GJRMI - Volume 5, Issue 5, May 2016 by Dr. Hari Venkatesh.K.Rajaraman

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ISSN 2277 â&#x20AC;&#x201C; 4289 www.gjrmi.com Editor-in-chief Dr Hari Venkatesh K Rajaraman

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Honorary Members - Editorial Board Dr Farhad Mirzaei Dr. Sabarinath Subramaniam Dr. Yogitha Bali

INDEX – GJRMI - Volume 5, Issue 5, May 2016 INDIGENOUS MEDICINE Ayurveda – Kaya Chikitsa PHARMACOGNOSTICAL AND PHARMACEUTICAL ASSAY OF GOMUTRA HARITAKI WITH SPECIAL REFERENCE TO THREE DIFFERENT SAMSKARAS (KLINNA, SWEDANA AND BHAVANA) Shivam Joshi*, Mandip Goyal, Harisha CR, VJ Sukla

146–157

Ayurveda – Dravya Guna A COMPARATIVE CLINICAL TRIAL TO ASSESS THE ADD ON EFFECT OF ASVAGANDHA [WITHANIA SOMNIFERA (L.) DUNAL] IN MOTOR FUNCTIONS OF CHILDREN WITH MOTOR DEVELOPMENTAL DISORDERS Preethy AS*, Dinesh KS, R Remadevi

158–172

Ayurveda – Moulika Siddhanta – Review CONCEPT OF NITYA SEVANIYA AAHARA DRAVYA Saylee Deshmukh*, M K Vyas

173–182

Cover Page Photography: Dr. Hari Venkatesh K.R. Plant ID: tender branch of Karamarda (Carissa carandas L.)* of the family Apocynaceae; Place: Koppa, Chikkamagalur District, Karnataka, India *Botanical Name validated from www.theplantlist.org as on 31/05/2016

Research article PHARMACOGNOSTICAL AND PHARMACEUTICAL ASSAY OF GOMUTRA HARITAKI WITH SPECIAL REFERENCE TO THREE DIFFERENT SAMSKARAS (KLINNA, SWEDANA AND BHAVANA) Shivam Joshi1*, Mandip Goyal2, Harisha CR3, VJ Sukla4 1

Ph.D. Scholar, Department of Kayachikitsa, IPGT & RA, Jamnagar, Gujarat, India Assistant professor, Department of Kayachikitsa, IPGT & RA, Jamnagar, Gujarat, India 3 Head, Pharmacognosy laboratory, IPGT & RA, Jamnagar, Gujarat, India 4 Head, Pharmaceutical chemistry, IPGT & RA, Jamnagar, Gujarat, India *Corresponding Author: E-mail: ayuholistichealth@gmail.com; Mobile: +919427230432 2

Received: 14/03/2016; Revised: 08/05/2016; Accepted: 10/05/2016

ABSTRACT Samskara is defined as Gunantaradhanam (transformation) of the Swabhavika Guna (inherent attributes) of a substance which leads to the addition of new properties. The changes in finished product because of Samskara can be perceived at pharmacognostical, phytochemical levels. Present study was aimed to assess the role of Klinna (soaked), Swedana (boiled) and Bhavana (trituration) Samskara on Gomutra Haritaki, on basis of Phyto-Pharmacognostical, HPTLC and UV-VIS-NIR Reflectance (180–2500 nm) study. Total 3 samples were prepared of Haritaki with Gomutra viz. Klinna Gomutra Haritaki (GH-2), Swedita Gomutra Haritaki (GH-3), Bhavita Gomutra Haritaki (GH-4). All the samples showed changes at pharmacognostical, pharmaceutical, HPTLC densitogram and UV-VIS-NIR Reflectance study level. Swedita Gomutra Haritaki (GH-3) had highest variation in all study level. Powder microscopy of GH-3 showed presence of clumped epicarp cells, squashed mesocarp cells (not clear), parenchyma cells with brown content with dark cellular content and crystalline material etc. Phytochemical parameters showed pH of 7.0, loss on drying value of 9.303% w/w, ash value 15.84% w/w, water soluble extract 57.2% w/w and alcohol soluble extract 43.5% w/w. HPTLC showed eight peaks at 256 nm and 366 nm. In UV-VIS-NIR reflectance also, GH-3 had higher variation and different profile from GH-2, GH-4. It indicates that both Swedana Samskara and Gomutra as media had unique role in preparation of Gomutra Haritaki.

KEY WORDS: Bhavana, Gomutra Haritaki, Haritaki, HPTLC, Klinna, Pharmacognosy, Phytochemical, Samskara, Swedana, UV-VIS-NIR

Cite this article: Shivam Joshi, Mandip Goyal, Harisha C R, VJ Sukla (2016), PHARMACOGNOSTICAL AND PHARMACEUTICAL ASSAY OF GOMUTRA HARITAKI WITH SPECIAL REFERENCE TO THREE DIFFERENT SAMSKARAS (KLINNA, SWEDANA AND BHAVANA), Global J Res. Med. Plants & Indigen. Med., Volume 5(5): 146–157

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INTRODUCTION: Paradi Gunas are a group of Guna which are regarded as key to successful treatment (Acharya Y.T. 2009). Among Paradi Gunas, Samskara (transformation) and Samyoga (combination) play important role in pharmaceutics. Kalpanas (formulations) of medicinal drugs as to ensemble the desired condition of patient and disease can be produced as and when required on the basis of various Samyoga and Samskaras (Acharya Y.T., 2009). Samskara is defined as Gunantaradhanam (transformation) of the Swabhavika Guna (inherent attributes) of a substance which leads to the addition of new properties. Various methods of Samskaras (Acharya Y.T., 2009) are mentioned in Ayurveda pharmaceutics such as Toyasannikarsa (Dilution), Agnisannikarsa (heat application), Saucha (cleaning), Manthana (churning), Desha (storing in a specific place), Kala (maturing), Vasana (container or preservation), Bhavana (impregnation) etc. Bhavana is one of the important Samskara mentioned in classics which is a wet trituration process frequently used in Rasaushadhi Kalpana as well as Kasthaaushadhi Kalpana. It has multidimensional pharmaceutical and therapeutic implications as stated in Charaka Samhita, this preparation results in quicker and amplified action with minimum dosage. Haritaki (Terminalia chebula Retz.), one among the most commonly used herbs, is extensively used in preparation of Ayurveda medicine either for preventive or curative aspect. Gomutra Haritaki is a multidimensional formulation among combined formulations of Haritaki described in various contexts, In Charaka Samhita, Gomutra Haritaki is referred mainly in Kaphaja Shotha (odema having Kapha dominance), Kaphaja Arsha (Hemorrhoids having Kapha dominance) and Kaphaja Pandu (Anemia having Kapha dominance) (Acharya Y.T., 2009). In Sushruta Samhita, it is used mainly in Sushka Arsha Chikitsa (Acharya Y.T. 2012). Dalhana has indicated that Gomutra Haritaki should be used

constipated bowel and diminished status of Agni (Gadhavitaksya Mandagni). In Ashtangahridaya, it is described in Kaphaja Pandu, Kaphaja Shotha, Mukharoga (oral cavity disorder) (Hari Sadasiva Sastri Paradakara Bhisagacharya, 2014). From above mentioned classical references, it can classified that Gomutra Haritaki can be prepared mainly with 3 Samskaras i.e., 1. Gomutra Klinnita (saturated-soaked) Haritaki 2. Gomutra Swedita (boiled) Haritaki 3. Gomutra Bhavita (triturated) Haritaki Samskara Guna is of three types, Vega (velocity), Bhavana (trituration) and Sthitisthapakatva (capacity of a drug to maintain its original form) (Dhundhiraj Shashtri 2007). Swedana is Agni Sannikarsa (processing with fire) while Bhavita and Klinna is Niragni Sannikarsa Samskara (processing without fire). Among the three Guna of Samskara, it can be stated that Vega, Bhavana and Sthitisthapakata is related with pharmaceutical process of Swedana, Bhavita and Klinna respectively. MATERIALS AND METHODS: Collection and Authentication of Raw Drugs Haritaki (Terminalia chebula Retz.) was collected from raw drug market, Jamnagar (Gujarat). Pharmacognostical authentication of drug was done in Pharmacognosy laboratory attached with institute while Gomutra (cow urine) was collected from Gaushala located in Dared village (Di. Jamnagar). Method of Preparation of 6 Samples Total 6 samples of Haritaki were prepared with different media i.e., [Jala (water), Gomutra (cow urine) and Eranda Taila (castor oil)] by different method of preparation i.e., (Samskara-Klinna, Swedana, Bhavana). The details of method of preparation of these six samples are;

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Global J Res. Med. Plants & Indigen. Med. | Volume 5, Issue 5 | May 2016 | 146–157

