoxide radical and TBARS as percent inhibition, FRAP as trolox equivalent (mg/g) and total phenolic content as gallic acid equivalent (mg/ g) were used as variables using XLSTAT 2011 software.

3.

Results and discussion

Sensory parameters such as appearance, color, texture, aroma and taste along with the microbiological status are very crucial attributes that affect the consumer acceptance of a produce. Sensory and microbial analyses were therefore carried out for optimizing the radiation dose with a view to improve the shelf life of AC.

3.1.

Microbiological analysis

Microbial status of irradiated and non-irradiated samples is shown in Table 1. The counts for non-irradiated samples remained acceptable up to six months of storage. Beyond this period the counts increased significantly above the acceptable limit (<5  105 cfu/g) prescribed for herbal medicines. Total bacterial and fungal load reduced with increasing irradiation dose. No bacterial load was detected in the radiation processed samples when exposed to 7.5 kGy and 10 kGy either immediately or up to 18 months of storage. After 18 months the microbial load increased beyond the acceptable limit. Yeast and mold counts also demonstrated a similar trend with a decline in population with increasing radiation dose (Table 1). At 7.5 and 10 kGy, a drastic reduction in yeast and mold count was observed immediately after irradiation. The counts were maintained low during the entire storage period in these samples as compared to control. Thus radiation treatment at 7.5 and 10 kGy was highly effective in reducing total aerobic,

yeast and mold counts in AC. The data obtained from microbiological analyses thus suggests that a lower dose of 7.5 kGy was sufficient to maintain microbial quality within acceptable limits and extend shelf life up to 18 months. Hence the effect of irradiation (7.5 kGy) on other parameters was investigated only up to a storage period of 18 months.

3.2.

Sensory analysis

Table 2 shows the effect of radiation processing on the sensory quality of AC. No significant changes in the sensory attributes like color, aroma, texture, taste and overall acceptability was noted by the panelists when exposed to various doses. The sensory attributes also remained unchanged up to a dose of 7.5 kGy during the entire storage period of 18 months. Recently Ramathilaga et al. (Ramathilaga & Murugesan, 2011) have shown that electron beam irradiation did not change sensory properties of chyavanaprash, an Ayurvedic poly herbal formulation. The data obtained from sensory analyses thus further supports the results of microbiological analyses on the acceptability of the herbal drug stored up to 18 months.

3.3.

Antioxidant activity

Radiation treatment has been reported to alter the antioxidant status of plant produce depending on the dose delivered, exposure time, and the raw material used (Alothman et al., 2009). Solvents used in the extraction process have also been shown to play a key role in influencing the effect of irradiation on scavenging activity and total phenolic content (Khattak, Simpson & Ihasnullah, 2008). Perez, Calderon, and Croci (2007) demonstrated that the g-radiation resulted in higher antioxidant activity in ethanol and water extract of rosemary leaf. A corresponding increase in total phenolic content in water extract was also noted. The antioxidant status is also dependent on whether the assay was carried out immediately after the radiation treatment or certain duration after treatment (Song et al., 2006). Therefore, evaluating the antioxidant status of the herbal formulation just after radiation processing and on subsequent storage assumes importance. No studies on the antioxidant capacities of AC have been explored so far. In the present study, we carried out various assays on antioxidant activities of AC extracts at every three month interval

Table 1 e Microbiological quality of radiation processed AC powder during long term storage. Storage time

0 month 3 months 6 months 9 months 12 months 15 months 18 months 21 months

Yeast & mould count (cfu/g)

Bacterial load (cfu/g) NI 4

4

2  10 ± 0.3  10 2.5  104 ± 0.1  104 3.4  105 ± 0.5  105 3.7  107 ± 0.4  107 >109 >109 >109 >109

I (7.5 kGy)

I (10 kGy)

NI

0 0 0 0 0 70 ± 6 2.6  103 ± 0.2  103 8.4  105 ± 0.2  105

0 0 0 0 0 50 ± 4 1.2  102 ± 11 2.8  104 ± 0.3  104

78 ± 7 200 ± 17 404 ± 32 960 ± 53 2  103 ± 0.1  103 1.4  104 ± 0.09  104 2.2  105 ± 0.04  105 8.8  105 ± 0.13  105

I (7.5 kGy) 0 0 0 0 12 33 52 79

±2 ±2 ±4 ±5

I (10 kGy) 0 0 0 0 0 16 ± 2 29 ± 3 42 ± 3

NI- Nonirradiated. I-Irradiated.

