A biochemical approach to understand the concept of Snigdha-Ruksha Guna
Deepa Anserwadekar1*,Pratik Belambe2, Dhirajkumar Ahire2 & Rashmi Tupe2*
1Department of Samhita Sanskrita Siddhant, College of Ayurved, Bharati Vidyapeeth University, Pune-46
2Biochemical Sciences Division, Rajiv Gandhi Institute of IT and BT, Bharati Vidyapeeth University, Pune-46 E-mail: rashmitupe@gmail.com, deepanser@gmail.com
Received 22.05.13, revised 19.08.13
According to Ayurveda, all food is medicine because all food affects the body. However, these two subjects are rarely studied to find out precise biochemical mechanism of the interrelation between food and their Gunas. Objectives of the present work are : 1) to associate the food properties in terms of antioxidant potential and macronutrient bioavailability with their known type of Snigdha and Ruksha Gunas, and 2) to evaluate the influence of roasting on this association. To achieve this, antioxidant potential of food extracts was measured by seven in-vitro methods and capacity of extracts to prevent H2O2
induced erythrocyte oxidative damage by estimating four cellular antioxidant parameters. By using an in-vitro digestion method; fat, carbohydrate and protein absorption was estimated. Results indicated that Ruksha samples have higher antioxidant potential and can better protect the erythrocytes from oxidative damage with more protein and triglyceride absorption as compare to Snigdha samples. In the comparison of raw and roasted rice samples, erythrocyte protection was superior in raw samples with enhanced triglyceride and carbohydrate intestinal absorption. These results clearly indicate the differential behavior of Ruksha and Snigdha samples at antioxidant, erythrocyte and intestinal absorption levels.
Keywords: Ruksha, Snigdha, Antioxidant potential, Bioavailability
IPC Int. Cl.8: C09K 15/00, A47G 19/26, A47J 39/02, A47J 37/00, A01D 20/00, C12
The term Ayurveda, a Sanskrit word, translates into knowledge (Veda) of life (Ayur); Veda also means science. Ayurveda deals with Human body, etiology of disease, herbal-mineral-metal drugs and food items. According to this Traditional Indian thought, all food is medicine because all food affects the body, and the effect of a food (its function) trumps arbitrary classifications based on its origin (its form) when being classified. For the treatment purpose according to Ayurvedic perspective Gurvadi Gunas are widely used where the increased elements, i.e. Gunas are treated by opposite Guna. So, if patient’s Ruksha Guna is high then it is to be cured by food with Snigdha Guna and vice-versa1,2,3,4. The functional activity of Snigdha food is Kledana (moistening), Mruduta (softness), Ardrata (Malleability) which is supposed to stimulate Kapha and Mala whereas regulates Vata. Likewise the functional activity of Ruksha food is dryness which increases Vata and regulates Kapha,, decreases Bala, creates vaivarnya (discoloration) as shown in Table 1.
In this study, the examples of food with the Snigdha
properties are rice, Jawas, Teela, wheat and udeed where as Ruksha property food are Yava, Mudga, Jawar, Chanaka and Nachani. Also it is assumed that with heat treatments like roasting causes minor change in their properties. Despite the intimate relationship between Ayurvedic Guna theory and food, these two subjects are rarely studied to find out precise biochemical mechanism of the interrelation between food and their Gunas.
Current nutritional knowledge lays emphasis on macronutrient quantity of food, i.e. protein, carbohydrate and lipids. The nutrients which are released from the food during digestion, only some portion (bioavailable fraction) will be available for their physiological functions or storage in body5. The bioavailability of these nutrients depends on the physical properties of the food matrix. Several in-vivo or in-vitro methods are used to estimate the bioavailability of a nutrient. However, in-vitro methods are preferred over in-vivo methods since the latter are expensive, difficult to reproduce and ethically controversial6. Secondly, it is not completely known which dietary constituents are responsible for medicinal property, but antioxidants appear to play a
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*Correspondence author
major role in the protective effect of plant foods7,8. This can be studied by estimating the antioxidant potential of food extracts as well as by assessing cellular antioxidant parameters using oxidative stress induced cell model.
Materials and methods Samples and extraction
Food samples of Snigdha – Ruksha Gunas mentioned by Charak Samhita were taken for biochemical analysis. The English names, Sanskrit names, botanical names of ten dietary samples are listed in Table 2. To study the effect of roasting of food samples six rice varieties were taken (Shashtishali, Raktashali, Basmati, Ambemohar, Kolam and local rice) and the raw and roasted forms were used for analysis. The samples were collected and powdered with a mechanical grinder and stored in air-tight containers.
