GJRMI - Volume 1, Issue VII, July 2012 by Dr. Hari Venkatesh.K.Rajaraman

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ISSN 2277 – 4289 www.gjrmi.com Editor-in-chief Dr Hari Venkatesh K Rajaraman

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Honorary Members - Editorial Board Dr Farhad Mirzaei Mr. Harshal Ashok Pawar

INDEX MEDICINAL PLANT RESEARCH Agriculture EFFECT OF GRADED LEVELS OF NPK ON HERB, OIL YIELD AND OIL COMPOSITION OF BASIL (OCIMUM BASILICUM L.) Alipour MANSOORKHANI Roghaye, SHAHRIARI Zolfaghar, MOHASELLI Vahid, OSFOORI Mohsen, SHAHRIARI Amir Ghaffar………………………………………………………………………………………………..…..258–264

Pharmacology WOUND HEALING POTENTIAL OF MELIA AZEDARACH L. LEAVES IN ALLOXAN INDUCED DIABETIC RATS Veda Vidya T, Srinivasan D, Sengottuvelu S…………………………………………………………………...265–271

INDIGENOUS MEDICINE Ayurveda IMPORTANCE OF KUNAPAJALA (TRADITIONAL LIQUID ORGANIC MANURE) OF VRIKSHAYURVEDA IN MEDICINAL PLANT CULTIVATION Bhat Savitha D, Ashok B K, Acharya Rabinarayan, Ravishankar B…………………………………….…….272–279

A COMPARATIVE ANTI-TUSSIVE ACTIVITY OF LAGHU GOKSHURA [Tribulus terrestris Linn.] AND BRIHAT GOKSHURA [Pedalium murex Linn.] PANCHANGA IN SWISS ALBINO MICE Methekar Chandrika, Patel B R, Aghera Hetal, Ashok B K, Ravishankar B……………………………….…280–285

IMPACT OF AYURVEDIC SHODHANA (PURIFICATORY PROCEDURES) ON BHALLATAKA FRUITS (SEMECARPUS ANACARDIUM LINN.) BY MEASURING THE ANACARDOL CONTENT Ilanchezhian R, Acharya R N, Roshy Joseph C, Shukla V J………………………………………………….286–294

QUALITY OF LIFE CONSEQUENCES IN DIABETIC POLYNEUROPATHY Niranjan Y, Santwani M A, Baghel M S……………………………………………………………...............295–300

MANAGEMENT OF NON HEALING WOUND WITH NYAGRODHADI KWATHA AND JATYADI GHRUTA (CLASSICAL AYURVEDIC PREPARATIONS) Lad Meenal D, Bandgar Satish B…………………………………………………………………..………….301–308

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – FLOWER OF BHARANGI, ROTHECA SERRATA (L.) STEANE & MABB, LAMIACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA

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Original Research Article EFFECT OF GRADED LEVELS OF NPK ON HERB, OIL YIELD AND OIL COMPOSITION OF BASIL (OCIMUM BASILICUM L.)

Alipour MANSOORKHANI Roghaye1, SHAHRIARI Zolfaghar2*, MOHASELLI Vahid3, OSFOORI Mohsen4, SHAHRIARI Amir Ghaffar5 1,2,3,4,5

Institute of Applied Scientific Education of Jahad-e-Agricultural, Fars Jahad-e-Agriculture Educational Center, Shiraz, Iran.,University of applied science and technology, Fars Branch, Shiraz, Iran. *Corresponding author: E-mail: Shahriari225@gmail.com Received: 26/05/2012; Revised: 27/06/2012; Accepted: 30/06/2012

ABSTRACT A field experiment was carried out using a randomized complete block design (RCBD) to evaluate the effect of four levels of N, P and K fertilizer (Kg h–1) including (N0, P0, K0), (N50, P25, K25), (N75, P50, K50) and (N100, P75, K75) on the herb, oil yield and oil composition of basil (Ocimum basilicum L.) in Sepidan, Iran. Data showed that the vegetative growth of basil, as indicated by plant height and plant fresh and dry weight was significantly increased by the addition of NPK fertilization rate. N75, P50, K50 was the best level of fertilizer application for vegetative growth of basil. The concentration of average essential oil in basil in both the cuts was not affected by the rate of NPK fertilization. Increasing rates of NPK fertilization significantly increased the biomass and consequently also the oil yield of basil in both cuts. Concentrations of oil in basil were significantly higher in the first cut than in the second cut at all levels of NPK. There was no significant correlation between concentrations of oil and vegetative growth traits included plant height, dry weight and wet weight of the basil. The main essential oil constituents were only slightly affected by fertilization in both cuts. KEYWORDS: Biomas, Fertilizer, Essence, Vegetative growth, Basil, Ocimum basilicum L.

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GJRMI, Volume 1, Issue 7, July 2012, 258–264

INTRODUCTION Basil (Ocimum basilicum L.) is an aromatic herb used extensively to add a distinctive aroma and flavor to food. The leaves can be used afresh or dried for use as a spice. Essential oils extracted from fresh leaves and flowers can be used as aroma additives in food, pharmaceuticals and cosmetics (Simon et al., 1999; Javanmardi et al., 2002; Senatore 1996). Traditionally, basil has been used as a medicinal plant in the treatment of headaches, coughs, diarrhea, constipation, warts, worms and kidney malfunction (Simon et al., 1999). It also possesses various beneficial effects, e.g., antiseptic, carminative, antimicrobial and antioxidative properties (Baranauskiene et al., 2003). The essential oil of basil is used as antifungal and insect-repelling agent. (Reuveni et al., 1984; Werner 1995). Among many plant growth factors, the nutritional requirements of the crop are considered to be the most important factor (Singh et al., 1989). An important factor affecting the quantity and quality of the harvested basil yield is to find the optimum level of fertilization (Hoflich et al., 1994). Nitrogen, Phosphorous and Potassium are macro nutrients and affect the growth, protein synthesis, reactions, development and reproduction (Sharafzadeh 2011). The aim of this study was to evaluate the influence of different levels of N, P and K on fresh and dry herb yield, oil yield and oil composition of basil. The main objective of this work was to evaluate a measure to decrease the excessive application of NPK fertilizers and to increase the quality of Basil (O. basilicum L.). MATERIALS AND METHODS This study was conducted on experimental field in Iran, Sepidan (30°16' N, 51° E; 2250 m above Mean sea level) with relative humidity (70%), mean annual temperature (14.9°C) and rainfall in the study area is distributed with an annual mean of 623 mm. Some physicochemical properties of experimental

soil are given in Table 1. The field experiment was carried out using a randomized complete block design (RCBD) with three replications. The treatments comprised of four levels of N, P and K fertilizers (Kg h–1) including (N0, P0, K0), (N25, P25, K50), (N50, P50, K75) and (N75, P75, K100). During the growing season two cuts were done. During each cut 10 plants were randomly selected for determining plant height and plant fresh and dry weights. Essential oil was isolated using a clevenger-type apparatus according to Marotti and Piccaglia (1992). 100 g of fresh Genovese basil herb were distilled for three hours in one liter water and then the percentage of essential oil per fresh weight was calculated. The dehydrated oil of each treatment was subjected to gas chromatography (Kandil et al., 2009). All data were subjected to analysis of variance using SPSS statistical software Ver. 17. Means were separated and compared using Duncan multiple range test (DMRT) at the 0.05 level of significance. Pearson correlation coefficient was calculated between variables (Assad 1997). RESULTS AND DISCUSSION The vegetative growth of basil, as indicated by plant height and plant fresh and dry weight has been shown in Table 2. There was significant difference in dry weight (p < 0.01), wet weight (p < 0.01) and plant height (p < 0.05) among different rate of NPK application especially between low and high rate in both cuttings. This data was in agreement with Alhasan and Alsafar (2009). The maximum plant height in the first cut was achieved on N75, P50, K50 which was significantly different when compared to control. In the second cut there was no significant difference between plant heights of N75, P50, K50 and N100, P75, K75 treatments. There was no significant difference between wet weight and dry weight of N75, P50, K50 and N100, P75, K75 treatments in both cuts. Results showed that N75, P50, K50 is the best level of fertilizer application for vegetative growth of basil.