Please cite this article in press as: Chatterjee, S., et al., Radiation processing: An effective quality control tool for hygienization and extending shelf life of a herbal formulation, Amritamehari churnam, Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.09.007

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Table 2 e Sensory analysis of irradiated and non-irradiated AC powder. Sensory attributes

NI (initial, 0 months)

I (after 18 months storage) 2.5 kGy

Color Aroma Taste Texture Overall acceptance

7.72 7.58 5.42 7.55 7.81

± 1.63 ± 1.11 ± 0.95 ± 1.21 ± 1.59

7.74 7.50 5.39 7.72 7.86

during eighteen months of storage. As the radical system used for the antioxidant evaluation is known to influence the experimental result, various assays representing different radical systems were employed to determine radical scavenging capacities. As phenolic compounds have been reported to greatly influence antioxidant activities, total phenolic content (TPC) of AC was initially determined.

3.4.

Total phenolic content

TPC of various extracts obtained from non-irradiated and irradiated AC samples showed that 80% aq. methanol extract had the highest TPC followed by methanol, water and hexane extracts (data not shown). The individual herbal constituents of this formulation namely P. emblica, S. reticulata, T. cordifolia and C. longa are reported to possess high concentration of phenolic antioxidants such as gallic acid, ellagic acid, curcuminoids. These compounds along with other constituent phenolics may act by protecting lipid peroxidation and quenching free radicals formed during radiation processing. Velioglu, Mazza, Gao, and Oomah (1998) reported a significant correlation between TPC and antioxidant activity in selected fruits, vegetables and grains. An appreciable content of phenolic compounds in the 80% aq. methanol extract of AC powder thus suggests the formulation to possess potent antioxidant activity. As the 80% aq. methanol extract from the non-irradiated and irradiated AC samples had the highest TPC, this extract obtained from the various stored samples was used for monitoring TPC. Radiation processing was not found to have any significant impact on the TPC of samples even after storage up to duration of 18 months (Table 3). Our findings are in agreement with the recent reports on g-irradiated chyavanaprash (poly herbal Ayurvedic formula), fenugreek and turmeric (Chatterjee, Variyar, & Sharma, 2009) wherein it was demonstrated that radiation processing does not alter the TPC in these products.

± 1.65 ± 1.10 ± 1.01 ± 1.14 ± 1.61

3.5.

5 kGy 7.79 ± 1.34 7.67 ± 0.88 5.41 ± 0.98 7.64 ± 1.51 7.77 ± 1.64

7.5 kGy 7.84 7.68 5.40 7.67 7.82

± 1.15 ± 1.12 ± 0.97 ± 0.88 ± 1.23

10 kGy 7.60 7.50 5.37 7.58 7.70

± 1.41 ± 1.36 ± 0.91 ± 0.65 ± 1.55

DPPH radical scavenging activity

Hexane, methanol, 80% aq. methanol and aqueous extracts of the formulation were tested for DPPH radical scavenging activity. DPPH radical scavenging activities were highest in 80% aq. methanol extract followed by methanol, water and hexane extracts (data not shown). Hence only 80% aq. methanol extract was used for all other antioxidant activity assays and also for storage studies. In another report (Chanda & Dave, 2009) it has been reported that among the various solvents are used for extraction of bioactive compounds from the plants, the most commonly used solvent was methanol. Both non-irradiated and irradiated 80% aq. methanol samples showed concentration dependent scavenging activity (Fig. 1) with no significant changes (p < 0.05) as a result of radiation processing. No significant difference was also observed between the samples when stored for one and half years. A positive correlation between the TPC and the ability of the extracts to scavenge DPPH radical was observed in the present study. Similar results were also reported in several foodstuffs (Velioglu, Mazza & Oomah, 1998). Q4

3.6.