1. GH-1: The fruits of Haritaki (without seeds) was powdered. 2. GH-2: Haritaki (whole fruit) was soaked in Gomutra for 24 hrs and then seeds were removed from soaked Haritaki, dried and powdered. 3. GH-3: Haritaki (whole fruit-1 part) was boiled with cow urine until cow urine (2.5 part) evaporated. Seed was removed, pulp was dried and powdered. 4. GH-4: Haritaki Churna was given Bhavana of Gomutra for 6 hrs and Bhavita Gomutra Haritaki was dried and powdered. 5. GH-5: Haritaki (whole fruit-1 part) was boiled with mineral water until water (2.5 part) evaporated. Seeds were removed, pulp was dried and powdered. 6. GH-6: Haritaki (without seeds) was roasted in Eranda Taila and thereafter powder was prepared. All the samples were sieved through 80 mesh and preserved in an air-tight glass vessel. Though, all the six samples were analyzed but for the present paper, the results of only three samples viz. GH-2 (Klinna Samskarita Gomutra Haritaki), GH-3 (Swedana Samskarita Gomutra Haritaki) and GH-4 (Bhavana Samskarita Gomutra Haritaki) are discussed. Samples were subjected to pharmacognostical, pharmaceutical, HPTLC and UV-VIS-NIR Reflectance analysis to compare the different implications the various methods (samskaras) show up on the final product. Pharmacognostical Analysis Pharmacognostical analysis of GH-2, GH-3 and GH-4 based on organoleptic characters, i.e., colour, odour, taste and texture were recorded. Microscopic studies was carried out dissolving drug (powder) in small quantity of distilled water, filtering through filter paper and then precipitated and then was studied with and without stain to find out the lignified materials along with other cellular constituents. The micro photographs were taken under Carl Zeiss

Trinocular microscope attached with camera (Khandelwal K.R., 2008). Pharmaceutical Analysis All the three samples were analysed with appropriate protocols for standard physicochemical parameters such as aqueous extractive, alcohol extractive, pH, total ash, loss on drying at the Pharmaceutical Chemistry Lab, I.P.G.T. & R.A., Jamnagar. In the HPTLC study samples, Methanol extract of drugs were spotted on pre-coated silica gel GF 60254 aluminium plates by means of Camang Linomate V sample applicator fitted with a 100 µL Hamilton syringe. The mobile phase consisted of Chloroform: Methanolin a ratio of 9:1 v/v. After development, densitometric scan was performed with a Camag T. L. C. scanner III in reflectance absorbance mode at 254 and 366 nm under control of Win CATS Software (V 1.2.1. Camag). Then, the plate was sprayed with Vanillin sulphuric acid followed by heating and then visualised in daylight (Stahl E, 1969). UV-VIS-NIR UV-VIS-NIR (180–2500) reflectance was carried out at SICART, Vallabh Vidhyanagar, Gujarat, India. Study was conducted with instrument model λ 19 UV/VIS/NIR, data interval 1.0000 nm, slit width 5.0000 nm, scan speed 240 nm/min and smooth bandwidth 8 nm. The unscramble software 9.7 used for calculation. RESULT OF PHARMACOGNOSTICAL STUDY: Organoleptic Characters The sample GH-2 was yellowish brown with predominant Kashaya (Astringent) taste and Gomutra Gandhi (cow urine smell). The sample GH-3 was brownish with pungent with piercing taste and Gomutra Gandhi. The sample GH-4 was brownish yellow with predominant Kashaya (Astringent) taste and Gomutra Gandhi [Table-1].

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Table 1: Organoleptic characters of the samples - GH-2, GH-3 and GH-4 Organoleptic characters Colour Odour Taste Touch

GH-2

GH-3

GH-4

Yellowish brown Gomutra Gandhi Astringent Fine coarse

Brownish Gomutra Gandhi pungent with piercing Fine coarse

Brownish yellow Gomutra Gandhi Astringent Fine coarse

Microscopic Characters Powder microscopy of GH-2 showed epicarp cell with tannin content (loosened), mesocarp cell wall ruptured, mesocarp cells with tannin content (loosened as compared to raw Haritaki), fibers with wide lumen (undisturbed), sclereids with wide lumen, group of sclereids, pitted sclereids with wide lumen, starch grains (unchanged), fragment of pitted vessels, pitted stone cells with wide lumen and parenchyma cells with starch grains. Powder microscopy of GH-3 showed epicarp cells, among them some of epicarp cells were found clumped, mesocarp cells were more squashed (not clear), fibers with lumen, pitted sclereids with wide lumen, simple starch grains with hilum, parenchyma cells with brown content, fragment of pitted vessels, pitted stone cells, cellular content was darker (brown) and crystalline material. Powder microscopy of GH-4 showed collapsed epicarp cells, disturbed

mesocarp cells, disturbed wall of fibers, sclereids were ruptured and opened, simple starch grains with hilum, opened pitted stone cell (walls are opened) and crystalline material (body) was less in amount [Plate 1 to 9]. Pharmaceutical Analysis All the three samples were analysed using various standard physicochemical parameters at the pharmaceutical chemistry lab. The pharmaceutical parameters such as aqueous extractive, alcohol extractive, pH, total ash and loss on drying were presented in Table 2. HPTLC On performing HPTLC, the chromatogram of samples GH-2, GH-3 and GH-4 showed peaks with Rf values at 254 nm and 366 nm. [Table 3, Plate 10]

Table 2: Phytochemical parameters of the samples - GH-2, GH-3 and GH-4 Investigation pH Loss on drying Ash value Water soluble extract Alcohol soluble extract

GH-2 7.0 1.086% w/w 4.90% w/w 64.3% w/w 54.8% w/w

GH-3 7.0 9.303% w/w 15.84% w/w 57.2% w/w 43.5% w/w

GH-4 7.0 7.196% w/w 8.82% w/w 67.1% w/w 44.8% w/w

Table 3: Rf value at 254nm and 366nm of samples - GH-2, GH-3 and GH-4 Samples- HPTLC 1. Haritaki soaked with Gomutra (GH-2) 2. Haritaki Boiled with Gomutra (GH-3) 3. Haritaki Bhavana with Gomutra (GH-4)

Rf value at 254 nm 0.04, 0.26, 0.35, 0.41, 0.50, 0.66, 0.89, 0.95 0.09, 0.36, 0.42, 0.48, 0.56, 0.68, 0.80, 0.93 0.14, 0.26, 036, 0.42, 0.60, 0.67, 0.80, 0.90, 0.97

Rf value at 366 nm 0.17, 0.35, 0.43, 0.56, 82, 0.88 0.11, 0.21, 0.36, 0.42, 0.49, 0.57, 0.79, 0.86 0.14, 0.36, 0.42, 0.56, 0.77, 0.89

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Plate 1 to 9: Comparison of Microphotographs of GH-2, GH-3 and GH-4

Plate 1: Epicarp cell of GH-2, GH-3 and GH-4

(a) Epicarp cell with tannin content and loosened wall of GH-2

(b) Clumped epicarp cell of GH-3

Plate 2: Mesocarp cell of GH-2, GH-3 and GH-4

(a) Mesocarp cell ruptured of GH-2

(a) Mesocarp cell more squeezed of GH-3

(b) Disturbed mesocarp cell of GH4

Plate 3: Fibers of GH-2, GH-3 and GH-4

(a) Fiber unchanged as Haritaki

(b) Fiber with narrow lumen of GH-3

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Plate 4: Sclereids of GH-2, GH-3 and GH-4

(a) Sclereids with wide lumen of GH-2

(b) Groups of sclereids of GH-3

Plate 5: Pitted vessels of GH-2 and GH-3

(a) Pitted vessels of GH-2

(b) Fragment of pitted vessels of GH-3

Plate 6: Simple starch grains of GH-2, GH-3 and GH-4

(a) Simple starch grains unchanged as Haritaki of GH-2

(b) Simple starch grains with hilum of GH-3

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Plate 7: Stone cell of GH-2, GH-3 and GH-4

(d) Pitted stone cell with wide lumen of GH-2

(e) Pitted stone cell of GH-3

Plate 8: Paranchyma cell of GH-2 and GH-3

(a) Paranchyma cell with starch grains of GH-2

(b) Paranchyma cell with brown content of GH-3

Plate 9: Crystalline Material of GH-3 and GH-4

(a) Crystalline material of GH-3 (may be from Gomutra)

(b) Crystalline material of GH-4 (less compare to GH-3)

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Global J Res. Med. Plants & Indigen. Med. | Volume 5, Issue 5 | May 2016 | 146–157

Plate 10 : Peak display of Rf value GH-2 GH-3 and GH-4 at 254nm and 366nm

figure a: Peak display of Rf value GH-2 at 254nm and 366nm

figure b: Peak display of Rf value GH-3 at 254nm and 366nm

figure c: Peak display of Rf value GH-4 at 254nm and 366nm

UV-VIS-NIR All the 6 samples were used for UV-VISNIR reflectance study with different media and different preparation methods. Results of three samples GH-2, GH-3, GH-4 are discussed in the present article. In principal component analysis (PCA), PC-I- 92.6%, PC-II – 98.25%, PC-III – 99.84% data was obtained. GH-2 and GH-4 samples appeared similar. GH-2 and GH4 were similar in Near IR region but they were

different from all other samples. GH-3 was totally different from other samples and overall spectrum intensity was lower. GH-3 had higher variation while other sample had higher leverage. Chemical profile of GH-3 was changed. Plate-11 [Figure (a), (b) and (c)] shows raw spectrum of NIR reflectance of spectra, 1st difference NIR reflectance of all samples after conversion into log [1/% R] preprocessing and NIR UNSCR respectively.