The aqueous and methanolic extracts of the food samples were prepared by adding 1 gm of dry powders of selected materials in 10 ml water /methanol, further stirring at 150 rpm (steelmet incubator shaker, India) at ambient temperature for 3 hrs. Insoluble residues from the solutions were removed by centrifugation at 8,000 gm for 10 min (SuperspinR- V/FM, Plasto craft, India), filtration by Whatman No. 1 and the clear supernatants were used
for analysis. The extracts were stored at 4°C in plastic vials, till further use9. All the estimations were performed in triplicates.
In-vitro estimation of antioxidant potential of extracts
Total polyphenols estimation
Total polyphenols of extracts was determined using the Folin–Ciocalteu reagent10. 1% methanolic sample extracts (300 µl) were mixed thoroughly with 1.5 ml of freshly diluted 10% Folin–Ciocalteu reagent.
1.2 ml of sodium carbonate solution (7.5%) was added to the mixture and incubated for 30 minutes in dark and the absorbance was measured at 765 nm.
The concentration of total phenolic compounds in the extracts was determined using an equation obtained from the standard gallic acid (0.002-0.02 mg/ml), and expressed as mg of gallic acid equivalents (GAE) per gm dry weight of plant material. The gallic acid standard curve used was y= 45.5x +0.031 (R² = 0.983), where y is the absorbance at 765 nm and x is the concentration of gallic acid.
DPPH radical scavenging ability
This activity of extracts was measured by the decrease in absorbance of methanolic solution of DPPH11. The reaction mixture contained 5 ml DPPH working solution (3.3 mg of DPPH in 100 ml
Table 1—Five Snigdha and five Ruksha food samples
Sr. No Common name of the samples Scientific name Sanskrit name Snigdha/Ruksha
1 Rice Oryza sativa Shali Varga Snigdha
2 Jawas (Flaxseed) Linum ustitatissmum Atasi /Uma Snigdha
3 Teela (Sesame ) Sessamum indicum Teela Snigdha
4 Gahu (Wheat) Triticum sativa Godhum Snigdha
5 Udeed (Black gram) Phaseolus mungo Masha Snigdha
6 Yava / Sattu (Barley) Hordeum vulgare Yava Ruksha
7 Mudga/ Muga (Green gram) Phaseous aureus Mudga Ruksha
8 Jawar (Sorghum) Sorghum vulgare Yavanaala Ruksha
9 Chana (Red gram) Cajanus cajan Chanaka Ruksha
10 Nachani (Finger millet) Eleusine coracana Madhuli Ruksha
Table 2—Action of Guna and its applied aspect by Ayurvedic perspective
Property Ayurvedic perspective Causative factors for conditions
Snigdha
Kledana (Moisten), Snehana (oleate), Mardava (softens), Balya (Srengthens), Varnya (improves
complexion), Vrushya (improves fertility)
Kapha dominant conditions like (hyperlipidemia), Prameha (Diabetes) Arsha, Hrudaroga (cardiac disease), Shiro-roga Arsha, Hrudaroga
(cardiac disease), Shiro-roga Ruksha
Shoshan (absorption), Lekhana / Khar (scraping), dryness, Sthambha (restrain), Kathina (hardness), creates roughness and hampers Bala and Varna
Vata dominant conditions like Urusthambha (restricted activity of lower extremity), Krushta, Kshata-Kshina (physical activity more than-Krushta, Kshata-Kshina (physical activity more than owns capacity), Vatarakta (Gout)
methanol) and 1ml of the sample extract. Ascorbic acid at different concentrations (5-50 µg/ml) was used as standard. The mixture was shaken and incubated for 30 min. in the dark at room temperature.
The absorbance was read at 517 nm relative to the control (as 100%) using a spectrophotometer.
The percentage of radical-scavenging ability was calculated by using the formula: % DPPH scavenging activity = [(A0 − A1)/A0 × 100], where A0 was the absorbance of control (without sample) and A1 was the absorbance of extracts or standards.
The DPPH solution without sample solution was used as a control.
ABTS radical scavenging ability
The ABTS radical cation scavenging assay was analyzed following a modified method of Re et al.12. For the assay of test samples 980 µl of ABTS reagent was mixed with 20 µl of the sample extract/standard.