FRAP activity

The ferric reducing ability in terms of Ascorbic acid equivalent capacity (AEAC) of 80% aq. methanol extracts obtained from non-irradiated and irradiated samples during the entire

Table 3 e Total phenolic contents (mg gallic acid/g of extract) of 80% aq. methanol extract of non-irradiated and irradiated AC powder during storage. Storage time (months) 0 3 6 9 12 15 18

Non-irradiated 283.79 ± 0.82 283.21 ± 0.97 283.10 ± 0.84 N.A. N.A. N.A. N.A.

Irradiated 283.53 283.77 283.55 283.57 283.59 283.95 282.33

± 1.05 ± 0.61 ± 0.36 ± 74 ± 71 ± 0.89 ± 2.28

Fig. 1 e DPPH radical scavenging assay. Extract concentration used was 0.01% (A) and 0.1% (B). Activity expressed in terms of %DPPH radical scavenged. Data represented as mean ± S.D (n ¼ 9).

Please cite this article in press as: Chatterjee, S., et al., Radiation processing: An effective quality control tool for hygienization and extending shelf life of a herbal formulation, Amritamehari churnam, Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.09.007

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storage period is shown in Fig. 2. Appreciable reducing capacity was noted in both control and radiation processed samples and remained constant throughout the entire storage period for radiation processed samples.

3.7.

SO radical scavenging activity

Although superoxide anion is a weak oxidant, it generates the powerful and damaging hydroxyl radicals as well as singlet oxygen, both of which contribute to oxidative stress (Meyer & Isaksen, 1995). Percentage of inhibition of 80% aq. methanol extract in various stages of storage for non-irradiated and irradiated samples is shown in Fig. 3. No significant change (P < 0.05) was noted due to irradiation during the entire storage period.

3.8.

OH radical scavenging activity

The hydroxyl radical is one of the potent reactive oxygen species in the biological system. It reacts with polyunsaturated fatty acid moieties of cell membrane phospholipids and causes damage to cell (Halliwell & Gutteridge, 1981). This assay shows the ability of the extract to inhibit hydroxyl radical-mediated deoxyribose degradation in a Fe3þ-EDTA and H2O2 reaction mixture (Fig. 4). At 0.1% concentration the percent inhibition value of the herbal drug was 63%. Gamma irradiation did not bring about any changes in the scavenging activity. The Hydroxyl radical scavenging activity also remained unaltered in the products when stored up to 18 months.

3.9.

Lipid peroxidation activity

The ability of the extracts to prevent oxidative damage to membrane lipids was checked by measuring the lipid peroxides (in terms of TBARs). Oxidative damage to rat liver mitochondria was induced by ascorbate-Fe2þ system that

Fig. 2 e Ferric reducing antioxidant power (FRAP assay). Extract concentration used was 0.01% (A) and 0.1% (B). Activity expressed in terms of mM Ascorbic acid equivalent antioxidant capacity (AEAC). Data represented as mean ± S.D (n ¼ 9).

Fig. 3 e Superoxide radical scavenging assay. Extract concentration used was 0.01% (A) and 0.1% (B). Activity expressed in terms of % superoxide radicals scavenged. Data represented as mean ± S.D (n ¼ 9). generates OH-radical like species on incubation at physiological temperature of 37  C. As compared with oxidatively damaged mitochondria, the amount of lipid peroxides formed in the presence of the herbal formulation was significantly lowered. The n moles of lipid hydroperoxide formed was reduced effectively by 80% aq. methanol extract of AC. No significant change was observed between irradiated and nonirradiated samples up to a storage period of 18 months (Fig. 5). Though radiation treatments have been shown to either increase or decrease the antioxidant content of fresh plant produce (Alothman et al., 2009), there are also reports wherein irradiation treatment did not show any significant effect on antioxidant properties. Gamma irradiation doses up to 30 kGy did not induce any significant changes in flavonoids, tannins and phenolic contents in Artichoke (Koseki et al., 2002). In another study the antioxidant activity remained unaltered in

Fig. 4 e Deoxyribose degradation assay. Extract concentration used was 0.01% (A) and 0.1% (B). Activity expressed in terms of % inhibition of hydroxyl radical formation. Data represented as mean ± S.D (n ¼ 9).