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Plate 11: UV VIS NIR Reflectance of the samples

Figure (a) : raw spectrum of NIR reflectance of spectra

Figure (b): NIR PCA score plot [GH-1: Haritaki Churna, GH-2: Klinna Sankarita Gomutra Haritaki, GH-3: Gomutra Swedita Haritaki GH-4: Gomutra Bhavita Haritaki, GH-5: Jala Swedita Haritaki, GH-6: Eranda Bhrista Haritaki]

DISCUSSION: Organoleptic characters Rupa (Colour) is Guna of Agni Mahabhuta (fire element). Colour of raw Haritaki was golden yellow which was changed as brown after Swedana process with Gomutra (GH-3) while yellowish brown and brownish yellow

was observed in Klinna (GH-2) and Bhavana (GH-4) samples respectively. It indicates that Agni Mahabhuta was increased in all three Samsakarita Gomutra Haritaki samples. It suggests that Agni Tatwa (Ushna-hot, Tikshnapenetrating, Ruksha-dry, Vishada-cleansing, Sukshma-micro Guna) (Acharya Y.T., 2012) increase during process of Klinna, Bhavita and

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Swedita Gomutra Haritaki. Gandha (Odour) is Guna of Prithvi Mahabhuta (earth element). Odour of Haritaki was characteristic which was changed as Gomutra Gandhi GH-2, GH-3, GH4 samples but GH-3 sample had most Tikshna Gomutra Gandha. Due to Ushna, Sukshma Guna of Swedana Karma and Ushna, Tikshna, Drava Guna of Gomutra, it may have decreased the qualities of Parthiva Ghataka of Haritaki and increased Mriduta (Dravata) and Sukshmata in Gomutra Haritaki leading to Gomutra Gandhi character. Rasa (Taste) is Guna of Jala Mahabhuta (water element). Taste of raw Haritaki was Kashaya (Astringent) which was changed as pungent with piercing taste in GH-3 sample. Taste was changed in only Swedita Gomutra Haritaki (GH-3). It is due to Ushna and Tikshna (penetrating) Guna of Swedana and Bhedana (piercing) Karma of Gomutra. Klinna (GH-2) and Bhavita Gomutra Haritaki (GH-4) were Kashaya as simple Haritaki (GH-1). Sparsha (Touch) is Guna of Vayu Mahabhuta (air element). Touch of GH-2, 3, 4 samples were fine coarse but fineness was most increased in GH-3 which may be due to boiling effect of Gomutra on Haritaki. Pharmacognostical Epicarp cells were changed as loosened epicarp cells (GH-2), clumped epicarp cell (GH-3) and collapsed epicarp cells in comparison with normal Haritaki. Mesocarp cell in GH-2, GH-3, GH-4 samples were changed as ruptured mesocarp cells (cluster crystal are not observed), squeezed mesocarp cells and disturbed mesocarp cells respectively. In GH-2 sample, due to only soaking, wall of epicarp cell were loosened and mesocarp cells were ruptured. In GH-3, due to Swedana Samskara, Ushna-Tikshna Guna and Bhedana (Shivprasad Sharma, 2012) Karma of Gomutra, epicarp cells were clumped and mesocarp cells were squeezed. In GH-4 may be due to mechanical pressure of Bhavana Samskara, epicarp cells were collapsed and mesocarp cells were disturbed. In GH-2, fibers of simple Haritaki were unchanged while GH-3 and GH4 were changed as disturbed wall fibers. In GH4 sample, sclereids were ruptured and disturbed

due to Bhavana Samskara. All samples were found with pitted stone cells but GH-4 was found with opened wall stone cells. GH-3 had changed parenchyma cells with brown content. Probably, it may be due to Pittala Guna (Acharya Y.T., 2012) and Dipana, Pachana and Bhedana Karma of Gomutra (Shivprasad Sharma, 2012). GH-3 and GH-4 were found with crystalline material but it was less in Bhvana Samskarita Gomutra Haritaki (GH-4) in comparison to Swedana Samskarita Gomutra Haritaki (GH-3). Presence of crystalline material may be due to Saksharatvata of Gomutra (SS, Sutra Sthana 45/220-221, pp 213) (Acharya Y.T., 2012). Changes in the intra cellular structures were also found which may be due to increase bio-availability of intra cell nutrients due to Swedana Samskara of Gomutra. This finding also indicates that Gomutra is bio-availability enhancer (Gurpreet Kaur Randhawa, 2010). Pharmaceutical Loss on drying in Klinna (soaked) Gomutra Haritaki (GH-2) was on lower side (1.086% w/w) while Swedita Gomutra Haritaki (GH-3) was on higher value (9.303% w/w). It suggests moisture content was more in GH-3. It can be stated that Haritaki absorbs Gomutra materials highest in Swedana followed by Bhavana and Klinna Gomutra Haritaki. Total Ash value of GH-3 was 15.84% w/w which was more from other samples. Ash value indicates inorganic component of drug. While comparing GH-2, GH-3, GH-4, Ash values were higher side in GH-3 (15.84% w/w) followed by GH-4 (8.82% w/w) and GH-2 (4.90% w/w) samples. It means that after Swedana Samskara, Gomutra Haritaki had highest Gomutra Ksharabha Tatwa while Klinna Gomutra Haritaki had lower. Values of samples indicate that role of Gomutra in Swedana effect over Haritaki. pH was 3.0 in samples GH-1 i.e., plain Haritaki. After Gomutra processed Haritaki GH-2, GH-3 and GH-4, pH were changed to 7.0. Average pH value of Gomutra is near by 7.6 to 8.2. Haritaki is Kashaya Rasa (dominant of astringent taste) Pradhana Dravya (Prithvi and Vayu Mahabhuta Pradhanya) and Gomutra is Katu Rasa (dominant of pungent taste)

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Pradhana Dravya (Vayu and Agni Mahabhuta Pradhana). Haritaki is Amla Pradhana Dravya (dominant of sour) while Gomutra is Kshara Pradhana Dravya (dominant of caustic). Amla and Kshara Samyoga creates Madhuryama (Acharya Y.T. 2009). After process with Gomutra, all three Samskarita sample had similar pH. Variation in pH value in plain Haritaki and Samskarita Haritaki indicate Madhurikarana process. Water soluble extract GH-2 (64.3% w/w), GH-3 (57.2% w/w), GH-4 (67.1% w/w) were increased in comparison to plain Haritaki (49.2% w/w). The water soluble extractive of GH-3 was less in comparison to GH-2 and GH-4 samples. The water soluble contents dissolve and separate from the raw Haritaki during Samskara, this phenomenon occurs by molecular diffusion mechanism (Carter SJ, 2000), and follows the principles of mass transfer. Mass transfer increases as the viscosity of the liquid decrease. The alcohol soluble extractives were 54.8% w/w in GH-2, 43.5% w/w in GH-3 and 44.8% w/w in GH-4. Comparison showed that Klinna Gomutra Haritaki (GH-2) had high value than GH-3, GH-4. This indicates removal of the content during the procedure of Swedana and Bhavana. HPTLC HPTLC study of the GH-2 has yielded a standard fingerprint of the formulation consisting of 8 and six peaks on short and long UV, respectively with common Rf value of 0.35. HPTLC study of the GH-3 has yielded a standard fingerprint of the formulation consisting of 8 and eight peaks on short and long UV, respectively with common Rf value of 0.36, 0.42. HPTLC study of the GH-4 has yielded a standard fingerprint of the formulation consisting of 9 and six peaks on short and long UV, respectively with common Rf value of 0.14, 0.36, 0.42. In GH-2, 0.64 Rf value had high absorbance and 0.82 Rf value was common in GH-2, 3, 4 samples. [Table 3, Plate 10]

UV-VIS-NIR Considering all UV-VIS-NIR, total 2302 data points were found (UV 200–400 λ, VIS 401–800 λ, NIR 801–2500 λ), 99.84% data was found in 3 PC. Samples GH-2 and GH-4 were similar on basis of spectrum performance and had similar types of chemical profile but low level of soaking generate shift of quadrate among two samples. GH-3 had higher variation while other sample had higher leverage. Gomutra Swedita Haritaki (GH-3) had apparent different profile from Klinna (Soaked) Gomutra Haritaki (GH-2) and Bhavita Gomutra Haritaki (GH-4). It clears that Swedana with Gomutra of Haritaki had different effect over Haritaki. Hence, it may have higher potency and also penetration than other two preparation methods. CONCLUSION: Changes in pharmacognostical, pharmaceutical and UV-VIS-NIR reflectance (180–2500 nm) findings of Gomutra Swedita Haritaki (GH-3) had highest variation as compared to plain Haritaki sample. Among three methods of Gomutra Haritaki preparation, Swedana Samskrita Gomutra Haritaki had highest potency, Sharira Dhatu Samanyatwa and Anabhishyandi property due to Ushna, Tikshna, Sukshma, Pittala Guna and Dipana, Pachana, Bhedana, Ksharana Karma of Gomutra. Changes reported in intra cellular structures indicate towards increased bioavailability of intra cell nutrients due to Swedana Samskara. This finding also indicates that Gomutra is a bio-availability enhancer. Ultimately, it proves that Agni Mahabhuta increases after Swedana process with Gomutra and Haritaki. However correlation between these changes in all three samples (GH-2, GH3, GH-4) and clinical efficacy need to be assessed independently.

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REFERENCES: Acharya YT (2009), Charaka Samhita, with Ayurveda Dipika commentary by Chakrapani, Chaukhambha Surbharati Prakashana, Varanasi. Acharya YT (2012), Sushruta Samhita, with Nibandhasamgraha commentary by Dalhana, Chaukhambha Surbharati Prakashana, Varanasi. Carter SJ (Ed.) (2000), Cooper and Gunn’s Tutorial Pharmacy. New Delhi: CBS Publishers and distributors.

241. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articl es/PMC3117312/ (02.08.2015) Hari Sadasiva Sastri Paradakara Bhisagacharya (2014), Astanga Hridaya with Sarvangasundari and Ayurveda Rasayana commentary by Arunadatta and Hemadri, Chaukhambha Surbharati Prakashana, Varanasi. Khandelwal K.R. (Ed.) (2008), Practical Pharmacognosy techniques and experiments. Pune: Nirali Prakashan.

Dhundhiraj Shastri (Ed.) (2007), PrashtapadaPadartha dharmasangraha, Varanasi: Chowkhamba Publications.

Shivprasad Sharma, Astanga Samgrah, with Shashilekha commentary by Indu, Chaukhambha Sanskrit Series Office, Varanasi.

Gurupreet Kaur Randhawa (2010), Cow urine distillate as bio enhancer. J Ayurveda integr Med, Volume 1 (issue 4) 240–

Stahl E (Ed.) (1969), A Laboratory hand book. Berlin: Springer-Verlag.

Source of Support: NIL

Conflict of Interest: None Declared

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Research article A COMPARATIVE CLINICAL TRIAL TO ASSESS THE ADD ON EFFECT OF ASVAGANDHA [WITHANIA SOMNIFERA (L.) DUNAL] IN MOTOR FUNCTIONS OF CHILDREN WITH MOTOR DEVELOPMENTAL DISORDERS Preethy AS1*, Dinesh KS2, R Remadevi3 1

MD. Holder, Dept. of Dravyaguna Vijnana, V.P.S.V. Ayurveda College, Kottakkal, Kerala, India Associate Professor & Head, Department of Kaumarabhritya, V.P.S.V. Ayurveda College, Kottakkal, Kerala, India 3 Retd. Professor & Head, Dept. of Dravyaguna Vijnana, V.P.S.V. Ayurveda College, Kottakkal, Kerala, India *Corresponding Author: E-mail address: dr.preethy.cinnish@gmail.com 2

Received: 10/04/2016; Revised: 30/04/2016; Accepted: 20/05/2016

ABSTRACT Motor Developmental Disorders are caused by an impairment or interruption in the normal growth and development of a child in motor grounds. Asvagandha [Withania somnifera (L.) Dunal]. was found to be an apt drug in this condition theoretically, which is balya (strengthening), brmhana (nourishing), rasayana (rejuvenating), medhya (brain stimulant) and also proved to have effect in the neuromuscular level. The study was planned as a Comparative Clinical Trial to assess the add on effect of Asvagandha along with the conventional treatment adopted by the Department of Kaumarabhritya (Paediatrics) at V.P.S.V. Ayurveda College Hospital, Kottakkal, Kerala, India. Twenty participants diagnosed with motor developmental disorders were selected and were divided into study and control groups. Duration of drug administration was three months. Assessment of both the groups was carried on the basis of four subjective parameters such as muscle bulk, power, tone and deep tendon reflexes. Statistical procedure used was R M ANOVA. Study group had an upper hand over the control in most of the parameters especially in the field of ‘power’ and ‘tone’. But statistically the result was not significant. KEY WORDS: Asvagandha [Withania somnifera (L.) Dunal], Motor developmental disorders, Comparative clinical Trial, Power, Tone

Cite this article: Preethy AS, Dinesh KS, R Remadevi (2016), A COMPARATIVE CLINICAL TRIAL TO ASSESS THE ADD ON EFFECT OF ASVAGANDHA [WITHANIA SOMNIFERA (L.) DUNAL] IN MOTOR FUNCTIONS OF CHILDREN WITH MOTOR DEVELOPMENTAL DISORDERS, Global J Res. Med. Plants & Indigen. Med., Volume 5(5): 158–172

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INTRODUCTION Developmental disorders are those which are caused by an impairment or interruption in the normal growth and development of a child. All these conditions are encephalopathies and cerebral palsy can be an umbrella term which comprises of all the symptoms of these disorders. The underlying reason may be due to a defect at the levels of axonal and dendritic development, the rate of pruning (neurological regulatory processes), synaptogenesis, the quality of neuro transmission or myelination (Sara.J.Webb and Christopher S.Monk, 2001). These are very prevalent nowadays, estimated to be 1.5–2.5 per 1000 live births (Forfar and Arneils, 2008). The common clinical features include a defect in gross motor development, defect in fine motor development, a decrease in muscle bulk, a decrease in muscle power, an increase or decrease in muscle tone, co ordination of movement will be impaired, deep tendon reflexes will be exaggerated or diminished, abnormal movements will be usually present. Recent advances in the modern management of the cerebral palsy include intrathecal baclofen pump, multilevel orthopedic bony and soft tissue surgery and direct injection of botulinum toxin (Botox) into the affected muscles for decreasing the muscle spasticity (Odle, Teresa, 2006). They may also trigger significant side effects, such as drowsiness, and their enduring effects on the developing nervous system are largely unknown. It is seen that good outcome is obtained by ayurvedic management in the motor developmental disorders (Roshni Anirudhan, 2009). The query to find out better medicines for existing disease paved way to the drug Asvagandha [Withania somnifera (L.) Dunal] which is specifically mentioned as a rasayana (rejuvenating) drug for children along with its brmhana (nourishing),balya (strengthening) and medhya (brain stimulant) (Hari Sadasiva Sastri Paradakara, 2011) properties. But the

drug has not been tried in the motor developmental disorders yet. Therefore keeping in mind aforesaid things, present study was planned with the following objective: To reveal the unknown pharmacological effect of Asvagandha [Withania somnifera (L.) Dunal] in motor functions of children with motor developmental disorder. MATERIALS AND METHODS The study was approved by Institutional Ethics Committee. Approval no: IEC/Cl/009/11 dated 07.04.11 The study design was a comparative clinical trial. The trial drug Asvagandha curna [powder of Withania somnifera (L.) Dunal] was obtained from Arya Vaidya Sala, Kottakkal (a GMP certified company). Twenty participants diagnosed with motor developmental disorders were selected and were divided into two groups, study and control by randomization, using random number table. Here the investigator was kept blind. Both the groups received conventional treatment adopted by the Department of Kaumarabhritya (Paediatrics). In addition study group received Asvagandha curna [powder of Withania somnifera (L.) Dunal] for a period of three months. Time of administration was morning and evening with cow’s milk as adjuvant which was provided readily in the hospital for all patients and the subjects were strictly asked to follow the same way for the next two months also. The dose was decided according to Cowlings rule. [child dose = adult dose × (age+1)/12 ]. adult dose is 3–6 g (API, 2001). Assessment of both the groups was carried out at before treatment period, after treatment and during monthly follow ups for five months.

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Setting for study

OBSERVATION

I.P.D of Dept. of Kaumarabhrtya, V.P.S.V Ayurveda College Hospital, Kottakkal, Kerala, India

In the criteria muscle bulk, that of bilateral arm, fore arm, thigh and calf was measured. The criteria power covered that of bilateral upper and lower limbs. In deep tendon reflexes, those of bilateral biceps tendon, triceps tendon, patellar tendon (knee) and achilles tendon (ankle) were elicited. In the criteria tone, that of bilateral shoulder, elbow, wrist, hip, knee and ankle were assessed.

Study Population Selection of participants was done irrespective of gender, caste, religion and economic status from population between ages 2 to 12 years. Participant’s recruitment and screening Participants were selected according to the inclusion and exclusion criteria. a)

Inclusion criteria

Diagnosed patients as per the modified Denver developmental screening test for gross & fine motor functions within the age group 2–12 years with proper consent from the parent. b)

Exclusion criteria

The children in whom oral administration of the drug was difficult, with other associated degenerative diseases, above 10 years of age and has not taken any treatments so far and with contractures were also excluded. Common clinical features found in motor developmental disorders are a defect in gross motor development, defect in fine motor development, a decrease in muscle bulk, a decrease in muscle power, an increase or decrease in muscle tone, impaired co ordination, exaggerated or diminished deep tendon reflexes (Kenneth F.Swaiman, 2007). So for the research purpose, changes in muscle bulk, muscle power, muscle tone and in deep tendon reflexes were selected as criteria for assessment (Dinesh.K.S., Associate Professor & Head, Department of Kaumarabhritya, V.P.S.V. Ayurveda College, Kottakkal, personal communication).

DATA ANALYSIS Data outcome were tabulated. Repeated Measures of ANOVA was performed using the statistical package (SPSS) version 16.0 to find out level of significance for all the scales. within-the subjects effects, between-the subjects effects and estimates were calculated for the criteria muscle bulk, power and tone. Here, for the easy comparison of estimates, the rate of change is calculated, making the baseline data ‘zero’. And the statistical analysis was done on this data. In the case of deep tendon reflexes One sample ‘t’ test was performed for the comparison of the variable with the specific value. Then repeated measures of ANOVA were performed to calculate between-the subject’s effects and estimates. In the case of ‘power’ the degrees of freedom are adjusted to attain sphericity (sphericity is an assumption of an ANOVA with a repeated measures factor and violation of this assumption can invalidate the analysis’ conclusions. Sphericity relates to the equality of the variances of the differences between the levels of the Repeated Measures Factor). RESULTS Results can be explained well with the help of graphs obtained from statistical package (SPSS) version 16.0 and the tabulated data generated by Repeated Measures of ANOVA.