The decrease in absorbance was recorded at 0 min (initial reading) and after 6 min (final reading) at 734 nm. Ascorbic acid (0.0176-0.176 mg/ml) was used as reference standard. % Radical scavenging = (Initial reading-final reading)/Initial reading X 100 %.
NO scavenging activity
Scavenging activity of NO by the plant extract was determined by the method of Garratt13 .10 mM of 2 ml SNP, 0.5 ml of phosphate buffer saline (pH 7.4) and 0.5 ml of sample extract / standard was incubated at 25°C for 150 min. 1 ml of Griess reagent A (1% sulfanilamide in 5% phosphoric acid) was added into 0.5 ml of the above reaction mixture and it was allowed to stand for 5 minute for diazotization. 1 ml of Griess reagent B (Napthylethylenediamine dihydrochloride 0.1% w/v) was added in the above mixture and incubated at 25°C for 60 min. The absorbance was recorded at 540 nm. Curcumin (50-500 µg/ml) was used as reference standard.
The percentage of radical-scavenging ability was calculated as DPPH radical Scavenging activity.
H2O2 scavenging activity
Scavenging activity of H2O2 by the herbal extract was determined by the method of Ruch et al.14. 4 ml sample extract was mixed with 0.6 ml of 4 mM H2O2
solution prepared in phosphate buffer (0.1 M, pH 7) and incubated for 10 min. BHT (0.02-0.08 mg/ml) was used as reference standard. The absorbance of the solution was taken at 230 nm against blank solution containing the plant extract without H2O2. The
percentage of radical-scavenging ability was calculated as DPPH radical Scavenging activity.
LPO assay
A modified thiobarbituric acid reactive species was used to measure the lipid peroxide formed using egg yolk homogenate as lipid rich media15 Egg yolk homogenate (0.5 ml of 10% v/v) and 0.1 ml of methanolic sample extracts were added and volume made up to 1 ml with distilled water; further 0.05 ml of FeSO4 (0.07 M) was added to induce LPO and incubated for 30 min. Then, 1.5 ml of 20% acetic acid (pH 3.5) and 1.5 ml of 0.8% (w/v) thiobarbituric acid in 1.1% SDS were added, vortexed and heated at 95°C for 60 min. After cooling, 5 ml of butan-1-ol were added and centrifuged at 3000 rpm for 10 min.
The absorbance of the upper organic layer was measured at 532 nm. BHT (100-1000 mg/ml) was used as reference standard. Inhibition of LPO percent by the extract was calculated as :[(1-E)/C] x 100 %, where C is the absorbance value of the fully oxidized control (without sample) and E is the absorbance in presence of herbal extract.
RP estimation
The RP of the aqueous extract was evaluated according to the method of Oyaizu16 The mixture containing 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of K3Fe(CN)6 (1% w/v) was added to 1ml of the extract. The resulting mixture was incubated at 50°C for 20 min, followed by the addition of 2.5 ml of TCA (10% w/v). The mixture solution (2.5 ml), mixed with distilled water (2.5 ml) and 0.5 ml of FeCl3 (0.1%, w/v). BHT (0.02-0.08 mg/ml) was used as reference standard and optical density at 700 nm was measured against sample blank.
Prevention of H2O2 induced erythrocyte oxidative damage by food extracts
Aqueous extracts of the food samples were used in erythrocyte studies. Sheep blood was obtained from local butcher shop in EDTA coated tubes and centrifuged at 3000 rpm for 10 minutes to remove plasma, followed by washing with 0.15 M sodium chloride solution (thrice). Cells were then suspended in phosphate buffered saline (pH 7.3) so that final yield of RBC suspension was 1× 108 RBCs per ml.
For treatment of different samples on erythrocytes, 2ml erythrocytes and 2 ml extract was mixed and 1 ml (500 µm) H2O2 to induce oxidative shock to the cells17. The purpose of this was to study the capacity of samples in preventing oxidative damage
to the erythrocytes. This mixture was kept for gentle shaking at room temperature for 2 hrs then centrifuged at 5000 rpm for 10 min. The treated erythrocytes hemolysate was used for analysis of reduced glutathione (GSH), catalase activity, total cellular antioxidant potential and lipid peroxidation (LPO).