Please cite this article in press as: Chatterjee, S., et al., Radiation processing: An effective quality control tool for hygienization and extending shelf life of a herbal formulation, Amritamehari churnam, Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.09.007

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0.15 0.20 0.22 0.19 0.16 0.25 0.23 8.19 ± 8.11 ± 8.01 ± 7.95 ± 8.10 ± 7.99 ± 8.04 ±

NI I NI I NI I

8.12 ± 0.22 7.92 ± 0.18 7.98 ± 0.24 8.02 ± 0.26 ± 0.15 ± 0.20 ± 0.22 ± 0.19 ± 0.16 ± 0.25 ± 0.23 0 3 6 9 12 15 18

9.81 9.83 9.79 9.80

I NI I NI I NI I NI I

± 1.47 10.12 ± 1.35 9.6 ± 0.17 9.71 ± 0.12 ± 1.51 9.98 ± 1.28 9.30 ± 0.27 9.38 ± 0.23 ± 1.39 10.09 ± 1.37 8.92 ± 0.18 8.91 ± 0.18 8.89 ± 0.14 ± 1.43 9.94 ± 1.41 10.03 ± 1.50 8.95 ± 0.13 9.92 ± 1.53 8.85 ± 0.06 9.89 ± 1.47 8.89 ± 0.12

2.78 2.77 2.80 2.72

± 0.17 ± 0.19 ± 0.22 ± 0.15

2.81 2.73 2.76 2.75 2.72 2.78 2.74

± 0.21 2.53 ± 0.11 2.46 ± 0.14 ± 0.18 2.45 ± 0.07 2.46 ± 0.09 ± 0.18 2.32 ± 0.13 2.37 ± 0.10 ± 0.16 2.21 ± 0.09 ± 0.19 2.21 ± 0.03 ± 0.20 2.18 ± 0.10 ± 0.19 2.16 ± 0.14

8.12 7.92 7.98 8.02

± 0.22 ± 0.18 ± 0.24 ± 0.26

8.19 8.11 8.01 7.95 8.10 7.99 8.04

NI

8.12 ± 7.92 ± 7.98 ± 8.02 ±

0.22 0.18 0.24 0.26

8.19 ± 8.11 ± 8.01 ± 7.95 ± 8.10 ± 7.99 ± 8.04 ±

HPLC Mangiferin

Densitometry HPLC

Ellagic acid

Densitometry HPLC

Gallic acid

Irradiation has been shown to bring about changes in level of phytochemicals that in some cases can bring about a desirable impact on the physicochemical properties of the herbal produce. A good number of reports are available on the effect of gamma irradiation on the phytochemicals (Alothman et al., 2009). Irradiation can influence the levels of phytochemicals depending on the dose applied, exposure time, and the sensitivity of the phytochemicals towards irradiation and the nature of the raw material used. Hence monitoring both qualitative and quantitative changes in bioactive phytochemicals after irradiation and storage is of importance. As selectivity of antioxidants extracted depends on the nature of solvents used, (Alothman et al., 2009), the samples were successively extracted with hexane, methanol, 80% aq. methanol and water. No significant differences (p < 0.05) were noted in the yield of extracts isolated from non-irradiated and radiation processed samples either immediately after irradiation or after 18 months of storage (data not shown). The chemical quality of the irradiated samples was also studied based on the quantification of the major chemical markers namely gallic acid, ellagic acid, three curcuminoids (viz., curcumin, demethoxy curcumin and bis-demethoxy curcumin) and mangiferin using TLC and HPLC amalyses. Extractability of all these constituents was found to be higher in 80% aq. methanol

Densitometry

Chemical analysis

Storage (months)

3.10.