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Table 1. Results of some physicochemical properties of experimental soil. Sand %

Silt %

Clay %

EC dsm-1

N (ppm)

P (ppm)

K (ppm)

7.7

0.25

Table 2. Effect of increasing NPK fertilization rate on growth parameters and essential oil content of Basil (Ocimum basilicum L.) grown in the Sepidan, Fars province, Iran. Treatment

Plant Height (cm)

Wet weight (g)

Dry weight (g)

N0, P0, K0 N50, P25, K25 N75, P50, K50 N100, P75, K75 N0, P0, K0 N50, P25, K25 N75, P50, K50 N100, P75, K75 F Test CV%

First Cut

25.33c 41.83b 58.33a 49.5b

341.66d 554.33b 1076a 1110a

64c 116.33b 196a 221.66a

Essential oil (g/100 g dry matter) 0.81a 0.69a 0.72a 0.67a

Second Cut

30c 45b 61a 60a

400c 605.1b 1210a 1220a

72c 121b 202.3a 222.4a

0.42b 0.4b 0.37b 0.39b

* 9.94

** 9.4

** 8.85

* 10.23

Columns with the same letter(s) were not significantly different according to Duncan multiple range test at p ≤ 0.05. * and ** significant at 0.05 and 0.01, respectively.

Table 3. Simple correlation coefficients between vegetative traits and essential oil of Basil (Ocimum basilicum L.). Traits

Height

Wet Weight

Dry Weight

Height (cm)

Wet Weight (g)

0.916

Dry Weight (g)

0.896

0.991**

Essential oil

–0.348

–0.257

–0.365

Essential oil

**Correlation is significant at the 0.01 level (2-tailed).

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GJRMI, Volume 1, Issue 7, July 2012, 258–264

Compound (%)

Table 4. Effect of NPK fertilization rates on essential oil composition (%) of the first cut of Basil (Ocimum basilicum L.) grown in the Sepidan, Fars Province, Iran.

Treatments

N0P0K0

N50 P25K25

N75 P50K50

N100P75K75

Essential oil (g/100 g dry matter)

0.81

0.69

0.72

0.67

α-pinene 6-methyl-5-heptene-2-one Limonene 2,2-dimethyl-3,4-octadienal Cis-verbenol Birneol P-mentha-1,5-dien-8-ol Methyl chavicol n-octyl acetate Nerol (z)-citral Geraniol (E)-citral Neryl acetate Geranyl acetate Methyl eugenol Trans-caryophyllene Trans-α-bergamotene α-humulene Germacrene D Trans-α-bisabolene Caryophyllene oxide α-cadinol

0.25 1 0.2 0.5 1.2 1 1 24.15 0.25 2 24 3 30.25 0.25 0.25 0.7 3 1 2 1.5 1.5 0.5 0.25

0.5 1 0.25 0.25 1 1 1 31 0.25 2 20 1.5 25 0.5 0.25 1 3 1 1.5 1.5 2 0.5 0.25

0.25 1 0.25 0.25 1 0.5 1 46 0.5 3 14.5 3 18 0.25 0.25 0.5 3 0.5 1.25 1.5 2 0.5 0

0.25 1 0.25 0.25 1 0.5 1 46 0.5 3.25 14.5 3.5 17 0.25 0.25 0.5 3 0.5 1.25 2 2 0.25 0

The concentration of average essential oil in basil in each cut was not affected by the rate of NPK fertilization. Increasing rates of NPK fertilization significantly increased the herb yield and consequently also the oil yield per square meter of basil in both cuts. These data were in agreement with Kandil et al., 2009. There was no significant correlation between concentrations of oil and vegetative growth traits including plant height, dry weight and wet weight of the basil (Table 3). Concentrations of oil in basil were significantly (p < 0.05) higher in the first cut than in the second cut at all levels of NPK. These differences could probably be attributed

to differences in environmental factors i. e., temperature, moisture and light (Ali et al., 1986; Elbalal et al., 1983; Omer et al., 1994; Kandil et al., 2009). The percentage and composition of essential oil could be markedly affected by the geographical environment, places that plants are grown, physical and chemical characteristics of soil, seed source, plant age, parts of plant that which is used for oil isolation and oil isolation method (Sharafzadeh 2011). Data in Tables 4 and 5 show the effect of NPK fertilization rate on the compositions of essential oil of the first and second cut of basil,

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GJRMI, Volume 1, Issue 7, July 2012, 258–264

respectively. The main essential oil constituents were only slightly affected by fertilization rate in both cuts. Data was in agreement with Kandil et al., (2009). Twenty three components were identified in the essential oil of Basil (Ocimum basilicum L.) at N0P0K0 and N50P25K25, However twenty two components were identified in the essential oil of Basil at N75P50K50 and N100P75K75. α-

Cadinol is not seen in the essential oil of Basil at N50P50K75 and N75P75K100. The major components in both cuts were Methyl Chavicol (24–46%), (Z)-Citral (14.5– 24%), (E)-Citral (18.25–30.25%), Geraniol (1.5–3%), Trans-caryophyllene (2.9–3.2) and Nerol (1.5–3.25%). Other components were present in amounts less than 2%.

Compound (%)

Table 5. Effect of NPK fertilization rates on essential oil composition (%) of the second cut of Basil (Ocimum basilicum L.) grown in the Sepidan, Fars Province, Iran. Treatments

N0P0K0

N50 P25K25

N75 P50K50

N100P75K75

Essential oil (g/100g dry matter)

0.42

0.4

0.39

0.37

0.2 1.02 0.23 0.52 1.25 1 1 24 0.25 1.5 23 2.8 29 0.28 0.26 1.5 3.2 1.1 1.9 2.05 2 0.55 0.25

0.44 1.1 0.22 0.23 0.9 1 1 30 0.21 2.3 21 1.6 26 0.5 0.19 1 3.2 0.8 1.5 1.5 2 0.43 0.28

0.25 0.8 0.24 0.22 1.01 0.53 1 43 0.48 3 16 3 18.9 0.23 0.26 0.5 3.1 0.5 1.3 1.42 1.9 0.54 0

0.26 1.15 0.2 0.22 1 0.5 1 44 0.51 3.2 15 3 20 0.25 0.25 0.5 2.9 0.5 1.2 1.8 2 0.25 0

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CONCLUSION

ACKNOWLEDGMENTS

The concentration of average essential oil in basil in both cuts was not affected by the rate of NPK fertilization. Increasing rates of NPK fertilization significantly increased the biomass and consequently also the oil yield of basil. The main essential oil constituents were only slightly affected by fertilization.

The authors should give special thanks to Hamidreza Mahmodian Fard, Head of Fars Jahad-e-Agriculture Educational center for financial support of this work.

REFERENCES Ali AA, Makboul MA, Assaf MH, Anton R. (1986). Constituents of essential oil of Egyptian marjoram. Bull. Fac. Sci. Assuit Univ. 15:79–87. Alsafar MS, Alhasan YM. (2009). Efect of nitrogen and phosphorous fertilizers on growth and oil yield of indigenous Mint (Menta longifolia L.). Biotechnol. 8:380–384. Assad

M T. 1997. Agricultural field experiments design and analysis. Shiraz University Press. 543 pages.

Baranauskiene R, Venskutonis PR, Viskelis P, Dambrauskiene E. (2003). Influence of nitrogen fertilizers on the yield and composition of thyme (Thymus vulgaris). J. Agric. Food Chem. 51:7751–7758. Elbalal SA, Mandour MS, Nofal MA, Tawfik MSH. (1983). Physiological homeostasis of essential oil production in lemon grass (Cymbopogon citrates L.). Proc. IX International Congress of essential oils. Singapore, 13–17 March 1983, 9:147–151. Hoflich G, Wiehe W, Kohn G. (1994). Plant growth stimulation with symbiotic and associative rhizosphere microorganisms. Experientia 50:897– 905.

Javanmardi J. Khalighi A, Kashi A, Bais HP, Vivanco JM. (2002). Chemical characterization of basil (Ocimum basilicum L.) found in local accessions and used in traditional medicines in Iran. J. Agric. Food Chem. 50:5878– 5883. Kandil MAM, Khatab ME, Ahmed SS, Schnug E. (2009). Herbal and essential oil yield of enovese basil (Ocimum basilicum L.) grown with mineral and organic fertilizer sources in Egypt. Journal für Kulturpflanzen, 61:443–449. Marotti M, Piccaglia R. (1992). The influence of distillation conditions on the essential oil composition of three varieties of Foeniculum vulgare Mill. J. of Essent. Oil Res. 4:569–576. Omer EA, Khatab ME, Ibrahim ME. (1998). Production and volatile oil of new four cultivars of basil (Ocimum basilicum L.) cultivated in Egypt. Indian Perfumer 42:49–57. Reuveni R, Fleischer A, Putievsky E. (1984). Fungi-static activity of essential oils from Ocimum basilicum chemotypes. Phytopath. Z. 110:20–22. Senatore F. (1996). Influence of harvesting time on yield and composition of the essential oil of a thyme (Thymus pulegioides L.) growing wild in Campania (southern Italy). J. Agric. Food Chem. 44:1327–1332.

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Shahram S. (2011). Effect of Nitrogen, Phosphorous and Potassium on Growth, Essential Oil and Total Phenolic Content of Garden Thyme (Thymus vulgaris L.). Advances in Environmental Biology, 5(4): 699–703.