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MUSCLE BULK Table No 1: comparison of 20 participants on the rate of change of circumference of arm, fore arm, thigh and calf (R M ANOVA) Within –the subjects effects Parameter

Rt.arm Lt.arm Rt. fore arm Lt. fore arm Rt.thigh Lt.thigh Rt.calf Lt.calf

Group

F(6,54)

Study

0.497

>0.05

Control Study Control Study Control Study Control Study Control Study Control Study Control Study Control

0.443 0.816 0.548 0.469 0.325 0 1.325 2.528 1.695 2.528 1.695 0.595 2.062 1.425 0.333

>0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05

Between the subjects- effects

Estimates

F(1,18)

Mean effect

case

control

0.313

>0.05

0.035

0.017

0.198

>0.05

0004

-0.003

0.012

0.056

>0.05

0.026

0.030

0.014

2.094

>0.05

-0.0017

0.035

0.012

0.313

>0.05

0.029

0.026

0.016

0.198

>.05

0.037

0.008

0.019

0.416

>0.05

0.015

0.032

0.018

0.590

>0.05

0.037

0.015

0.021

The table (Table No: 1) shows that, in the rate of change of circumference of right and left arm, both the Study and control groups have all the p values (both Within –the subjects effects and between the subjects- effects) >0.05, which means that they are insignificant. The Table No: 1 shows that, in the rate of change of circumference of right and left fore arm, both the study and control groups have all the p values >0.05, which means that they are not significant.

The Table No: 1 shows that, in the rate of change of circumference of right and left thigh, both the study and control groups have all the p values >0.05, which means that they are not significant. The table No: 1 shows that, in the rate of change of circumference of right and left calf, both the study and control groups have all the p values >0.05, which means that they are insignificant.

Graph No 1: comparison of 20 participants on the rate of change of circumference of arm

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Graph No 2: comparisons of 20 participants on the rate of change of circumference of fore arm

Graph No 3: comparisons of 20 participants on the rate of change of circumference of thigh

Graph No 4: comparisons of 20 participants on the rate of change of circumference of calf

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POWER Table No 2: comparisons of 20 participants on the rate of change of power of upper limb and lower limb (R M ANOVA) Within -subjects Effect Parameter

Group

Between - subjects effects

Estimates

F(1,18)

Mean effect

Study

control

Rt.UL

Study

(5,35)

4.590

<0.01

0.436

>0.05

0.220

0.158

0.067

Lt.UL

Control Study

(6,42) (6,54)

4.009 4.522

<0.01 <0.001

0.034

>0.05

0.135

0.122

0.053

Control

(6,54)

4.402

<0.001

Study Control Study Control

(5,35) (5,35) (5,35) (6,36)

2.758 2.625 4.590 1.233

<0.05 <0.05 <0.01 <0.01

1.428

>0.05

0.030

0.083

0.032

3.836

>0.05

0.036

0.118

0.029

Rt.LL Lt.LL

[df - degrees of freedom,F-Fisher’s co-efficient, Rt.UL- Right Upper Limb, Lt.UL- Left Upper Limb, Rt. LL – Right Lower Limb

Graph No 5: comparisons of 20 participants on the rate of change of power of upper limb

Graph No 6: comparisons of 20 participants on the rate of change of power of lower limb

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The above table (Table No: 2) shows that in the parameters of the rate of change of power of right upper limb and left upper limb, all the p values of within- subjects effects were <0.05,indicating that there was significant change in the power of upper limbs in both groups. But in the case of between - subjects effects, the p values were >0.05, indicating insignificance. The Table No: 2 suggests that, for the parameter rate of change of power of a significant change in the power of right lower limb in both groups in within the - right lower limb, both the study and control groups shows

p <0.05,which means that, there is subjects effects. On the contrary, in the case of power of left lower limb, the study group only showed p <0.05 (in within the -subjects effects), which means that there is a significant change in the power of left lower limb in this group. But in the case of rate of change of power of left lower limb, control group showed p >0.05, which indicates that, there is no significant change, in within the subjects – effects. In the case of between the subjects- effects, the p values were >0.05, which implies that, there is no significant change in power of lower limbs in both the groups on comparison.

TONE Table No 3: comparisons of 20 participants on the rate of change of tone of shoulders, elbow and wrist (R M ANOVA) Within –the subjects effects df

Parameter

Rt. shoulder

Study

(6,42)

2.493

<0.05

Control

(6,42)

2.719

<0.05

Lt. shoulder

Study

(6,42)

10.548

<0.001

Control

(6,42)

2.406

<0.05

Rt.elbow

Study

(6,42)

0.460

>0.05

Control Study Control Study Control Study Control

(6,42) (6,42) (6,42) (6,42) (6,42) (6,42) (6,42)

0.518 1.197 2.109 1.137 1.124 1.137 2.358

>0.05 >0.05 >0.05 >0.05 >0.05 >0.05 <0.05

Lt. elbow Rt.wrist Lt. wrist

The output of R M ANOVA shows that, in the case of rate of change of tone of shoulders, both the study and control groups have shown p values <0.05 within-the subjects effects, which means that there is significant difference in the rate of change of tone of shoulders in both the groups . But the p values were >0.05, in between- the subjects effects, indicating there was no significant difference between the two groups in the case of rate of change of tone of shoulders.

Betweeneffects F(1,18)

the

subjects

Estimates Mean effect Study

control

0.111

>0.05

-0.271

-0.330

0.125

3.769

>0.05

-0.330

-0.323

0.121

0.722

>0.05

0.219

-0.076

0.016

>0.05

-0.115

-0.156

0.066

>0.05

-0.146

-0.167

0.195

1.116

>0.05

-0.042

-0.302

0.174

In the case of rate of change of tone of elbows (Table No: 3), both the study and control groups were found to have p values >0.05 in within- the subjects effects, indicating that there was no significant change in both the groups. Between- the subjects effects also shows the p values were >0.05, confirming that there was no significant difference in the tone of elbows when the two groups are compared.

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In the case of rate of change of tone of wrists (Table No: 3), the output of R M ANOVA indicates that, in within–the subjects effects, the p values are >0.05, which means that no significant change has happened in the rate of change of tone of right wrist, in both groups. While coming to the rate of change of

tone of left wrist, in within–the subjects effects, the control group has shown a p<0.05, indicating statistically significant change. But when it comes to between- the subjects effects, both the p values were >0.05, which means that there is no significant difference in rate of change of tone, when the groups are compared.

Graph No 7: comparisons of 20 participants on the rate of change of tone of shoulders

Graph No 8: comparisons of 20 participants on the rate of change of tone of elbows

Graph No 9: comparisons of 20 participants on the rate of change of tone of wrists

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Graph No 10: comparisons of 20 participants on the rate of change of tone of hips

Table No 4: comparisons of 20 participants on the rate of change of tone of hip, knee and ankle (R M ANOVA) Within –the subjects effects df

Parameter Rt.hip

Lt.hip

Rt.knee

Lt.knee

Rt.ankle

Lt.ankle

Study

(6,54)

1.495

>0.05

Control

(6,54)

0.510

>0.05

Study

(6,54)

0.534

>0.05

Control

(6,54)

2.101

>0.05

Study

(6,42)

2.075

>0.05

Control

(6,42)

0.960

>0.05

Study

(6,42)

0.389

>0.05

Control

(6,42)

1.467

>0.05

Study

(6,42)

1.556

>0.05

Control

(6,42)

1.890

>0.05

Study

(6,42)

2.064

>0.05

Control

(6,42)

0.715

>0.05

Between- the subjects effects

Estimates

F(1,18)

Mean effect

Study

control

0.076

>0.05

0198

-0108

0.231

0.936

>0.05

-0.104

-0.406

0.221

0.128

>0.05

-0.212

0.111

0.224

0.010

>0.05

0.125

0.135

0.210

1.388

>0.05

-0.080

0.319

0.240

0.895

>0.05

0.205

-0.012

0.144

Within–the subjects effects of rate of change of tone of hip (Table No: 4) suggests that, since all the p values being >0.05, the effect of treatment in the rate of change of tone of right and left hips are not significant. Between-the subjects effects also suggest the same, i.e., no statistically significant difference is seen between the two groups, for the p values are >0.05.

Within–the subjects effects of the rate of change of tone of knees (Table No: 4) implies that, all the p values are >0.05, and so the effect of treatment is not statistically significant within the study as well as in the control group. In between- the subjects effects too, the p values being >0.05, suggests no significant change has seen on comparing the two groups.

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Withinâ&#x20AC;&#x201C;the subjects effects of rate of change of tone of ankles (Table No: 4) implies that, all the p values are >0.05, and so the effect of treatment is not statistically significant within the study as well as in the

control group. In between- the subjects effects too, the p values are >0.05, suggesting no significant change has happened on comparison of the two groups.