HPLC

Three curcurminiods

C. longa after gamma irradiation at a dose of 10 kGy (Chatterjee, Desai, & Thomas, 1999). Murcia et al. (2004), in a study on the antioxidant properties of seven dessert spices, revealed that irradiation up to 10 kGy had no effect on the antioxidant properties of the spices. Diehl (1995) reported that in food matrices a mutual protection by the different constituents could result in a lower impact of radiation. This could possibly explain the unaltered antioxidant status of the radiation processed AC.

Densitometry

Fig. 5 e Lipid Peroxidation by TBARs in rat liver mitochondria. Extract concentration used was 0.05% (A) and 0.1% (B). Activity expressed in terms of n moles of TBARs formed/mg protein in rat liver mitochondria damaged by ascorbate-Fe2. Data represented as mean ± S.D (n ¼ 9).

Table 4 e Compounds (mg/gm) estimated by densitometry and HPLC.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

7

0.15 6.30 ± 0.11 6.31 ± 0.15 0.20 6.30 ± 0.11 6.35 ± 0.14 0.22 6.19 ± 0.11 6.22 ± 0.18 0.19 6.20 ± 0.08 0.16 6.19 ± 0.13 0.25 6.17 ± 0.14 0.23 6.22 ± 0.09

J o u r n a l o f R a d i a t i o n R e s e a r c h a n d A p p l i e d S c i e n c e s x x x ( 2 0 1 5 ) 1 e1 0

Please cite this article in press as: Chatterjee, S., et al., Radiation processing: An effective quality control tool for hygienization and extending shelf life of a herbal formulation, Amritamehari churnam, Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.09.007

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extract. Gallic acid and ellagic acid were separated and identified by comparing their retention times with that of authentic standards injected under identical conditions using solvent system 1. Similarly solvent system 2 was used for separation and estimation of curcuminoids where as solvent system 3 was used for estimation of mangiferin as described in Material and Methods section. Quantitative data of these major chemical markers as monitored by TLC densitometry and HPLC was shown in Table 4.

3.11.

Principal component analysis (PCA)

Quantitative data obtained from HPLC and antioxidant assays was analyzed using PCA to identify qualitative and quantitative changes in major chemical constituents and antioxidant activity of herbal drug due to radiation processing and subsequent storage for a period of 180 days. First two principal components explained 31.38% and 16.79% of data variance (F1 and F2, respectively). Loading and score plots are depicted in Figs. 6 and 7 respectively. The score distribution from first two PCs (Fig. 6) demonstrated two separate groups in samples analyzed. First group had both control and irradiated samples in initial storage period (up to 6 months) and was located on positive side of F1. Samples both control and irradiated stored beyond a period of six months constituted second group located on negative side of F1. Interestingly, no segregation was obtained between control and irradiated samples. From loading plot (Fig. 7) it can be concluded that major chemical constituents like gallic and ellagic acid, mangiferin, curcuminoids along with total phenolics content, OH and superoxide SO radical scavenging activity correlated with the first group while FRAP activity was positively correlated to second group. It can be concluded that during storage there was a decrease in the content of phenolic acids, mangiferin, curcuminoids, OH and SO radical scavenging activity. However, an increase in FRAP activity was observed during storage. No effect on TBARS and DPPH activity was observed with radiation treatment or storage time. Although a decrease in phenolic compounds, curcuminoids, mangiferin, OH and SO radical scavenging activities

Fig. 6 e PCA Loading plots for Amritamehari churnam powder samples during storage.

Fig. 7 e PCA Score plots depicting the effect of radiation on Amritmehari Churnam during storage, blue color applies to non-irradiated whereas red color applies to irradiated samples.

was observed during storage, but analysis of actual values revealed that decrease was less than 10 and 5% for chemical constituents and antioxidant activities respectively. These results suggest radiation treatment of herbal drug for shelf life extension and hygieneization to be a practical proposition.

3.12.