Singh KPR, Singh JP. (1989). Effect of Nitrogen and inter and intra row spacing on herb and oil yield of Transplanted Japanese Mint (Mentha arvesis L.). Ann. Agric. Res. 10:258– 261.

Simon JE, Morales MR, Phippen WB, Vieira RF, Hao Z. (1999). A source of aroma compounds and a popular culinary and ornamental herb. In J. JANICK (Ed.): Perspectives on new crops and new uses. Alexandria, VA, ASHS Press. pp. 499–505.

Werner RA. (1995). Toxicity and repellency of 4-allylanisole and monoterpenes from white spruce and tamarack to the spruce beetle and Eastern larch beetle (Coleoptera scolytidae). Environ. Entomol. 24:372–379.

Source of Support: Nil

Conflict of Interest: None Declared

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Original Research article WOUND HEALING POTENTIAL OF MELIA AZEDARACH L. LEAVES IN ALLOXAN INDUCED DIABETIC RATS Veda Vidya T1, Srinivasan D2, Sengottuvelu S3* 1,2

Department of Pharmacology, Karpaga Vinayaga Institute of Medical Sciences & Research Center, Chinna Kolambakkam, Palayanoor PO, Madurantagam Tk, Kancheepuram Dist-603308 3

Department of Pharmacology, Nandha College of Pharmacy & Research Institute, Koorapalayam Pirivu, Perundurai Main Road, Erode-638001, Tamil Nadu, India. *Corresponding Author: E-mail: sengt@rediffmail.com; Mobile: 09994426689

Received: 18/05/2012; Revised: 18/06/2012; Accepted: 21/06/2012

ABSTRACT The present study was conducted to evaluate the wound healing effect of Melia azedarach L. leaf extract in alloxan induced diabetic rats. The methanolic leaf extract of Melia azedarach L (2% and 5%) were topically applied and the wound healing potential was evaluated in alloxan induced diabetic rats. The wound healing efficacy was studied by using excision wound model, which was inflicted by cutting away 500 mm2 of the skin on the anterio–dorsal side of the alloxan induced diabetic rats. Povidone iodine ointment was used as reference standard. Wound healing property, based on wound contraction & percentage of wound contraction was measured and assessed upto 18 days. The topical application of methanolic leaf extract of Melia azedarach L (2% and 5%) had promoted wound healing in diabetic rats. There was significant contraction of wound observed with topical application of methanolic leaf extract of Melia azedarach L in alloxan induced diabetic rats. Further phytochemical studies are required to isolate the active compounds responsible for these pharmacological actions.

Keywords: Melia azedarach L, Diabetic mellitus, wound healing

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INTRODUCTION Diabetes mellitus is one of the major contributors to chronic wound healing problems. When diabetic patients develop an ulcer, they are at high risk to develop major complications, including infection and amputation. Diabetes mellitus is a condition which is known to be associated with a variety of connective tissue abnormalities (Abdollah et al., 2010). The collagen content of the skin is decreased as a result of reduced biosynthesis and or accelerated degradation of newly synthesized collagen. These abnormalities contribute to impaired wound healing in diabetics. One of the most outward and debilitating complications of diabetes is the

development of chronic non-healing foot ulcerations, occurring in 15% of diabetics (Goodson and Hunt., 1977). Many Ayurvedic medicinal plants play a very important role in the process of wound healing. Plants are more potent healers because they promote the repair mechanisms in the natural way. Melia azedarach L (Photo slides I) belonging to the family Meliaceae is one of the most useful traditional medicinal plants like Azadirachta indica. M. azedarach L. is native to tropical Asia. It is wide spread and naturalized in most of the tropics and subtropical countries. It was introduced and naturalized in Philippines, United States of America, Brazil, Argentina, many African and Arabian countries (Adnan and AL-Rubae., 2009).

Photo Slides I: (a,b,c) displaying different parts of Melia Azedarach L

a.Branch of M. azedarach L

b. Leaves of M. azedarach L

Leaves are used in leprosy, scrofula etc. It is also a known anthelmintic, antilithiatic, diuretic, deobstruent and resolvent. Seed oil is the most active medicinal product of the plant and used as an antiseptic for sores and ulcers that show no tendency to heal. Fresh leaf extract is applied externally for burns. Fresh leaf extract is used as a mouth wash in gingivitis. 5 ml of Leaf extract is administered orally thrice a day for treating piles (Abdul Viqar Khan et al., 2011). Seeds of Melia azedarach L have been scientifically reported to exert antimalarial (Nathan et al., 2006), anti fungal (Carpinella et al., 2005), ovicidal (Maciel et al., 2006) insecticidal (Akthar et al., 2008), antifeedant (Charleston et al., 2005), rodenticidal (Roop et al., 2005) activities invitro and in-vivo studies.

c. Fruits of M. azedarach L

Studies have been reported that Azadirachta indica leaf which is closely related species of Melia azedarach L possess hypoglycemic (Murty et al., 1978) and wound healing activity (Chaw et al., 1994). It was also reported that Azadirachta indica leaf exhibited wound healing effect in alloxan induced diabetic rats (Sengottuvelu et al., 2007). In view of the ethno-botanical uses and medicinal properties of M. azedarach L, it is surmise that this plant might possess wound healing properties. Therefore, the present study was aimed to evaluate the wound healing activity of the leaf extract of M. azedarach L in alloxan induced diabetic rats.

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MATERIAL AND METHODS Chemicals and Reagents Alloxan procured from LOBA Chemie (Mumbai). All other chemicals and reagents used in this study were of analytical grade. Plant material The leaves of Melia azedarach L were collected from outskirts of Erode, in the month of May. The leaves of M. azedarach L were identified and authenticated by the botanist, Botanical Survey of India, Agricultural University, Coimbatore. The (voucher no:45/212) specimen had been deposited in the herbarium for future reference. Preparation of extract 100 g of powdered drug was soaked in 250 ml of 95% methanol solution for 24 h followed by cold maceration for further 48 h with occasional shaking. The mixture was filtered using muslin cloth followed by removal of excess of solvent by means of rotatory evaporator. The dried extract was used for the study. Preparation of formulation 2 g and 5 g of dried extract was admixed to 99 g of simple ointment base to obtain 2% and 5% of M. azedarach L ointment respectively. Animals Male Wistar Albino rats weighing between 150–220 g were used for the study. The animals were obtained from animal house, IRT Perundurai medical college, Erode, India. On arrival the animals were placed at random and allocated to treatment groups in polypropylene cages with paddy husk as bedding. Animals were housed at a temperature of 24 ± 2oC and relative humidity of 30–70 %. A 12:12 light: dark cycle was followed. All animals were allowed free access to water and fed with standard commercial pelleted rat chaw (Hindustan Lever Ltd, Mumbai). All the experimental procedures and protocols used in this study were reviewed by the Institutional

Animal Ethics Committee (688/2/C-CPCSEA) and were in accordance with the guidelines of the CPCSEA. Induction of diabetes Alloxan monohydrate (SD Fine chemicals Ltd, Boisar.) was used to induce diabetes in rats. The base line plasma glucose levels were determined prior to alloxan administration. Diabetes was induced in rats by a single intraperitoneal injection of alloxan monohydrate (120 mg/kg) in sterile saline. After 72 h of alloxan injection, the diabetic rats (blood glucose > 250 mg/dl) were used for the study (Ravi et al., 2003). Excision wound model An excision wound was inflicted by cutting away approximately 500 mm2 full thickness of shaved skin at a predetermined area on the anterio dorsal side of the alloxan induced diabetic rats under pentobarbitone (30 mg/kg., i.p) anesthesia. The entire wound was left open. Animals were closely observed for any infection and those which showed signs of infection were separated, excluded from the study and replaced (Udupa et al., 1994). Experimental protocol Totally 30 animals were used in this study. The rats were divided into 5 groups of 6 animals each. Excision wound was inflicted in all the rats of 5 groups. Group I, non diabetic animals were treated with simple ointment base. Group II, diabetic control animals were treated with simple ointment base. Group III and IV, diabetic animals were treated with 2% and 5% of M. azedarach L methanolic extract in simple ointment base respectively. Group V, diabetic animals were treated with standard povidone iodine ointment. All the test drugs were applied topically on the wound, twice daily for 18 days. Assessment of wound contraction Wound contraction was monitored by metric measurement of the wound area once in 3 days upto 18th day. This was studied by

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tracing the raw wound area on a transparent polythene paper and the traced area was measured by using a graph paper. The wound contraction was measured as a percentage decrease of original wound size of 500 mm2 for each animal of a group. Statistical analysis Results were expressed as mean ± SEM. The data were analyzed by using one way analysis of variance (ANOVA) followed by Dunnet’s t test. P values < 0.05 were considered as significant. RESULTS The results of wound healing activity and % (percentage) of wound contraction of Melia azedarach L leaf extract in alloxan induced diabetic rats are showed in Table I & Figure I respectively. Topical application of both concentrations 2% and 5% of Melia azedarach L leaf extract promotes the contraction of wound in diabetic rats, when compared to

diabetic control. The percentage of wound contraction after topical application of test drugs on 6th day of observation shows that in diabetic control it was 3.29%. In 2% and 5% of M. azedarach L leaf extract it was 9.29% and 10.79% respectively, whereas in standard drug povidone iodine ointment it was 9.69%. Observation shows that on 9th day onwards, the topical applications of both the concentrations M. azedarach L leaf extract promoted the wound contraction faster than diabetic control. Wound contraction progressed much faster in M. azedarach L leaf extract from 12th day than the normal control and diabetic control. There was significant (p < 0.001) contraction of wound, observed with M. azedarach L leaf extract and standard povidone iodine ointment on 12th to 18th day when compared with diabetic control. Topical applications of M. azedarach L leaf extracts promoted wound contraction better than povidone iodine treated groups and non diabetic control groups.