Estimation of GSH
This was estimated using the method of Ellman18. 0.25 ml erythrocytes hemolysate was added to 0.5 ml of precipitating solution composed of 5% TCA in 1 mM EDTA. The samples were centrifuged at 2000 rpm for 10 min. 0.1 ml fraction of the supernatant was mixed with 2.5 ml of 0.1 M phosphate buffer (pH 8.0) followed by addition of 0.1 ml, 0.01% DTNB. Absorbance (412nm) was measured within 10 min. against a blank sample without erythrocytes. The GSH concentration was calculated by using the molar extinction coefficient of 13600 M−1 cm−1. The results were expressed as nM hemolysate.
Measurement of catalase activity
Catalase activity was assayed by the method of Chance as modified by Aebi19 .The activity of catalase was estimated by mixing 0.1 ml of hemolysate with 0.567 ml of 50 mM sodium phosphate buffer (pH 7.0) and the reaction was started by addition of 0.33 ml of 30 mM Hydrogen Peroxide (H2O2) prepared in the same buffer. The blank for this assay was composed of 0.1 ml hemolysate and 0.9 ml buffer with no addition of H2O2. Absorbance (240 nm) was measured at 0 and 60 secs. The results were expressed as International Unit (IU).
FRAP assay
Antioxidant capacity of plasma was estimated by the Ferric Reducing Antioxidant Power assay (FRAP)20. For this, 0.1 ml of hemolysate was mixed with 3 ml of working FRAP reagent (acetate buffer pH 3.6, FeCl3.7H20 and tripyridine triazide - 10:1:1) and incubated at 37°C. After vortexing, absorbance (593 nm) was measured at 0 min. and 4 min. Ascorbic acid standard (100µM-1000µM) was processed in the same way. The results were expressed as µmol of reduced ferrous by comparing with standard.
Estimation of LPO
Malondialdehyde (MDA), end product of LPO was assessed. 200ul of treated erythrocytes were mixed with 1 ml of 10% TCA and centrifuged at 2000 rpm
for 15 min. 1 ml of supernatant was transferred to another test tube to which 2 ml thiobarbituric acid was added (0.67% in 0.25 M HCl). Tubes were mixed and kept in boiling water bath for 25 min followed by 10 min incubation in ice cold water. Absorbance was measured at 535 nm and concentration of MDA was calculated using molar extinction coefficient of 153000M-1cm-1. The results were expressed in nM of MDA per mg of proteins21.
In-vitro dialysibility of carbohydrate, protein and triglyceride from samples
For studying in-vitro dialysibility of the sample, traditional cooking methods are followed for the samples before treating them with gastric and intestinal enzymes. 0.5 gm samples are introduced into 20 ml ultrapure water. To create an acidic environment, its pH is set to 2; 0.25ml pepsin solution is added in above mixture and is kept for incubation for 2 hrs on a shaker. After 2 hrs, 2 ml bile–pancreatin solution is added into the mixture. At the same time, dialysis tubes are filled with sodium bicarbonate are dipped in above mixture. This setup is again kept for incubation for 2 hrs on a shaker. After incubation, dialysate (tube content) and undialysate are removed in separate tubes. Estimation of biomolecules such as triglycerides (by using lipoprotein lipase), proteins (by Lowry) and carbohydrates (by phenol – Sulfuric acid method) is done from both dialysate and undialysate22,23,24.
Results and discussion
In-vitro antioxidant potential of samples
The antioxidant potential of the aqueous and methanolic extracts of the samples belonging to Snigdha and Ruksha Gunas are shown in Tables 3 & 4. It is found that in both methanolic samples having Ruksha property showed higher antioxidant potential than samples with Snigdha property. Similarly the aqueous extracts of sample with Ruksha property demonstrated superior antioxidant activity than samples with Snigdha property with the exception of two assays, i.e. H2O2 and NO scavenging activities, where samples with Snigdha property showed higher antioxidant property. Total polyphenol content of the methanolic extracts was found to be higher in samples showing Ruksha property as compared to samples showing Snigdha property. Reducing potential of samples showing Ruksha property is higher than samples showing Snigdha property in both methanolic as well as aqueous extracts. Samples having Ruksha
property namely Yava and Nachani showed comparatively higher antioxidant activity than other Ruksha samples in both the extracts. Sample with Snigdha property like Teela and jawas has shown highest antioxidant activity in many assays in both the extracts.