EPR spectroscopy

In continuation with biological and chemical approaches physical technique of EPR spectroscopy was employed to study the radiation induced changes in the matrix of the AC before and after radiation treatments. Fig. 8 exhibits the superimposed EPR spectra of non-irradiated, irradiated (7.5 kGy) and thermally treated samples. The EPR spectra, recorded using a magnetic field sweep width of 30 mT, clearly demonstrated a broad singlet line with unresolved hyperfine splitting. The EPR spectrum of non-irradiated sample was characterized by a singlet with g ¼ 2.0050 ± 0.0002 and

Fig. 8 e EPR spectra of non-irradiated, thermally treated and irradiated (7.5 kGy) samples of Amritamehari churnam powder.

Please cite this article in press as: Chatterjee, S., et al., Radiation processing: An effective quality control tool for hygienization and extending shelf life of a herbal formulation, Amritamehari churnam, Journal of Radiation Research and Applied Sciences (2015), http:// dx.doi.org/10.1016/j.jrras.2015.09.007

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DBpp ¼ 0.766 mT. This g value obtained compares well with those reported in literature (Desrosiers & McLaughlin, 1989). Several reports have suggested these free radicals to be those of semi-quinones produced by the oxidation of plant polyphenolics (Scewartz, Bolton, & Brog, 1972) or lignin (Tabner & Tabner, 1994). It is known that the polyphenols are mainly responsible for the antioxidant activity of the plant products. The broad line width of the naturally available EPR singlet showed a high concentration of polyphenolic paramagnetic centers responsible for the antioxidant activity of the samples. After exposure to ionizing radiation the EPR lines (g ¼ 2.005) originated from polyphenols exhibited a considerable increase in signal intensity and no radiation specific EPR signal was observed. Enhancement of naturally present signal intensity and line width of the radical species in irradiated food matrix has been reported in literatures (Sanyal et al., 2009). The effect of thermal energy in the sample was also investigated by heating the sample at 100  C for 1 h. The EPR spectrum (Fig. 8) of thermally treated sample was almost comparable with that of control one. No increase in polyphenolic paramagnetic centers was observed after thermal treatment. Fig. 9 depicts the effect of increasing radiation dose from 2.5 to 10 kGy on the spectra of the herbal medicine. The integral intensities of the EPR signals were obtained by double integration of the spectrum and the integral intensity was observed to be significantly related to the radiation dose. However, at higher doses saturation response was observed because the rate of recombination of the radiation induced radicals was enhanced due to increased radical concentration. The measurement was performed 2 d after irradiation. In order to study the stability of the radiation induced paramagnetic signals the EPR spectra of non-irradiated and gamma radiation processed AC sample were monitored till 50 days of storage in normal laboratory condition (temperature 26  C; relative humidity 72%). In case of irradiated sample, an exponential decline of EPR signal intensity was observed, as is illustrated in Fig. 10 for samples treated at a dose of 7.5 kGy. The storage time after radiation treatment substantially influenced EPR spectra of gamma irradiated samples, as the EPR spectra of non-irradiated sample remained intact. The fading kinetics of the irradiated sample was observed to be

9

Fig. 10 e Time kinetics of the EPR signal during storage of non-irradiated and irradiated (7.5 kGy) samples of Amritamehari churnam powder.

similar to other irradiated food and allied products (Sanyal, Chatterjee, Variyar, & Sharma, 2012). The results suggested that the radiation induced enhancement of the phenolic free radicals in the sample was unstable and became comparable with non-irradiated samples within 15e20 days. In addition EPR studies were unable to detect any radiation specific paramagnetic centers confirming indirectly the unchanged properties of the sample after irradiation as obtained from other approaches.

4.

Conclusion

Radiation processing does not significantly affect the chemical composition, physical property, antioxidant activity and sensory quality while improving microbial quality of the herbal formulation, Amritamehari churnam. Hence the extent of changes, if any, depends on the nature of the herb and the conditions of post-harvest handling and storage radiation processing can thus be effectively used to improve shelf life and quality of herbal products.

Uncited reference Lopez-Rubira et al., 2005, Lowry et al., 1951, Roginsky and Lissi, 2005, Saroj et al., 2006, Yusof et al., 2007. Q5

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Fig. 9 e EPR spectra of dose dependence of EPR central line (g ¼ 2.0050).

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