Table I: Effect of Melia azedarach L leaf extract on excision wound in alloxan induced diabetic rats. Wound Area (mm2) Diabetic animals Group I Non-Diabetic control

Group II Diabetic control

Group III Melia azedarach 2%

Group IV Melia azedarach 5%

Group V Povidone iodine

486.15 ± 23.12

496.20 ± 24.60

481.72 ± 26.11

480.11 ± 27.71

482.22 ± 21.56

460.53 ± 26.25*

483.51 ± 19.22

453.56 ± 20.61*

446.06 ± 22.33**

451.52 ± 22.60*

303.22 ± 24.50**

475.34 ± 25.78

293.40 ± 19.63**

253.13 ± 17.54***

260.66 ± 15.42***

206.20 ± 18.21***

462.78 ± 17.95

193.21 ± 12.29***

167.09 ± 10.15***

199.57 ± 12.53***

113.36 ± 8.61***

440.51 ± 20.32

98.63 ± 5.13***

72.16 ± 5.63***

103.17 ± 6.61***

17.94 ± 1.12***

410.06 ± 21.06

14.31 ± 1.03***

11.58 ± 0.96***

18.56 ± 1.08***

Post Wound ing Days

Values are in Mean ± SEM; (n = 6) *P < 0.05, **P < 0.01, *** P < 0.001 Vs Diabetic Control

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Figure I: Percentage wound contraction of Melia azedarach L leaf extract on excision wound in alloxan induced diabetic rats. 120

% Wound Contraction

100

0 3

DAYS

Non Diabetic Control

Diabetic Control

Melia Azedarach 2%

DISCUSSION The present study was undertaken to evaluate the wound healing activity of Melia azedarach L leaf extract in alloxan induced diabetic rats. Wound healing deficits with Diabetes mellitus believed to be largely caused by some basic mechanisms (Sidhu et al., 1999). (i) Increased blood sugar that impairs blood flow and the release of oxygen. (ii) Impaired local immune and cell defenses and (iii) Microbial infections. M. azedarach L. leaves possess hypoglycemic activity, immunomodulatory activity, anti fertility activity and anti microbial activity (Kausik Biswas et al., 2002). In this study it has been proven that the topical application of M. azedarach L. leaf extract promotes wound healing in diabetic rats and its effect was comparable with standard

Melia Azedarach 5%

Povione Iodine

povidone iodine. The delay in the healing of wound in diabetic rats may be due to enhanced blood glucose, which favors the microbial growth. The Melia azedarach L leaf extract enhanced the wound healing in diabetic rats in our study which may be due to its antimicrobial activity. CONCLUSION From the result it could be concluded that the topical application of methanolic leaf extract of Melia azedarach L shows significant wound healing activity in alloxan induced diabetic rats. Further phyto-chemical studies are required to isolate the active compounds responsible for wound healing activity which could be a major contribution to prove the claims in Indian systems of medicine.

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REFERENCES Abdollah Ghasemi Pirbalouti, Shahrzad Azizi, Abed Koohpayeh, Behzad Hamedi (2010). Wound healing activity of malva sylvestris and punica granatum in alloxan-induced diabetic rats. Acta Poloniae Pharmaceutica. Drug. Res. 67: 511–516. Abdul Viqar Khan, Qamar Uddin Ahmed, Ramzan Mir M, Indu Shukla, Athar Ali Khan (2011). Anti bacterial efficacy of the seed extracts of Melia Azedarach against some hospital isolated pathogenic bacterial strain. Asian. Pac. J. Trop. Biomed. 1: 452–455. Adnan Y. AL-Rubae (2009). The potential uses of Melia Azedarach L. as pesticidal and medicinal plant, Review. Am. Eurasian J. Sustain. Agric. 3: 185–194. Akthar Y, Young YR, Isman MB (2008). Comparative bioactivity of selected extracts from Meliaceae and some commercial botanical insecticides against two noctuid caterpillars, Trichoplusia ni and Pseudoletia unipunctat. Phytochem Rev. 7: 77–88. Carpinella MC, Ferrayolic CG, Palacios SM (2005). Antifungal synergistic effect of scopoletin, a hydroxycoumarin isolated from Melia azedarach L fruits. J. Agric. Food. Chem. 53: 2922–2927. Charleston DS, Kfir R, Vet LE, Dicke M (2005). Behavior responses of diamond back moth Plutella xylostela (Lepidoptera: Plutellidae) to extracts derived from Melia azedarach L and Azadirachta indica. Bull. Entomol. Res. 95: 457–465. Chaw AS, Manoj Kumar S, Bansal I (1994). Chemical constituents and biological activity of neem. A review. Ind. Drug. 32: 57–62.

Goodson WH, Hunt TK (1977). Studies of wound healing in experimental diabetes mellitus. J. Surg. Res. 22: 221–227. Kausik Biswas, Ishita Chattopadhyay, Ranajit K. Banerjee, Uday Bandyopadhyay (2002). Biological activities and medicinal properties of neem (Azadirachta indica). Curr. Sci. 82: 1336 –1345. Maciel MV, Morais SM, Bevilaqua CML, Camurea vasconcelos ALF, Costa CTC, Castro CMS (2006). Ovicidal and larvicidal activity of Melia azedarach L extracts on Haemonchus contortus. Vet. Parasitol. 140: 98–104. Murty KS, Rao N, Rao DK, Murty LBG (1978). A preliminary study on hypoglycemic and antihyperglycemic effects of Azadirachta indica. Ind. J. Pharmacol. 10: 247–250. Nathan SS, Savitha G, George DK, Marmadha A, Suganya L, Ghung PG (2006). Efficiency of Melia azedarach L. extract on malarial vector Anopheles stepheni Listone (Dipetra: Culicidae). Bioresour. Technol. 97: 1316–1323. Ravi

V, Moni K, Sarita G (2003). Hypoglycemic effect of aqueous extract of Ericostemma littoral on alloxan induced diabetes mellitus in rats. Ind. J. Exp. Biol. 8: 781–784.

Roop JK, Dhaliwal PK, Guraya SS (2005). Extracts of Azadirachta indica and Melia azedarach seeds inhibit folliculogenesis in albino rats. Braz. J. Med. Biol. Res. 38: 943–947. Sengottuvelu S, Srinivasan D, Vasudevan M, Nandhadumar J, Duraisamy R, Karthikeyan D, Sivakumar T (2007). Wound healing effect of aqueous extract of Azadirachta indica leaves in alloxan induced diabetic rats. The. Antisep. 104: 377–379.

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Sidhu GS, Mani H, Gaddipati JP, Singh AK, Seth P, Banaudha KK, Patnaik GK, Maheshwari RK (1999). Curcumin enhances wound healing in streptozotocin induced diabetic rats and genetically diabetic mice. Wound. Rep and Regener. 7: 362–374.

Source of Support: Nil

Udupa SL, Udupa AL, Kulkarni DR (1994). Studies on the anti-inflammatory and wound healing properties of Moringa oleifera and Aegle marmelos. Fitoterap. 65: 119–123.