In Ayurveda, Gunas are described as the way of presentation of characteristics of elements. However, these observational reports are difficult to experimentally elucidate. An attempt was made by Mishra et al.25 to evaluate the effect of Ruksha Guna drugs on Snigdha Guna of the body in hyperlipidemic patients (assumed to be a condition with increased
Snigdha Guna). In Ruksha Guna drugs treated group, up to 52.13% reduction in Snigdha Guna was observed concluding that Ruksha Guna had a good action on Snigdha Guna of body. In modern science, the increased cellular oxidative stress is established to be a pathophysiological process behind diseases. The samples with higher antioxidant potential are generally supplemented to manage the enhanced oxidative stress. For this purpose, we estimated antioxidant activities of the samples to determine the difference in them with respect to Gunas. The antioxidant potential of the food extracts are
Table 3—Antioxidant activity of samples in methanolic extracts of five Snigdha and five Ruksha samples Food
samples
ABTS (% Scavenging)
FRAP (nmols)
DPPH (% scavenging)
Total polyphenols (GAE/gm dry wt.)
H2O2
(% scavenging)
RP (BHT equivalents)
NO Scavenging (% scavenging)
LPO (nmoles MDA/ml
*10-5) Rice 22.99±38.18 19.003±33.5 61.13±23.65 0.93±0.76 87.40±15.01 0.094±0.097 40.46±8.22 74.82±8.88 Jawas 83.32±5.55 53.16±1.33 79.96±0.12 3.22±0.09 92.003±1.20 0.222±0.003 29.44±11.89 62.93±13.10 Teela 18.47±0.64 7.75±2.58 68.31±5.05 1.11±0.03 79.96±0.83 0.093±0.0004 47.20±5.94 76.03±3.96 Wheat 0±1.55 0.25±2.51 55.71±2.24 2.21±0.06 79.77±3.79 0.046±0.003 53.74±1.32 76.54±1.13 Udeed 17.67±2.67 0.88±0.06 57.09±5.29 0.89±0.02 103.56±1.76 0.061±0.001 33.41±5.61 56.24±9.95 Yava 75.33±2.34 38.60±2.11 88.92±2.16 3.11±0.09 102.31±0.28 0.203±0.01 49.53±5.28 66.17±2.68 Mudga 12.76±0.85 5.06±4.29 73.82±0.64 0.82±0.024 100±0.76 0.013±0.0008 45.56±6.93 50.11±7.92 Jawar 17.91±1.23 10.93±1.98 95.55±0.36 3.22±0.03 92.48±2.25 0.100±0.0009 42.06±3.96 30.21±0.80 Chana 17.92±1.05 11.81±5.92 95.41±0.12 1.11±0.04 103.56±1.36 0.056±0.07 40.19±15.86 28.95±0.97 Nachani 82.17±3.73 38.59±1.23 90.68±1.83 2.21±0.01 75.43±9.47 0.17±0.01 39.49±13.54 61.21±8.25 Values are mean ± SD. Abbreviations: GAE= Gallic Acid Equivalent; RP= Reducing Potential ; LPO= Lipid peroxidation;
DPPH= 2,2’-diphenyl-1-picrylhydrazyl; ABTS= 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid); NO= Nitric oxide;
H2O2 = Hydrogen peroxide; FRAP = Ferric reducing antioxidant potential
Table 4—Antioxidant activity of samples in aqueous extracts of five Snigdha and five Ruksha samples Food samples ABTS
(%Scavenging)
FRAP (nmols)
DPPH (%Scavenging)
H2O2
(%Scavenging)
RP (BHT Equivalents)
NO Scavenging (%scavenging)
pH value
Rice 12.46±7.65 7.17±6.44 59.16±26.39 56.47±24.76 0.037±0.02 55.59±4.59 6.36 Jawas 16.17±0.41 10±1.15 67.51±4.05 94.07±0.82 0.023±0.0005 58.87±2.50 5.97 Teela 29.31±1.83 8.69±0.80 55.14±2.73 63.33±5.50 0.040±0.0002 62.42±1.14 5.89 Wheat 17.73±1.05 3.26±1.13 37.63±2.61 69.49±9.17 0.011±0.0009 58.23±5.70 6.34 Udeed 23.09±0.28 4.91±1.21 32.09±1.69 43.04±2.43 0.014±0.0010 61.13±0.22 6.50 Yava 23.83±18.64 14.76±0.20 77.71±1.13 58.53±6.66 0.080±0.0012 49.19±15.23 5.48 Mudga 22.98±2.75 7.24±0.56 41.07±0.61 92.64±1.22 0.013±0.0003 66.61±1.54 6.42 Jawar 26.38±1.21 4.70±0.56 61.70±0.62 39.52±4.63 0.036±0.004 59.52±2.96 6.48 Chana 24.77±0.27 22.63±1.93 50.08±0.62 57.77±6.52 0.017±0.0001 60.97±0.45 6.31 Nachani 73.02±2.58 14.04±1.49 89.72±1.07 28.86±11.76 0.135±0.0089 5.32±5.24 6.28 Values are mean ± SD.
influenced by various factors and largely depends on both the composition of the extract and the analytical test system. Therefore, it is necessary to perform more than one type of antioxidant capacity measurements to take into account the various mechanisms of antioxidant action which will give more reliable results. Overall results indicated that samples with Ruksha Gunas have better antioxidant potential and can scavenge the increased reactive oxygen species efficiently as compare to Snigdha Guna samples.