Graph No 11: comparisons of 20 participants on the rate of change of tone of knees

Graph No 12: comparisons of 20 participants on the rate of change of tone of ankles

DEEP TENDON REFLEXES Graph No 13: comparisons of 20 participants on the deep tendon reflexes of ankles

2 1 0

Left Ankle Jerk

case 1 2 3 4 5 6 7 visit

control

grade

Right Ankle jerk 2 1 0

case 1 2 3 4 5 6 7 visit

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control

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Graph No 14: Comparisons of 20 participants on the Deep Tendon Reflexes of Knees 3

Left knee jerk

Right Knee Jerk

grade

case

5 0

case 1

control

0 1

4 visit

control

visit

Graph No 15: Comparisons of 20 participants on the Deep Tendon Reflexes of Biceps left biceps jerk

2 0

grade

right biceps jerk case 1 2 3 4 5 6 7

2 0

case 1

control

visit

control

visit

Graph No 16: comparisons of 20 participants on the deep tendon reflexes of triceps right triceps jerk

2 0

case 1

grade

grde

left triceps jerk 2 0

case 1 2 3 4 5 6 7

control

visit

control

visit

Table No 5: comparisons of 20 participants on the deep tendon reflexes (R M ANOVA) Area of assessment

Rt. Knee Lt.knee Rt.ankle Lt.ankle Rt. biceps Lt.biceps Rt.triceps Lt.triceps

Mean effect

Standard error

Study

Control

1.958 1.854 0.979 0.937 1.062 1.042 0.958 0.979

2.146 2.062 1.021 1.187 1.042 1.062 1.062 1.062

0.236 0.048 0.058 0.098 0.048 0.048 0.053 0.056

Test of between subjects effects F(1,18)

0.316 0.093 0.255 3.252 0.093 0.093 1.923 1.098

>0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05 >0.05

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One sample ‘t’ test was performed for every parameters of deep tendon reflexes (Graphs 13-16) and it was found that, in within-the subjects effects all the p values were >0.05, which denotes that there is no significant difference between the variable and the specific constant. Repeated measures of ANOVA was applied for the variables of deep tendon reflexes to test the between-the subjects effects (Table No: 5). All the p values were >0.05, which means there was no significant difference between the study and control group. DISCUSSION Muscle bulk The estimates of the repeated measures of ANOVA suggest that study group is having higher mean effect than the control group in the case of rate of change of circumference of arm, thigh and calf (table No:1) In the case of rate of change of forearm circumference (graph No: 2), the right forearm shows a hike in the measurement than the corresponding side, but it is not so in the case of circumference of left forearm. So it is difficult to come to a conclusive statement about the effect of Asvagandha in the rate of change of fore arm circumference. The graphs (graph No: 3) for the rate of change of circumference of thigh indicate an increasing tendency of the parameter in both the groups. Though the p values (Table No: 1) for within – the subjects effects and between the subjects effects are >0.05, which indicate insignificance, the estimates shows that the study group is having higher mean effect than the control group. The graphs (graph No:4) for the rate of change of circumference of calf, indicates that there is an increasing tendency in the circumference of both right and left calf regions, when the study group is considered.

But the change is not consistent when control group is judged. To conclude on muscle bulk, it can be said that, in 62.5% of assessment areas, study group showed higher mean effect than the control. Previous research work on Asvagandha [Withania somnifera (L.) Dunal] also substantiates this finding (Meena.S.L., 2001). Power The graphs (Graph No: 5) for the rate of change of power imply the increasing tendency of power of upper limbs in both the groups. The table (table No: 2) shows that, in within – the subjects effects for the rate of change of power of upper limb all the p values are <0.01, indicating statistical significance. But in between - the subjects effects, both p values are >0.05 indicating insignificance, but the estimates suggest a higher mean effect of study group than control, which says that, obviously, there is increase of power in the study group compared to the control group, but it is not consistent. In the case of rate of change of power of lower limb, the graphs (graph No: 6) indicate an increasing tendency in both the groups. Within-the subjects effects of rate of change of power of lower limbs suggest statistical significance for both the groups. But study group only had significance regarding the rate of change of power of left lower limb (table No: 2). Both the groups didn’t show any significance in between - the subjects effects. In within – the subjects effects of right lower limb, though both the p values (Table No: 2) are <0.05, indicating its significance, on comparison, the control group is having higher mean effect than the study group. To conclude on Power, it can be said that, in 75% of assessment areas, study group showed a higher mean effect than control. This can be read along with the effect of Asvagandha[Withania somnifera (L.) Dunal] in enhancing locomotor functions (Natsuki Nakayama et al., 2007).

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Tone All the participants of this research were having spastic cerebral palsy. So spasticity or hyper tonicity was present in every one. Reduction of tone is the favorable outcome here. The graph (graph No:7) for the rate of change of tone of shoulders illustrates a decreasing tendency of tone in both the groups; but the effect is more marked in the case of study group. On further analysis, it is found that, both the groups are having, within-the subjects effects significance for p<0.05 (table No: 3). In the case of left shoulder, as far as the study group is concerned, within–the subjects effects showed p<0.001, which is extremely significant. But in between-the subjects effects, both the p values were >0.05, conveying there is no significant difference between the two groups on comparison. But the mean effect was favorable for the control group, when rate of change of tone of Right shoulder is concerned, but it was favorable for the study group in the case of rate of change of tone of left shoulder. The favorable outcome of the control group in the case of rate of change of tone of left shoulder can be due to the fact that, right upper limb was affected in more participants of the study group (57.1%) than control (42.9%). The graph (graph No: 8) illustrating the rate of change of tone of elbow suggests that there is a decreasing tendency of tone in both the groups, but it is more evident in the case of left elbow may be because of the fact that right upper limb (58.3%) was more affected by the disease than the left one (41.7%). Here the control group showed more favorable mean effect (Table no: 3) than the study group. The graphs (Graph No: 9) for the rate of change of tone of wrist, shows that the rate of change of tone of wrist is favorable for both the groups as far as left wrist is concerned. Compared to right wrist, left wrist got more outcomes; the reason may be the same as

mentioned above. On analysis, for, within–the subjects effects of control group showed p<0.05 (table No: 3), which is significant. Here also the mean effect is more favorable in the case of control group. The graph (graph No: 10) for the rate of change of tone of hip suggests there is a decreasing tendency of tone in both the groups. Though the within–the subjects effects and between-the subjects effects have all the p values >0.05 (table No: 4), which are not significant, the mean effect shows, study group is having favorable value in the case of rate of change of tone of right hip while control group is having favorable value in the case rate of change of tone of left hip. The graphs (graph No: 11) for the rate of change of tone of knee, suggest there is a decreasing tendency of tone in both the groups. Within–the subjects effects and between - the subjects effects have all the p values >0.05 (table No: 4), which are not significant. But in the estimates, the mean effect shows that study group is having desirable mean effect than the control. The illustrations (graph No: 12) for the rate of change of tone of ankle suggest that there is a decreasing tendency for the rate of change of tone of left ankle for both the groups. But in the case of rate of change of tone of right ankle, study group only showed a decreasing tendency. Anyway, the within–the subjects effects and between-the subjects effects have all the p values >0.05 (table No: 3), which indicates these differences are not statistically significant. But the mean effect is more favorable for the study group. On tone, in 50% of assessment areas, study group showed favorable mean effect than the control and this includes the tone of the ankle joint, the tackling of which has a higher clinical relevance. The neuroinhibitory effect especially GABAergic property of the drug can be accountable for this (Mehta, A.K et al., 1991).

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Deep Tendon Reflexes (DTR) The results of One sample ‘t’ test for deep tendon reflexes pointed out that there is no significant difference between the variable and the specific constant. This consistency of deep tendon reflexes can be justified by comparing these values with that of tone of the corresponding body part, for tone and reflexes are inter related and directly proportional (Guyton and Hall, 2005). For this mean value of tone and deep tendon reflexes were calculated and it was found to be comparable. The graphs (No:13 and No:14) for the deep tendon reflexes of lower limbs suggests that, they (right and left knee jerks and right and left ankle jerks) are somewhat approaching the desired value. But the between-the subjects effects shows all the p values are >0.05 (table No: 5), indicating insignificance. But the R M ANOVA procedure for between-the subjects effects shows that, though the p values are >0.05(table No: 5), the study group shows favorable mean effect than the control group, in the case of deep tendon reflexes of right and left knees and left ankle. The mean effect was similar for both the groups for the deep tendon reflex ofr ankle. The graphs (No: 15 and No: 16) for the deep tendon reflexes of upper limbs suggests that the DTR of triceps is approaching normalcy in due course, while that of biceps is not showing any tendency towards the desired value. Tests of between-the subjects effects showed all the p values are >0.05 (table No: 5), which implies insignificance, but the mean effect says that the values are more favorable in the study group, when the parameters, DTR of right and left triceps and left biceps are considered. But it was favorable for the control group in the case of right biceps jerk.

control. Axon- or dendrite-predominant outgrowth induced by constituents from Ashwagandha [Withania somnifera (L.) Dunal] is already proved, which goes in hand with the action of the same in Deep Tendon Reflexes (Kuboyama T, 2002). CONCLUSION The add-on effect of Asvagandha in motor developmental disorders was measured using specific tools like muscle bulk, power, tone and deep tendon reflexes. Since there is a wide variation among the results, a common conclusion cannot be drawn. So to conclude on muscle bulk, it can be said that, in 62.5% of assessment areas, study group showed higher mean effect than the control. On power, it can be said that, in 75% of assessment areas, study group showed a higher mean effect than control, but it is statistically not significant. On tone, in 50% of assessment areas, study group showed favorable mean effect than the control and this includes the tone of the Ankle joint, the tackling of which has a higher clinical relevance. On DTR in 75% of assessment areas, study group showed favorable mean effect than the control. The inference from the research is that Asvagandha [Withania somnifera (L.) Dunal] is having positive effect in motor developmental disorders. But statistically, it does not possess add on effect Asvagandha in the motor functions of children with motor developmental disorders under the specified setting. Since the drug shows a positive inclination in all the parameters, either-dose, duration, Sample size may be increased and can be further evaluated for more conclusive results. The changes happened to other comorbid conditions like epilepsy and other psycho social factors can be monitored in further studies.