Conflict of Interest: None Declared

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Review article IMPORTANCE OF KUNAPAJALA (TRADITIONAL LIQUID ORGANIC MANURE) OF VRIKSHAYURVEDA IN MEDICINAL PLANT CULTIVATION Bhat Savitha D1*, Ashok B K2, Acharya Rabinarayan3, Ravishankar B4 1

*Lecturer, Department of Dravyaguna, Muniyal Institute of Ayurveda Medical Sciences, Manipal, Karnataka. 2 Research Assistant, Pharmacology laboratory, Dhanvantri Mandir, IPGT & RA, Gujarat Ayurved University, Jamnagar 3 Associate professor, Department of Dravyaguna, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India - 361008 4 Director, SDM Centre for Research in Ayurveda and Allied Sciences, Kuthpady, Udupi, Karnataka - 574118 *Corresponding Author: E- Mail: anudivas@yahoo.co.in, Mobile: 09727083816

Received: 14/05/2012; Revised: 20/06/2012; Accepted: 22/06/2012

ABSTRACT Medicinal plants play an important role not only as traditional medicine but also as trade commodities. With depleting natural resources, cultivation of medicinal plants has become the need of the hour to meet the growing demand of herbal drug requirement in the field of medicine. But using chemicals in the form of pesticide and fertilizers may have undesirable impact on the quality of these plants as well as human health. Recently various state governments in India have come up with different schemes to cultivate medicinal plants through organic farming under Good Agricultural Practices (GAP). “Vrikshayurveda” a sub branch of Ayurveda describes a particular type of liquid organic manure called “Kunapajala” for this purpose. Scattered references are found regarding the effect of Kunapajala on medicinal plant cultivation. This review highlights the importance of organic farming and results of research works carried out on the effect of Kunapajala on medicinal plant cultivation.

KEY WORDS: Kunapajala, Vrikshayurveda, Organic manure, Medicinal plants, Fertilizers

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INTRODUCTION Recently herbal health care sector is gaining impetus due to the shift in the mindset of people towards herbal medicines which are considered safe and having wider biological activity (Alok Sharma et al., 2008). Growing demand for plant based medicines and health care products have led to an increase in the number of herbal drug manufacturers. With around 25,000 licensed herbal drug manufacturers in India it has become a lucrative business with a global market currently standing over $ 60 billion annually and is expected to get higher at 6.4% average growth rate. In Indian herbal industry, about 1000 single drugs and 3000 compound preparations have been registered so far. Except few plant species like Opium, Senna, Psyllium, Periwrinkle, Cinchona etc., which are obtained from commercial cultivation, majority of other plant species are collected from the natural sources (Inamdar et al., 2008), (Sheetal Verma and Singh, 2008; Kamboj, 2000). Over exploitation of these natural resources due to increase in population, decrease in forest cover have made many medicinal plants endangered. According to the Red list of Threatened Plants, 19 species are already extinct and 1236 species are facing various degrees of threat across different biogeography regions in the country. (Ramprasad Naik, 2012). Hence cultivation of alternative supply sources of such species is therefore essential. Cultivation of plant species using organic manure dates back to 1000AD in India. It is dealt in Vrikshyayurveda, which forms a part of Ayurvedic history and is treated as a separate subject owing to its importance and extensive nature. It is an age old agro practice which is of great relevance even today in sectors like agriculture and horticulture. It not only deals with pest and disease management of plants but also encompasses study areas like storage of seeds, sowing, germination, plant propagation, manuring etc. For nourishment of plants, use of a biofertilizer called ‘Kunapajala’ has been mentioned. References of which can be traced in the manuscript of ‘Vrikshayurveda’ written by Surapala, around 1000 AD. The details of

Kunapajala are also found in a chapter called Upavanavinoda of an anthropological compilation called ‘Sharangadhara Paddhati’ written by Acharya Sharangadhara, belonging to the thirteenth century (Lakshmipathi, 2004; Nene, 2006). The literary meaning of the Sanskrit word Kunapa is “smelling like a dead body or stinking” and the name is apt for the liquid manure which is prepared using excreta, bones, flesh and marrow of animals, fish, decayed plant products etc (Williams Monier, 2002). In this paper an attempt has been made to review studies carried out in this regard, highlighting the importance of Kunapajala in the field of medicinal plant cultivation. Benefits of organic farming versus chemical fertilizers in medicinal plant cultivation: India has varied agro-climatic conditions which make it suitable for growing a wide range and variety of valuable medicinal plants. But the higher cost of production as compared to the material collected illegally from the forests, unstable demand for the produce, slow growth rates and low prices paid for traditional medicines hardly make cultivation of medicinal plants a profitable exercise. These factors are luring the farmers towards use of chemical fertilizers to increase the yield in a short span of time (Cunningham, 1993). Though, chemical fertilizers increase the yield, they pose certain serious health threats to human beings especially infants, pregnant and nursing mothers (Vermeer et al., 1998). Another concern for health is contamination of medicinal plants with toxic heavy metals like mercury, lead, cadmium, etc., through fertilizers, harmful industrial wastes contaminating the water sources etc. They can be absorbed into the plants and can cause disturbances in the kidneys, lungs, liver leading to several deformities like congenital paralysis, sensory neural defects and even cancer (Edward Someus, 2009; Dargan et al., 2008). Twelve cases of lead poisoning attributed to Ayurvedic medicines were reported to the centres for disease control (CDC, 2004) over a four year span. Also certain plants of Brassicaeae family tend to accumulate lead in

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larger quantities. (Adriane 2009). A study report by Kushwaha et al. (1999) showed that chemical fertilizer was generally associated with higher damage potentials than any of the animal wastes. In contrast, organic manures are considered to be safe and yielding good produce by improving water penetration, waterholding capacity, improvement in soil structure, microbial biomass, nutrient availability, drought and heat stress resistance. It also helps in improving the soil pH which has an impact on plant growth and soil microbial activity (Amigo Cantisano, 2000). They not only act as a source of nitrogen but also increase the efficiency of applied nitrogen (Zhu et al., 1987; Satheesh and Balasubramanian, 2003). Organic certification of medicinal plants Bearing in mind the benefits and safety of organic farming, different state governments of Assam, Andhra pradesh, Bihar, Uttaranchal etc., under Government of India have established State Medicinal Plants Board and a national level body called National Medicinal Plant Board for promoting the growth and development of medicinal plants sector in the country. The guidelines quote that any herbal product used for global trade is to be free from heavy metals, toxic impurities and certified to be organic or Good Agricultural Practices(GAP) compliant. Also, innovative group of committed farmers have been promised assistance for cultivation of medicinal plants using organic farming technologies through eligible voluntary organisations that will provide technical and supervisory support (Anonymous, 2008, 2010). Organic certification methods are also seen in many other countries like Israel, Romania etc., aiming towards global acceptance for organic certified plants and plant produce (Anonymous, 2001; Camelia, 2007). Kunapajala as organic manure a. Classical method for preparation of Kunapajala: Flesh, fat and marrow of Pig, Deer, Fish, Sheep, Goat and Rhinoceros should be boiled

in water. When properly boiled, the mixture is transferred to an earthen pot. To this mixture milk, powder of Sesame oil cakes, well cooked Masha (Black gram), honey, ghee and hot water are added. There is no mentioned fixity for the quantity of ingredients. The pot is then kept in a warm place for a fortnight and the resultant solution is called Kunapajala (Shrikrishna Jugnu, 2004). In Vrikshayurveda of Surapala, different varieties of Kunapajala have been mentioned. The verse 101 mentions that excreta, bone marrow, brain, flesh and blood is mixed with water and kept as it is for some time to be known as Kunapajala. In the subsequent verses it is quoted that bones of Horses, dead Parrot, Fish, horns of Sheep and Goat, Cow dung cake should be boiled in water and later filled along with sufficient quantity husk in a previously oil smeared pot. Instead of boiling, it can also be roasted in an iron pot and mixed with sesame oil cakes and honey. Good quality black gram and ghee should be added in the end. The ingredients mentioned do not have specific measure but the prepared compound should be kept in a warm place (Sadhale Nalini, 1996). b. Need for modification of Kunapajala: Sharangadhara mentions that almost any animal waste can be used in preparing Kunapajala indicating the fact that a farmer has considerable flexibility in choosing the animals and their by products depending upon the crop. Since availability of flesh of Deer, Rhinoceros is not possible in the present days because of prohibition on killing of wild animals in India (Sanjay Upadhyay and Amruta Sane, 2009), Kunapajala would have to be modified using meat of other animals having similar properties. The Deer meat has nutrients like crude protein, Ca, P, Mg, Na, K, Cu, Zn, Fe, Mn etc. It can be substituted with chicken, pork and fish meat which are reported to be having the same nutrients and are also easily available (USDA, 1996; Zomborszky et al., 1996). Based on this rationality, preparation of Kunapajala was modified by Asha (2006) using meat of Rat (Rattus variegata), Fish