Additionally total polyphenolic contents are higher in Ruksha Guna which gives further scope to assess the relation between herbal drugs having Ruksha Guna and its polyphenolic contents in more specific methodology. Our results of phenolic contents in various samples are in agreement and supported by Sreeramulu et al.26 where comparative antioxidant activity of commonly consumed samples in India was done. More reducing potential of Ruksha Guna drugs indicates its Khara / Lekhana action which act as cleansing agent.
According to Ayurveda, heat treatment causes minor alteration in the Gunas of sample. This was evaluated in six varieties of rice in raw and roasted forms and results are indicated in Tables 5 & 6.
Overall the roasted rice samples illustrated better antioxidant activity than their normal counterparts.
Ayurveda believes that when roasted, property of sample changes, i.e. it becomes less Snigdha in case of samples with Snigdha property and more Ruksha in case of samples with Ruksha property. This was evaluated in six varieties of rice in raw and roasted counterparts. In case of local rice, we found that roasted sample was demonstrated higher antioxidant potential in all assays in both aqueous and methanolic extracts. Almost similar results were evident for other rice varieties where roasted forms indicated antioxidant activity in almost all assays in both the extracts. However, in Raktashali variety, sample with Snigdha property have shown more antioxidant potential than the roasted form in methanolic extracts and in aqueous extract sample with Ruksha property showed highest activity in most assays. These conflicting results in different extracts maybe due to differential extraction of the bioactive molecules in the water and methanol due to variation in polarity of the solvents. Ayurveda has stated Raktashali and Shashtishali are most healthy dietary food and advised in healthy as well as diseased conditions.
Collective analysis of raw and roasted rice samples
showed that roasted samples have better antioxidant activity in almost all the assays in both the extracts. It is also suggesting that with roasting the antioxidant potential of Snigdha Gunas can be enhanced.
Erythrocyte protection in presence of samples The Snigdha and Ruksha Gunas of food samples were further evaluated at cellular level using erythrocyte exposed to oxidative stress. The results of various cellular antioxidant parameters, i.e. catalase, FRAP and GSH showed that samples with Ruksha property are better in protecting erythrocytes from oxidative damage than Snigdha samples. The levels of catalase and total cellular antioxidant potential was higher and lipid peroxidation is found less in erythrocytes treated with Ruksha samples than with Snigdha samples (Fig. 1 a-d). It indicates Ruksha Guna shows cell membrane protection may be due to absorption of excessive accumulation obstructing the micro channels. Thus it leads to proper circulation of nutrients in body. Surprisingly the levels of GSH were significantly higher in Snigdha samples treated erythrocytes as compared to Ruksha samples. In H2O2
exposure, more reactive oxygen species will act on cell membrane causing membrane lipid peroxidation and decrease in cellular enzymatic and non-enzymatic antioxidants. It can be inferred after the analysis, that the samples with Ruksha property were better in protecting erythrocytes than samples with Snigdha property. These results supports above findings where collective antioxidant potential of Ruksha samples was higher. From the biochemical point of view, it can be concluded that scavenging (Lekhana) of free radicals which were causing cellular damage was more in extracts of Ruksha samples. Study of rice samples to understand the effect of roasting on cellular protection indicated that the raw samples were better in protecting erythrocytes compare to roasted one as shown in Fig. 2 (a-d).
Intestinal bio accessibility of macronutrients in samples
The macronutrients contents were found to be higher in the Snigdha samples as compare to Ruksha samples. The level of protein, carbohydrate and triglyceride in Snigdha sample was found to be 121.91 ± 47.33 µg/ml, 17.12 ± 0.2 mg/ml, 125.02 ± 37.33 mg/dl and in Ruksha samples 86.46 ± 38.13 µg/ml, 16.99 ± 0.22 mg/ml, 117.68 ± 41.28 mg/dl, respectively. For uniform comparison the bioavailability data of samples was expressed in terms of 100 % as shown in Fig. 3a.