On DTR in 75% of assessment areas, study group showed favorable mean effect than the

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REFERENCES Anonymous (2001), API - The Ayurvedic pharmacopoeia of India reprint 2005; The controller of publication, New Delhi, Part 1, Vol. 1, p.15.

Meena.S.L., (2001), Bala sosha rog par Ashwagandhadi yog ka Chikitsatmak adhyayan. [M D Dissertation], National Institute of Ayurveda, Jaipur.

Forfar and Arneils, Textbook of Paediatrics, 7th Edition (2008), Churchill Livingstone Elsevier Health Science,p-888

Mehta, A.K., P. Binkley, S.S. Gandhi, and M.K. Ticku. (1991). Pharmacological effects of Withania somnifera root extract on GABAA receptor complex. Indian J Med Res. Aug; 94:312–5

Guyton and Hall, (2005).Textbook of Medical Physiology, 11th Edition, Published by Elsevier p-678 Hari Sadasiva Sastri Paradakara, (2011). Vagbhata,‘Astanga hrdaya’ with the commentaries ‘sarvangasundara’ of Arunadatta and ‘Ayurveda rasayana’ of Hemadri, Varanasi: Chaukhambha Sanskrit Sansthan; Uttara sthana 39/159. Kenneth F.Swaiman, (2007). Pediatric Neurology, Principles & Practice, Volume I, Forth Edition, Publisher : Mosby Kuboyama T (2002). Axon- or dendritepredominant outgrowth induced by constituents from Ashwagandha. Neuroreport. Oct 7; 13(14):1715–20.

Natsuki Nakayama, Chihiro Tohda, (2007), Withanoside IV improves hindlimb function by facilitating axonal growth and increase in peripheral nervous system myelin level after spinal cord injury ,Neuroscience Research, 58 (2): June, Pages 176–182. Odle, Teresa (2006), Gale Encyclopedia of Medicine, 3rd ed. Polzin, Scott; Cerebral Palsy. Roshni Anirudhan (2009). A Clinical trial to study The efficacy of selected Ayurvedic treatment modalities In cerebral palsy in children below 8 years. [M D dssertation], University of Kerala, Thiruvananthapuram; p.17. Sara.J.Webb and Christopher S.Monk. (2001). Mechanism of post natal neurobiological developments, implications for human development. Developmental neuropsychology, Lawrance Erlbaran Associate Publishers; 19(2): 147–171

Source of Support: NIL

Conflict of Interest: None Declared

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Review article CONCEPT OF NITYA SEVANIYA AAHARA DRAVYA Saylee Deshmukh1*, M K Vyas2 1

Ph.D.Scholar, Department of Basic Principles, Institute of Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar- India 2 Professor, Department of Basic Principles, Institute of Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar- India. *Corresponding author: Email: dsaylee@ymail.com

Received: 10/02/2016; Revised: 05/03/2016; Accepted: 10/05/2016

ABSTRACT Ayurveda has great potential in the field of Preventive medicine. Diet and Lifestyle plays key role in preventing many diseases. Among them diet is most important as health depends upon the type of diet taken by an individual. In Ayurveda, some food items are advised to be taken for a long duration while some are prohibited which are termed as ‘Nitya sevaniya’ and ‘Nitya asevaniya’ aahara dravya. It depends on the beneficial and harmful properties present in them. Present study aims to explain the rationality behind the concept of ‘Nitya sevaniya aahara dravya’ i.e. diet items which can be consumed for long time.

KEY WORDS: Nitya sevaniya, diet, aahara dravya

Cite this article: Saylee Deshmukh, M K Vyas (2016), CONCEPT OF NITYA SEVANIYA AAHARA DRAVYA, Global J Res. Med. Plants & Indigen. Med., Volume 5(5): 173–182

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INTRODUCTION Ayurveda plays a very important role in prevention of disease, besides treatment of diseases which includes aahara (diet), vihara (lifestyle) and aachara (behavioral pattern). Amongst them aahara i.e. diet is the most important part. Saarata of dhatu which is the reflection of body’s immunity depends on the nature of diet taken. Therefore Acharyas have advised to take only those food items which are helpful to maintain the health (Brahmanand Tripathi, 2006). Nitya sevaniya and asevaniya aahara dravya have been enlisted in Charaka samhita (Brahmanand Tripathi, 2006), Ashtanga Hridaya (Brahmanand Tripathi, 2007), Ashtanga Samgraha (Shivprasad Sharma, 2012) and Kaiyyadeva Nighantu (Priyavrata Sharma et al., 2009). While in Sushruta samhita (Ananta Ram Sharma, 2008), list of ekanta hitakara and ahitakara aahara dravya has been given. These nitya sevaniya and ekanta hitakara dravya are those which can be taken regularly in healthy condition for maintenance of health because of their specific

properties. According to the dictionary of Monier William, word Nitya means ‘for long time’ (Monier Wiliams, 2005) Charaka has divided dravya in three categoriesdoshaprashamana, dhatupradushana and swasthavrittakara (Brahmanand Tripathi, 2006). Doshaprashamana dravya are those used with a therapeutic purpose, dhatupradushana dravya are those which are responsible for disease pathogenesis by vitiation of dhatu and while swasthavrittakara dravya are responsible for maintenance of health by keeping all the three dosha in equal state. Among that Nitya sevaniya aahara dravya can be taken under the third category. Following table shows the list of Nitya Sevaniya aahara dravya mentioned in Ayurveda classics. Table.1 Present study aims to explain the rationality behind the concept of ‘Nitya sevaniya aahara dravya’ in Ayurveda and researches of modern science.

Table.1: List of Nitya sevaniya aahara dravya S. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Substances Amalaka Antariksha jala Dadima Go Dugdha Go Ghrita Godhuma Jangala mamsa Jivanti Madhu Mridvika Mudga Mulaka Pathya/ Haritaki Patola Saindhava Shasthika Shali Sharkara Sunishannaka Vastuka Yava

English/botanical name Phyllanthus emblica L. Rain water Punica granatum L. Cow’s milk Cow’s Ghee Triticum aestivum L. Meat of animals in arid climate Leptadenia reticulata Retz. Honey Vitis vinifera L. Vigna radiata L. Raphanus sativus L. Terminalia chebula Retz. Trichosanthes cucumerina L. Sodii chloridum Oryza sativum L. Sugar Marsilea quadrifolia Linn. Chenopodium album L. Hordeum vulgare L.

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MATERIALS AND METHODS A critical review of texts of Ayurveda like Charaka Samhita, Sushruta Samhita, Ashtanga Hrudaya, Ashtanga Sangraha, Kaiyyadeva Nighantu and research articles related to this subject. DISCUSSION Nitya sevaniya aahara dravya include selected entities among each of the categories like shukadhanya, shamidhanya, shakavarga, phalavarga and aahara upayogi dravya. They have been said to be useful for maintenance of health. Among them mudga (Vigna radiata L.) has been described best in shamidhanya i.e. pulses (Brahmanand Tripathi, 2006) possesses madhura, kashaya rasa, katu vipaka and sheeta virya with laghu and vishada guna (Brahmanand Tripathi, 2006) have high nutritional value. 100g of it produces 334 Kcal of energy. It is rich in carbohydrate (56.7g/100g) and proteins 23.86g. It is very good source for minerals like Potassium (843 mg/100g), Magnesium (127 mg/100g), calcium (124 mg/100g), phosporous (326 mg/100g) and iron (4.4 mg/100g). vitamins like carotene, thiamine, niacin, riboflavin, ascorbic acid and folic acid are also present in mudga (Gopalan C et al., 2007). According to researches antioxidant, antihyperglycemic (Yao Y et al., 2013), detoxifying (Tran Van Hien et al., 2002), antianemic (Manikandaselvi S et al., 2014), antihyperlipidemic (Nobuhiko Tachibana et al., 2012) and anti-microbial (Siti Nazrina Camalxaman et al., 2013) activities have been found in mudga. Regular consumption of mudga can regulate enterobacterial flora of intestine, decrease absorption of toxic substances, reduce risk of hypercholesterolemia and coronary heart disease and prevent cancer (Tang et al., 2014). Mudga extracts were also found to have a potent scavenging activity against pro-oxidant species, including reactive oxygen species and reactive nitrogen species as well as an inhibitory effect on low-density lipoprotein oxidation. (Ill Min Chung et al., 2011)

Shashtika shali (Oryza sativum L.) has been described as best among shukadhanya i.e. cereals. It has kashaya, madhura rasa, madhura vipaka and sheeta veerya (Brahmanand Tripathi, 2006). It is a very good source of carbohydrate, vitamin B complex and minerals like Iron. (Basu S et al., 2012). According to modern researches it posses antioxidant (Priya Gurumoorthy et al., 2014), cytoprotective (Bunyada Jittorntrum et al., 2009), immunomodulatory (Yang L C et al., 2015), hepatoprotective (Sinthorn W et al., 2015) Godhuma (Triticum aestivum L.) possesses madhura rasa, madhura vipaka and sheeta virya with guru and snigdha guna (Brahmanand Tripathi, 2006). It is a very good source of carbohydrate, vitamins and minerals. According to the researches, godhuma has been reported to posses anti-oxidant (Pandey BR et al., 2012), anti-hyperlipidaemic (Ji-Young Im et al., 2015), anti-microbial (Athul Sundaresan et al., 2015) activity, protective to the skin (G. Balint et al., 2006) and gastric (T Lakshmi Srinivas et al., 2013), intestinal mucosa (E. Ben-Arye et al., 2009) and neuro-protective (Han HS et al., 2010) Godugdha (cow’s milk) possesses madhura rasa, madhura vipaka and sheeta virya with guru and snigdha guna. It has been described as aajanmasatmya (compatible from the birth), ojovardhaka (increases vital power of the body) (Brahmanand Tripathi, 2006). It is a source of good quality protein, calcium and vitamins particularly, vitamin A, riboflavin, niacin and folic acid. In addition milk contains several bio-protective molecules that ensure health security to humans. Godugdha has been reported to possess anti-oxidant (Santosh Kumar et al., 2013), immunomodulatory (Opatha Vithana et al., 2012), gut protective activity (Bohuslav Dvorak, 2010). Goghruta (cow’s ghee) has been described in Ayurveda texts to have many beneficial properties like it is rejuvenating, bestows luster and beauty, enhances memory and stamina, increases the intellect, promotes longevity, is an aphrodisiac and protects the body from