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(Rastreiliger kanagurta), Goat meat (Capra aegagrus), Chicken (Gallus gallus) and Goat blood where the modified Kunapajala showed the presence of nitrogen (4.8%), potassium (11.7%) and phosphorus (3.9%) and was used after 30 days of composting. Usually the raw organic matter decomposes into humus which will be further digested by soil microbes producing high levels of organic acids like humic, carbonic and fulvic acids and increases high cation (+) exchange capacity. This capacity is responsible for the mobilization of calcium, potassium and other plant nutrients. In order to obtain good results aerobic composting is said to be beneficial. Kunapajala also acts as organic manure composted under proper conditions to fulfil the requirements of nutrients like Nitrogen, Phosphorus and Potassium. The nitrogen which is very essential for plant growth is supplemented by blood, cottonseed, fish meal and emulsion etc, whereas compost from bird manures, bone meal etc are rich source of Phosphorus and Potassium which helps in regulating root, bud, flower and fruit formation, cell division, sugar formation in the sap, chlorophyll production and photosynthesis, increasing crop resistance to diseases etc. The other important micronutrients are Magnesium, Calcium, Zinc, Manganese, Copper, Iron, and Selenium (Amigo Cantisano, 2000) which are also supplemented by the organic compost Kunapajala. c. Experimental outcomes of Kunapajala: Many scientists have carried out extensive research work on Kunapajala with special emphasis on cultivation of medicinal plants. Some of those works have been mentioned below. Senna (Cassia angustifolia Vahl.): Senna was grown by Brajeshwar (2002) using both laboratory prepared and commercial Kunapajala. It was sprayed at the rate of 2 ml per litre of water. Readings taken after 45 days of sowing showed significant results in

parameters like height of the plants (cm), enhancement of leaf area index etc. Leaves being the main organ containing Sennoside, Kunapajala was helpful in substantially increasing it. The study also showed that fertilizer treated plants flowered very early but Kunapajala treated plants have delayed flowering resulting in harvesting of more foliage. Total Sennoside content per plant (g) was analyzed after 70 days which showed that Senna grown using laboratory prepared Kunapajala showed excellent results when compared to chemical fertilizer and control group. Langali (Gloriosa superba Linn.): Experimental studies on Langali by Asha (2006) showed that Kunapajala treated plants exhibited excellent result in terms of general growth of the plants and fruiting when compared to control group and chemical fertilizer group. Even though the yield of tuber was not significant enough, one of its active principle ‘Colchicine’ (methanol extract) was found in higher amount in Kunapajala treated plants. Mango, Coconut, Chilly and Kiwi: The first reported scientific experimentation with Kunapajala was by Ayangarya Sreenivasa (2004), who reported admirable results of Kunapajala, when applied to Mango and Coconut. He further tried an “herbal Kunapa” using naturally fallen sour Mango fruits and Soapnut (Sapindus emarginatus) and applied it on Chilly plants with excellent outcomes. In addition, he prepared Kunapajala from poultry (chicken) bird flesh and called it Kukkutakunapa (kukkuta=chicken), and used it very effectively in increasing Kiwifruit yield from 120 kg in November 2004 to 1700 kg in November 2005 (Ayangarya Sreenivasa, 2005). Brinjal (Solanum melongena): A unique variety of Brinjal locally called as ‘Mattu gulla’ in Udupi District of Karnataka state of India is being cultivated by applying a special type of organic manure like Kunapajala

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prepared from a local fish variety called ‘Bhutaai’. Locals claim that this treatment results in large number of branches, higher yield, fruits with lesser seeds and lower susceptibility to diseases when compared with plants grown with artificial fertilizer (Bhat Ramesh and Vasanthi, 2008). Paddy: Mishra (2007) studied the growth of paddy using Kunapajala specially prepared for growth of cereal plants. 10 g each of Barley (Hordeum vulgare Linn.), Black gram (Vigna mungo Linn.), Sesame seed (Sesamum indicum Linn.) and Green gram (Vigna radiata Linn.) powder was mixed in hot water and allowed to cool. This preparation was used to manure paddy plants for every 10, 15, 20 and 25 days. Reading for evaluating growth of rice plants were taken after 100 days. Mean of readings for two consecutive years were taken for interpretations. Kunapajala applied for every 10 days showed significant increase in growth parameters like plant height, leaf length, inflorescence length, number of grains per inflorescence etc Vegetables: Study report by Narayanan (2006) shows that improved modifications in the preparation of Kunapajala by mixing Panchagavya (Cow’s milk, Curd, Ghee, Urine, and Dung) show tremendous results when applied to vegetables. DISCUSSION Kunapajala, a liquid bio fertilizer is a natural organic product derived from animal and plant products containing a significant quantity of one or more of the primary nutrients like Nitrogen, Phosphorus, and Potassium which are necessary for plant growth. Oil cakes, blood meal, fish manure etc are said to be concentrated organic manures. These are also known as organic nitrogen fertilizers. Before their organic nitrogen is used by the crops, it is converted through bacterial action into readily usable ammoniacal nitrogen and nitrate nitrogen. These organic fertilizers are, therefore, relatively slow acting, but they

supply available nitrogen for a longer period (Neff et al., 2003). Chicken manure, feathers, fish contain more of nitrogen which is essential to increase the leaf area. This is evidenced by the study report of Senna in which there was increase in the amount of foliage after the application of Kunapajala. Animal manure, fish, blood and bone meal, hoof and horn meal contain ample amounts of Phosphorus and Potassium. Phosphorus is used to establish a strong root system in young plants, fruit and shrubs while Potassium helps the plant absorb and hold onto water. It increases protein synthesis from Nitrogen. This explains why it is particularly good to enhance fruit development. Larger amounts of Potassium help plants produce more flowers and fruit rather than leaves (Olson, 1987). This is supported by the version of Surapala that for obtaining good yield of fruits and flowers, Kunapajala should be mixed with decomposed excreta and flesh of animals (Shrikrishna Jugnu, 2004). This offers an inspiration to design different combination of ingredients for preparation of Kunapajala according to the need of the plant part to be used. Composting Kunapajala helps in breaking down the manure into simpler forms, making it available to plants faster than the traditionally applied organic matter. Also compost nutrients are released slowly, allowing them to stay in the soil for a longer period enhancing microbial action in the soil, allowing it to absorb and retain water and nutrients more efficiently. Being a liquid it is readily available for the roots in a short time (Prabha et al., 2008). CONCLUSION Kunapajala by virtue of its behaviour as plant growth regulator is readily available as a simple compost manure showing its potency in increasing the leaf area, higher yield of flowers and fruits as well as phyto-constituents. It also seems that Kunapajala has some plant growth regulatory actions through which it enhances the overall growth of plants. Being a liquid biofertilizer it is a more suitable form of manure

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and can be beneficial in growth of medicinal plants with probably minimal toxic effects on human body when compared to chemical fertilizer. Researchers suggest that application of the principles of Vrikshayurveda like

Kunapajala does produce phenomenal and interesting results. Since few research works have been carried out, this discipline of science needs to be developed through concerted R & D efforts to ascertain its utility.

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Amigo Cantisano (2000). Know your soil, a handbook for organic growers and gardeners in temperate and sub-tropical regions. Organic Ag Advisors, Colfax, CA, Edited and adopted by Julian Dumanski, RDV, World Bank, pp 1–2. Anonymous (2001). National standard for organically grown plants and their products, Plant Protection and Inspection Services, Ministry Of Agriculture and Rural Development. State of Israel, pp 1–38. Anonymous (2008). Centrally sponsored scheme of national mission on medicinal plants operational guidelines, National Medicinal Plants Board, Department of AYUSH, Ministry of Health & Family Welfare, Government of India, New Delhi, pp 1–37. Anonymous (2010–11). Action Plan, Central Sector Scheme for Conservation, Development & Sustainable Management of Medicinal Plants, Submitted by Chhattisgarh State Medicinal Plants Board, India, pp 1–16.

KV (2006). Comparative pharmacognostic and pharmacological evaluation of Langali (Gloriosa superba Linn. Ph D Thesis, Gujarat Ayurved University. Jamnagar, India.