Results of in vitro dialysibility of triglycerides and
protein showed that its amount in dialysate fraction was more than undialysate one, indicating its higher bioaccessibility in Ruksha samples as compare to Snigdha samples. Whereas in case of in vitro dialysibility of carbohydrates, it was found that absorption of carbohydrates in both Snigdha and
Ruksha samples is comparable. More absorption of triglycerides and protein indicates capacity to absorb lipids, liquid and other essential constituents after completion of digestion. It is important role of Samana Vayu. Disturbance in this function creates condition like diarrohea / dysentry.
Table 5—Antioxidant activity of samples in methanolic extracts of rice varieties with and without roasting Rice samples ABTS
(% Scavenging) FRAP (nmols)
DPPH (% scavenging)
Total polyphenols (GAE/gm dry wt.)
H2O2
(% scavenging) RP (BHT equivalents)
NO Scavenging (% scavenging)
LPO (nmoles MDA/ml
*10-5) Shashtishali 15.48±0.73 14.15±1.12 88.85±3.72 1.04±0.03 93.16±1.20 0.098±0.007 48.13±2.64 63.42±13.19 Raktashali 100±24.17 81.87±6.30 84.54±1.27 2.34±0.07 85.84±3.01 0.281±0.034 33.88±9.58 89.63±11.43 Basmati 13.87±0.29 13.41±0.64 70.64±0.68 1.04±0.03 106.94±10.28 0.089±0.006 50.23±6.93 68.73±3.30 Ambemohar 2.13±0.44 2.077±0.54 35.36±1.28 0.30±0.012 76.78±0.76 0.031±0.02 38.08±0.33 75.86±6.35 Kolam 1.98±0.63 2.51±0.49 36.06±4.47 0.42±0.008 96.82±27.21 0.018±0.001 43.22±30.06 73.91±3.30 Local rice 4.45±0.58 0±0.46 51.31±1.05 0.47±0.014 64.84±0.92 0.048±0.001 29.21±8.92 77.35±1.29 Shashtishali
(Roasted)
13.04±0.35 14.36±0.85 82.85±1.88 0.03±0.027 74.76±2.52 0.091±0.003 44.86±1.98 71.24±5.83 Raktashali
(Roasted)
100±1.55 76.42±2.51 89.84±0.84 0.10±0.099 81.02±3.09 0.29±0.029 43.46±1.98 87.60±6.00 Basmati
(Roasted)
10.22±0.60 10.34±0.59 63.73±1.89 0.02±0.021 115.80±1.69 0.062±0.0004 41.82±8.92 73.84±4.53 Ambemohar
(Roasted)
12.59±6.32 1.43±0.30 52.93±1.92 0.013±0.013 75.52±18.51 0.047±0.005 41.59±1.32 18.48±8.42 Kolam
(Roasted)
8.84±0.45 6.53±0.42 62.81±3.30 0.03±0.025 94.80±1.7 0.068±0.0002 39.02±0.99 80.78±5.72 Local rice
(Roasted)
7.49±0.65 0.44±0.03 76.36±1.29 0.017±0.017 86.03±2.04 0.059±0.0013 30.14±15.52 67.04±1.61 Values are mean ± SD.
Table 6—Antioxidant activity of samples in aqueous extracts of rice varieties with and without roasting
Rice samples ABTS
(% Scavenging)
FRAP (nmols)
DPPH (% Scavenging)
H2O2
(% Scavenging)
RP (BHT Equivalents)
NO Scavenging (% scavenging)
pH value Shashtishali 9.31±0.84 8.50±0.23 85.45±1.41 78.53±9.39 0.03±0.0003 55.65±7.07 6.65 Raktashali 25.37±2.84 17.26±1.47 89.64±4.51 85.35±8.01 0.090±0.0029 54.84±0.45 6.38 Basmati 17.33±4.80 11.56±0.18 61.86±3.99 69.12±6.09 0.05±0.0006 55.16±10.49 6.26 Ambemohar 4.35±1.26 1.09±0.36 30.94±1.32 26.12±7.57 0.012±0.0042 61.29±6.84 6.33 Kolam 7.63±1.29 1.64±0.51 26.64±0.49 47.33±4.23 0.018±0.0029 47.74±0.45 6.29 Local rice 10.77±3.97 3.009±0.11 60.40±4.72 32.38±19.67 0.026±0.0011 58.87±4.33 6.28 Shashtishali (Roasted) 14.75±11.89 5.83±0.20 61.62±0.75 85.16±1.53 0.028±0.0004 51.13±2.50 6.65 Raktashali (Roasted) 43.40±0.76 20.85±0.67 92.89±1.36 72.86±2.63 0.121±0.0045 55.65±1.59 6.28 Basmati (Roasted) 9.13±0.47 8.59±0.73 64.70±2.30 49.16±3.86 0.041±7.5 55.64±5.24 6.10 Ambemohar (Roasted) 9.03±1.10 3.87±0.36 55.89±0.97 52.82±1.76 0.026±0.0012 61.61±3.19 6.25 Kolam (Roasted) 19.56±1.04 3.07±0.28 40.28±1.06 55.64±8.27 0.026±0 57.26±5.70 6.33 Local rice (Roasted) 10.10±0.48 3.29±0.087 67.47±4.32 69.38±7.88 0.024±0.0002 65.32±0.22 6.80 Values are mean ± SD.