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various diseases (Brahmanand Tripathi, 2006). blood brain barrier and also to facilitate Ghee has been proved to posses anti-oxidant, building or supporting the biochemical and anti-atherogenic properties (Chinnadurai K activities of tissues such as neurons (Achliya et al., 2013), gastroprotective and adaptogenic GS et al., 2004, 2005). (S M S Samarakoon et al., 2011). It also Saindhava (Rock salt) has been advised to potentiates antimicrobial activity (Prasad V et take daily. It possesses lavana rasa and sheeta al., 2006), immunostimulant, antioxidant and virya (Brahmanand Tripathi, 2006). But hepatoprotective activity (Fulzele SV et al., worldwide common salt is being taken daily. 2002). So Ghee contains omega-3 fatty acids Chemical formula of both is same as NaCl but which have been proved to improve contents of both differ as rock salt contains cardiovascular health and also modulate disease some extra minerals like potassium which are processes, such as hypertension, coronary beneficial to the health (Neelesh Khandelwal et artery disease, and hypertriglyceridemia (Gerry al., 2012). Contents of rock salt and common Schwalfenberg, 2006). Medhya action of salt are given in Table. 2. goghruta may be due to ability to penetrate Table.2: Chemical component of rock salt and common salt Chemical component Rock salt (%) Common salt (%) 39.00 39.34 Na 0.12 − K − 0.03 Mg − 0.08 Ca 60.27 60.66 Cl − 0.27 SO4 99.77 100 Total Sodium has a contractile mechanism while potassium has proven to have a relaxing effect on the smooth muscle of the arterioles (Sunita Inderjit Singh et al., 1955). So intake of common salt for long time can be one of the most important causes for essential hypertension.

Sharma, 2008). Anti-oxidant activity and skin protecting activity against AGEs and UV-A (Hwa Lee et al., 2014) has been proved. Hepatoprotective (Kumar AK et al., 2015), immune-modulatory (Joseph MM et al., 2012), cytoprotective (Piero Sestili et al., 2007), embryo-protective (Kishore RK et al., 2009)

Amalaki (Phyllanthus emblica L.) has been described in Ayurveda texts to have rejuvenating property which promotes longevity (Brahmanand Tripathi, 2006). Amalaki by its amla rasa, sheeta virya, ruksha guna do not provoke vata, pitta and kapha successively and helps for their maintenance. Antioxidant (Bhattacharya A et al., 1999), immuno-modulatory (Madhuri S et al., 2011), hepato-protective (Karadka Ramdas Thilakchand, 2013) and cyto-protective (M Sai Ram et al., 2002).

Mridvika (Vitis vinifera L.) is also mentioned as pathyatama fruit by Acharya Sushruta (Ananta Ram Sharma, 2008). It has been proved to possess antioxidant (G.K. Jayaprakasha et al., 2001), hepato-protective (Pirinççioğlu M et al., 2012), immunomodulatory (Rajaa k. baker et al., 2014), vasoprotective (Schneider E et al., 2008), neuroprotective (Jin HY et al., 2013), anticancer (Kequan Zhou et al., 2012) properties. It is also protective for heart and kidney against toxicity (B. V. S. Lakshmi et al., 2014) and also an effective gastroprotective (V. M. Cuevas et al., 2011).

Dadima (Punica granatum L.) is also one of the Nitya sevaniya Aahara dravya and it is also mentioned as Pathyatama (Ananta Ram

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Madhu (honey) has been proved to have hepatoprotective (Ateeq M J et al., 2013), cardio-protective (Md. Ibrahim Khalil et al., 2015), cyto-protective (Faeza Abdel Mogib ElDahtory et al., 2011), immunomodulatory (Majtan J, 2014), protective against hepatotoxicity and nephrotoxicity (Wafaa M. AbdelMoneim et al., 2007) Haritaki (Terminalia chebula Retz.) has been described by Acharyas to possess strong Rasayana i.e. rejuvenating property (Brahmanand Tripathi, 2006) and alleviate all the three Dosha (Ananta Ram Sharma, 2008). It has been proved to have anti-oxidant and antimicrobial (Pai Aruna, Rao et al., 2012), antiaging (Manosroi A et al., 2010), immunomodulatory (H.N. Shivaprasad et al., 2006), cardio-protective (Suchalatha S et al., 2004), hepato-protective (Min-Kyung Choi et al., 2015) activities. Jivanti (Leptadenia reticulata Retz.) has been described as best among all the vegetables. (Brahmanand Tripathi, 2006) due to its property of ‘sarvadoshaghni’ (Ananta Ram

Sharma, 2008). It is a rich source of vitamin A (K. Martin et al., 2002) possesses anti-oxidant and free radical scavenging property (Mallikarjuna, B et al., 2011), hepatoprotective (Junapudi Sunil et al., 2015), anti-bacterial (Irimpan MT et al., 2011) and protection against inflammation (Louis, C Jelly, 2015). CONCLUSION Properties of Nitya sevaniya aahara dravyas given in texts of Ayurveda are such that they do not provoke any of the dosha and keep all the three in equal state. Most of these drugs are Laghu i.e. easy to digest which protects the body from formation of aama i.e. indigested food which is responsible for pathogenesis of number of diseases. According to the modern researches all the aahara dravyas possess anti-oxidant property and most of them have immuno-modulatory, cytoprotective, hepato-protective while some possess vasoprotective, anti-microbial properties with very good nutritional value which is beneficial to health.

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Kishor RK, Sudhakar D, Parthasarathy PR. (2009), Embryo protective effect of pomegranate (Punica granatum L.) fruit extract in adriamycin-induced oxidative stress; Indian J Biochem Biophys.; 46(1):106–11 Kumar

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Nobuhiko Tachibana (2013), Intake of Mungbean Protein Isolate Reduces Plasma Triglyceride Level in Rats; Functional foods in healyh and disease; 3(9):365–376.

Priya Gurumoorthy and Kavitha G Singh (2014) Quantitative analysis of Proteins and Antioxidants; during Stress on Oryza Sativa; Journal of Global Biosciences, 3(2):552–561

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Sinthorn W, Chatuphonprasert W, Chulasiri M, Jarukamjorn K (2015), Thai red rice extract provides liver protection in paracetamol-treated mice by restoring the glutathione system; Pharmaceutical Biology., 8:1–10 Siti Nazrina Camalxaman, Zuhaida, Md Zain, Zulkhairi Amom, Maimunah Mustakim, Emida Mohamed, Azlin Sham Rambely (2004) In vitro Antimicrobial Activity of Vigna radiata (L) Wilzeck Suchalatha S, Shyamala Devi CS; Protective effect of Terminalia chebula against experimental myocardial injury induced by isoproterenol; Indian J Exp Biol. ;42(2):174–8 Sunita Inderjit Singh, Inderjit Singh (1955), The action of sodium and potassium on blood vessels and its relation to hypertension; Proceedings of the Indian Academy of Sciences - Section B, October, 42(4):172–182 T Lakshmi Srinivas, S Mohana Lakshmi, S Neelufar Shama, G Koteswara Reddy, K R Prasanna (2013), Journal of Pharmacognosy and Phytochemistry;2(4):91–97 Tang D, Dong Y, Ren H, Li l, He C (2014), A review of phytochemistry metabolite changes and medicinal uses of the

Source of Support:

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common food Mungbean and its sprouts; Chemistry Central Journal; 18 (1): 4. Tran Van Hien (2002), Radioprotective effects of Vitexina for breast cancer patients undergoing radiotherapy with Cobalt60; Inergrative cancer therapies; 1(1):38–43. V. M. Cuevas, Y. R. Calzado, Y. P. Guerra (2011), Effects of grape seed extract, vitamin C, and vitamin e on ethanoland aspirin-induced ulcers; Advances in Pharmacological Sciences,1–6 Wafaa M. Abdel-Moneim, Hemmat H. Ghafeer (2007), The potential protective effect of natural Honey against Cadmiuminduced Hepatotoxicity and Nephrotoxicity; Mansoura J. Forensic Med. Clin. Toxicol.; 15(2) Yang LC, Hsieh CC, Lin WC (2015); Characterization and immunomodulatory activity of rice hull polysaccharides; Carbohydrate Polymer, 25; 124:150–6 Yao Y, Yang X, Tian J, Liu C, Cheng X, Ren G (2013), Anti-oxidant and anti-diabetic activities of black mung bean (Vigna radiata L.); J Agric Food Chem; 28;61(34):8104–9

Conflict of Interest: None Declared

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