Ayangarya Valmiki Sreenivasa (2005). Kiwifruit plant treatment on the Himalayas of India, A Vrikshayurveda experience. Paper presented in Bridging Gap between Ancient and Modern Technologies to Increase Agricultural Productivity, Central Arid Zone Research Institute., Jodhpur, India. Bhat Ramesh V , Vasanthi S (2008). Antiquity of the cultivation and use of Brinjal in India. Asian Agri-History. 12(3): 169– 178. Brajeshwar (2002). Effect of different agronomic practices on Senna (Cassia angustifolia Vahl.). M.Sc. dissertation, Gujarat Ayurved University, Jamnagar, India. Camelia Ioana Ucenic (2007). Increasing products’ value through ecological and organic certification. Proceedings of the 2nd IASME / WSEAS International Conference on Energy & Environment (EE'07). Portoroz, Slovenia, May 15– 17: 246–252. CDC (2004). Lead poisoning associated with Ayurvedic medications. MMWR. 53(26): 582–584. Available at

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http://www.cdc.gov/mmwr/preview/mm wrhtml/mm5326a3.htm. Cunningham AB (1993). An Africa-wide overview of medicinal plant harvesting, conservation and health care, WWF/UNESCO/New People and Plants initiative, South Africa, pp 116– 118. Dargan PI, Gawarammana IB, Archer JRH, House IM, Shaw D, Wood DM (2008). Heavy metal poisoning from Ayurvedic traditional medicines: an emerging problem? Int. J. Environment and Health. 2(3): 463–474. Edward Someus (2009), Cadmium and heavy metals in chemosynthetic fertilizers. Environmental technologies. 1–4. Inamdar N, Edalat S, Kotwal VB, Pawar S (2008). Herbal drugs in milieu of modern drugs. Int. J. Green Pharm.2 (1): 2–8. Kamboj VP (2000). Herbal Medicine. Current Science.78 (1): 35–39. Kushwaha S, Ochi JE, Abubakar MM, Ayoola GB (1999). Effect of chemical fertilizer and animal wastes application on environmental life support using the “delphi” technique. Journal of Sustainable Agriculture. 14(1): 91–98. Lakshmipathi A (2004). Text book of Ayurveda historical background, 1st edition, Choukhamba Sanskrit Pratisthan, New Delhi, pp 361–362. Mishra PK (2007). Effect of Kunapa Jalam Vrikshayurveda on growth of Paddy. Indian Journal of Traditional knowledge. 6(2): 307–310. Narayanan RS (2006). Application of Gunapajalam (Kunapajala) as liquid biofertilizer in organic farms. Asian Agri- History. 10:161–164. Neff JC, Chaplin FS, Vitousek PM (2003). Breaks in the cycle: Dissolved organic

Nene YL (2006). Kunapajala a liquid organic manure of antiquity. Asian AgriHistory. 10: 315–321. Olson RA (1987), the use of fertilizers and soil amendments, land transformation in agriculture. Edited by Wolman MG and Fournier FGA, John Wiley Ltd. pp 18. Prabha K, Padmavathiamma, Loretta Y, Usha Kumari (2008). An experimental study of vermin bio waste composting for agricultural soil improvement. Bio resource Technology. 99 (6):1672– 1681. Ramprasad Naik D, Rahiman SA, Kaizar Hossain (2012). Vulnerable endangered, threatened and rare species categories in the submergence area of Polavaram area. Euro. J. Exp. Bio. 2 (1): 288-296. Sadhale Nalini (1996). Surapala’s Vrikshayurveda - The science of Plant life by Surapala. Asian Agri-History Foundation, Secunderabad, India, 1:104. Sanjay

Upadhyay, Amruta Sane (2009). Conserving protected areas and wild life: A judicial journey. ELDF and WWF India, pp 1–289.

Satheesh N, Balasubramanian N (2003). Effect of organic manure on yield and nutrient uptake under rice - rice cropping system. Madras Agric. J. 90(1–3): 41– 46. Sheetal Verma, Singh SP (2008). Current and future status of herbal medicines. Veterinary World. 1(11): 347–350. Shrikrishna Jugnu (2004). Surapala’s edition, Vrikshayurveda, 1st Chowkamba Sanskrit series Office, Varanasi, pp 98–101.

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USDA (1996). USDA Nutrient Database for Standard Reference. Available at http://www.nal.usda.gov/fnic/foodcomp Vermeer IT, Pachen DM, Dallinga JW, Kleinjans JC, Van Maanen JM (1998). Volatile N-nitrosamine formation after intake of nitrate at the ADI level in combination with an amine-rich diet. Environ Health Perspect. 106(8): 459– 463.

Zhu Z, Liao X, Cai G, Wang Z (1987). On the improvement of the efficiency of nitrogen of chemical fertilizers and organic manures in rice production. Soil Sci. 135: 35–39. Zomborszky Z, Szentmihályi G, Sarudi I, Horn P, Szabó Cs (1996). Nutrient Composition of Muscles in Deer and Boar. Journal of Food Science. 61(3): 625–627.

Williams Monier (2002). A Sanskrit English Dictionary, Motilal Banarsidas Publishers Pvt. Ltd., Delhi, pp 289.

Source of Support: Nil

Conflict of Interest: None Declared

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Original Research Article A COMPARATIVE ANTI-TUSSIVE ACTIVITY OF LAGHU GOKSHURA [Tribulus terrestris Linn.] AND BRIHAT GOKSHURA [Pedalium murex Linn.] PANCHANGA IN SWISS ALBINO MICE Methekar Chandrika1*, Patel B R2, Aghera Hetal3, Ashok B K4, Ravishankar B5. 1

Post Graduate Scholar, Department of Dravyaguna, IPGT & RA, Gujarat Ayurved University, Jamnagar Assistant Professor, Department of Dravyaguna. IPGT & RA, Gujarat Ayurved University, Jamnagar 3 Lab technician, Pharmacology Laboratory. IPGT & RA, Gujarat Ayurved University, Jamnagar 4 Research Assistant, Pharmacology Lab. IPGT & RA, Gujarat Ayurved University, Jamnagar 5 Head, R&D centre, SDM College of Ayurveda, Laxminarayana Nagar, Kuthpady, Udupi, Karnataka-574118. *Corresponding Author: E-Mail: vd.chandrika@gmail.com; Mobile: +918866613638. 2

Received: 09/06/2012; Revised: 20/06/2012; Accepted: 24/06/2012

ABSTRACT Gokshura is one of the ingredients of Dashamoola (Group of ten root drugs) mentioned in Ayurveda. In routine Ayurvedic practice, in the name of Gokshura, two plants viz., Laghu Gokshura [Tribulus terrestris Linn.] and Brihat Gokshura [Pedalium murex Linn.] are used. Texts of Ayurveda mentioned Dashamoola and Gokshura for the treatment of Kasa. As it is used in the treatment of Kasa, in the present study a comparative anti-tussive activity of whole plant (Panchanga) of Laghu and Brihat Gokshura was evaluated against sulphur dioxide induced cough in mice. Both the varieties of Gokshura were collected from their natural habitat, authenticated and processed to fine powder form. The mice were used as experimental animals and were randomly divided in to three groups of 6 animals each. The test drugs were administered orally at a dose of 780 mg/kg. Recodex, which contains codeine phosphate (2 mg/ml) and chlorpheniramine maleate (0.8 mg/ml), was used as standard anti-tussive drug for comparison. The Panchanga of Laghu Gokshura and Brihat Gokshura have shown moderate anti-tussive activity, among them Brihat Gokshura was found to be better. Hence in non-availability of root samples of these plants and also to prevent destructive harvesting, whole plants can be used in the treatment of Kasa. Further detailed studies are required to prove this claim.

KEYWORDS: Panchanga, Laghu Gokshura, Tribulus terrestris, Brihat Gokshura, Pedalium murex, Anti-tussive, Cough.

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INTRODUCTION Cough is a protective reflex mechanism that removes foreign material and secretions from the bronchi and bronchioles. It can be inappropriately stimulated by inflammation in the respiratory tract or by neoplasia. In these cases, anti-tussive (or cough suppressant) drugs are some times used, for example in the dry painful cough associated with bronchial carcinoma or with inflammation of the pleura (Rang et al., 2003a). It should be understood that these drugs merely suppress the symptom without influencing the underlying conditions. In cough associated with bronchiectasis (suppurating bronchial inflammation) or chronic bronchitis, anti-tussive drugs can cause harmful sputum thickening and retention (Rang et al., 2003a). Hydration of respiratory tract by steam inhalation, demulcents are effective in reducing symptoms in majority of cases but, for uncontrolled cough, opioid central cough suppressants are used. But it has got the greatest disadvantages due to a range of unwanted effects it produces like sedation, constipation, depression of the respiratory center, nausea, vomiting, itching (due to histamine release), tolerance and dependence, euphoria etc. Their administration can lead to increased sputum viscosity, decreased expectoration and hypotension (Rang et al.,

2003b). Therefore, there is a need to have effective anti-tussives which can successfully alleviate chronic cough without side effects. Laghu Gokshura is mainly used in well known compound formulation sold in market under the name of “Dashamoola” (Group of ten root drugs) In one of the Indian medicinal text, “Bhavaprakash nighantu”, Acharya Bhavmishra has mentioned Kasaghna property for Laghu Gokshura (Bhavmishra, Bhavprakash Nighantu, 2006). However, due to inadequate availability of Laghu Gokshura [Tribulus terrestris Linn.] many a times Brihat Gokshura [Pedalium murex Linn.] is used as its substitute in Dashamoola (Kokte et al., 2006). Further in market samples along with root, whole plants (Panchanga) are also admixed for commercial purpose. So, substitution of a particular plant as well as part used is the need of the hour, not only to overcome the scarcity of the medicinal plants but also to preserve it. With this intention, the whole plant of both varieties of Gokshura was used for the study, to learn whether they are having Kasaghna property or not. As Dashmoola is known for its Kasaghna action, this study focuses on comparing the anti-tussive activity of whole plant of both the varieties of Gokshura in an experimental model. Further to the best of our knowledge this is the first attempt in this direction.