In the rice samples with raw and roasted forms, the higher levels of protein, carbohydrate and triglyceride was observed in raw samples 76.77 ± 33.22 µg/ml, 17.11 ± 0.17 mg/ml and 134.99 ± 6.19 mg/dl in comparison to roasted form 47.92 ± 29.55 µg/ml, 16.94 ± 0.22 mg/ml and 122.14 ± 14.58 mg/dl, respectively. Interestingly protein bioavailability was significantly higher whereas the absorbed triglycerides and carbohydrate fraction was lower in roasted forms compared to raw type (Fig. 3b).
Traditional significance of study to the society/researchers and some constructive recommendations
To our knowledge this is the first report where Snigdha and Ruksha Gunas of dietary samples were evaluated by different biochemical mechanism. In the present study comparison of the Snigdha and Ruksha gunas of samples on the bio molecules bioavailability, antioxidant and cell model were performed for their complete assessment at intestinal (absorption), cellular (post-absorption) levels.This research
Fig. 1—Effect of five Snigdha and five Ruksha samples on erythrocytes
Fig. 2—Effect of rice varieties on erythrocytes
provided the insight about their differential mode of action. The Ayurvedic practitioners have been recommending these food groups for various ailments therefore their scientific validation is important to understand their therapeutic potential. This study is very significant because these food samples are consumed every day and they play a pivotal role in the person’s health, hence Ayurvedic practitioners, researchers, nutritional biologists, society will get benefit from these findings.
As the Ruksha and Snigdha Gunas mainly differ with their capacity to bind or absorb respectively, the binding constant of these samples to various bio-molecules can be assessed in future to get insight of their binding affinities. Further studies at micronutrients levels with clinical interpretation and animal studies where the changes in metabolism, enzyme activities and gene expression after these sample consumption can be carried forward. Based on these preliminary studies, other concepts in Ayurveda such as Madhur- Amla- Katu Rasa -Vipaka, and other Gunas like Sheeta- Ushana and Guru – Laghu can be further assessed by various in vitro and in vivo methods to validate their differential behavior. The concept of functional food and nutraceuticals is gaining attention, in future our traditional foods can be promoted as indigenous functional foods and a nutraceutical product can be eventually developed as a complementary therapy for various diseases.
Conclusion
It may be concluded that Ruksha samples have higher antioxidant potential and can better protect the erythrocytes from oxidative damage as compare to Snigdha samples. At the level of intestinal bio accessibility, protein and triglyceride in Ruksha samples were more absorbed than the Snigdha samples. In the comparison of raw and roasted types of rice samples, though the roasted forms demonstrated slightly higher antioxidant potential but
erythrocyte protection was superior in raw samples with enhanced triglyceride and carbohydrate intestinal absorption. These results clearly indicate the differential behavior of Ruksha and Snigdha samples at antioxidant, erythrocyte and intestinal absorption levels. This signifies that by using different biochemical analysis we can distinguish the Ruksha and Snigdha properties of samples.
Acknowledgement
The research study was funded and supported by Bharati Vidyapeeth Deemed University, Pune. The authors are sincerely thankful to Prof G D Sharma, Principal and Dr S A Shaikh, Vice- Principal, Rajiv Gandhi Institute of IT and Biotechnology for providing facilities to conduct the research. We are thankful to Prof Dr A.B. Patil Principal and Dr Mrs YV Joshi, College of Ayurveda undertaking this research project and for constant encouragement.
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