Photo Slides 1 & 2: Displaying the Habit of 2 varieties of Gokshura

1. Laghu Gokshura [Tribulus terrestris Linn.]

2. Brihat Gokshura [Pedalium murex Linn.]

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MATERIALS AND METHODS Animals Swiss albino mice of either sex weighing 28 ± 4 g were used for the study. The animals were obtained from the animal house attached to the pharmacology laboratory, IPGT & RA, Gujarat Ayurved University, Jamnagar. Animals were exposed to natural day and night cycles with ideal laboratory conditions in terms of ambient temperature (22 ± 2°C) and relative humidity (50–60%). They were fed with Amrut brand rat pellet feed supplied by Pranav Agro Industries, Baroda and tap water given ad libitum. The experiment was carried out in accordance with the directions of the Institutional Animal Ethics Committee (IAEC) after obtaining its permission (Approval number; IAEC/09/11/25). Test drugs The whole plant of Laghu Gokshura [Tribulus terrestris Linn.] and Brihat Gokshura [Pedalium murex Linn.] were collected from adjacent areas of Sassoi botanical garden, near Jamnagar, Gujarat and were subjected to pharmacognostical studies in order to authenticate. They were authenticated by pharmacognosist of the institute. The plants were washed with water, cut into pieces and dried under shade. The dried whole plants were pulverized to powder and were sieved with mesh no. 120. They were coded as LGP (Laghu Gokshura whole plant) and BGP (Brihat Gokshura whole plant) respectively and used for experimental screening. Animal groupings and dose fixation The selected animals were divided into three groups of six animals each. First group received deionized water and served as control. The test drugs LGP and BGP were administered to second and third groups respectively. Dose of the test formulations for the animals was calculated by extrapolating the human dose (6 g/day) (Anonymous, 2007) to animals (780 mg/kg) based on the body surface area ratio by referring to the standard table of Paget and Barnes (1964). The test drugs were

suspended in 0.5% w/v aqueous CMC (Carboxy methyl cellulose) solution to suitable concentration and administered orally at a volume of 0.1 ml/10 g body weight with the help of gastric catheter of suitable size sleeved on to a syringe nozzle. Recodex (Wockhardt Ltd., Mumbai, India) which contains codeine phosphate (2 mg/ml) and chlorpheniramine maleate (0.8 mg/ml) in the dose of 0.05 ml/10 g was administered to Group IV per oral as standard control drug. The test drugs and standards were administered one hour before the SO2 exposure. Experimental design The anti-tussive effect of the test formulations was evaluated in mice against sulphur dioxide induced cough by following the procedure of Miyagoshi et al. (1986). In brief, the assembly comprises of a 500ml threenecked flask containing aqueous saturated sodium hydrogen sulphite (Na HSO3; Nice Chemicals Pvt. Ltd.) solution. Into this bottle, concentrated Sulphuric acid (H2SO4; Merck, India) is introduced drop by drop; the reaction involved is as follows: 2NaHSO3 + H2SO4 = 2SO2 + Na2SO4 + H2O SO2 is filled in the column of water manometer by opening the three-way cork such that the SO2 can enter the water manometer but without any exit way until the pressure generated reads 75 mm of water as recorded by the water manometer. Then the three-way cork is rotated in such a way that the volume of SO2 collected in the water manometer escapes into the desiccator and not into the flask containing sodium hydrogen sulfite solution. These procedures are operated in a drift. The mouse to be tested is placed in 1 litre desiccator and covered with the lid. Certain amount of SO2 (5 ml which was kept constant throughout the experiment) is introduced to the desiccators by this procedure. The mice, after exposure to SO2 for one minute in the desiccators, were taken out of the desiccators and confined in an upturned filter funnel. The free end of the funnel is attached to a stethoscope, by the help of which the cough reflex of the mice was heard

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and the number of cough episodes in 5 minutes was enumerated. To avoid the observer bias, cough episodes were independently counted by

two observers using digital counters and stopwatches.

Figure 1: displaying the Apparatus for antitussive evaluation A. Saturated sodium hydrogen sulphite in 500 ml capacity of three-necked flask. B. Concentrated sulfuric acid in burette. C. Gas Reservoir. D. Water Manometer. E. Dessicator.

Table-1: Effect of test drugs on SO2 induced cough episodes. Groups

Number of cough episodes per 5 minutes

% inhibition of cough episodes

Control 66.80 ± 4.11 LGP 57.20 ± 3.73 BGP 54.00 ± 4.61 RS 34.00 ± 2.92* Data: Mean ± SEM, *P < 0.05 (Unpaired‘t’ test) Vs control

-14.37 19.16 49.10

The results are presented as Mean ± SEM. The data generated during the study were subjected to unpaired Students‘t’ test as well as one way ANOVA with Dunnets’ multiple ‘t’ test as post-hoc test. The level of significance was set at P < 0.05.

and BGP remarkably inhibited the sulphur dioxide induced cough episodes in comparison to control group; however the observed inhibition in both the treated groups were found to be statistically insignificant. Pre-treatment with reference standard drug significantly inhibited the sulphur dioxide induced cough episodes when compared with control group.

RESULTS

DISCUSSION

Exposure of the mice to sulphur dioxide leads to production of cough reflexes as revealed in table – 1. Pre-treatment with LGP

Coughing is a normal physiological response to an irritation of the laryngo-tracheobronchial system caused by mechanical or

Statistical analysis

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chemical stimulation. It may be painful and fatiguing and requires suppression by antitussive drugs. In animals, coughing has been elicited by mechanical (Tedeschi et al., 1959) or chemical irritation (Turner RA, 1968) and by electrical stimulation (Cavanagh, 1976) of tracheal mucosa or by nerve stimulation (Pickering, 1979). Of all these methods, chemical or mechanical stimulation are more similar to the physiological event and are also the most preferred experimental models. Therefore the model adopted for screening of anti-tussive activity is Sulphur dioxide induced cough, which is a widely used model for evaluating this activity of a candidate compound. Recodex which was used as a reference standard drug contains codeine phosphate is one of the oldest and the most studied of all cough suppressants, and is a standard through which all other cough suppressants are measured. The British National Formulary lists seven opiate analogues as anti-tussives and the most commonly used contain codeine, pholcodeine and dextromethorphan. The opiates exert their pharmacological action via p opioid receptors. In the present study, the anti-tussive activity of

Laghu Gokshura and Brihat Gokshura have been compared with that of recodex against coughing-induced by sulphur dioxide gas. Both the test drugs showed moderate antitussive effect, among which, Brihat Gokshura administered group was found to be better. However both of them failed to inhibit the cough reflex to significant extent as recodex. Both the varieties of Gokshura are having sweet taste due to presence of sugar and mucilage; these phyto-constituents may be responsible for the observed moderate antitussive effect. The mechanism by which they exert anti-tussive activity is unknown, hence further studies are needed to explore exact mechanism involved in observed anti-tussive activity. CONCLUSION Whole plant of Laghu Gokshura and Brihat Gokshura are having moderate anti-tussive activity and among them the activity observed in Brihat Gokshura is found to be better. So in the non-availability of root samples of these plants and also to prevent destructive harvesting whole plants can be used in the treatment of cough.

REFERENCES Anonymous (2007). The Ayurvedic pharmacopoeia of India, Part I, Vol. I, 1st edition. Ministry of health and family welfare, Govt of India, New Delhi, 52. Bhavmishra. Bhavprakash Nigantu. Commentary by KC Chunekar, (2006) Edited by GS Pandey, Chaukhambha Bharati Academy, Guduchyadi Varga, 292. Cavanagh RL, Gylys JA, Bierwagen ME (1976). Antitussive properties of butorphanol. Arch In Pharmacodyn. 220: 258–68.

Kokte CK, Purohit AP, Gokhale SB (2006). Pharmacognosy. Nirali Prakashana, Pune. 36th edition, 2006. Miyagoshi M, Amagaya S, Ogihara Y (1986). Anti-tussive effects of L-ephedrine, amygdalin, and makyokansekito (Chinese traditional medicine) using a cough model induced by sulfur dioxide gas in mice. Plant Med 4:275–278. Paget GE and Barnes JM (1964). Evaluation of drug activities, In: Pharmacometrics, vol-I, edited by Lawrence D R and Bacharach A L, (Academic press New York).161.

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Pickering RW, James GWL (1979). The antitussive activity of a novel compound RU 20201. Drug Res. 29: 287–289. Rang H.P., Dale M.M., Ritter J.M. (2003a). Pharmacology. Edinburgh: London, Churchill Livingstone, An imprint of Elsevier, 5th edition. 350. Rang H.P., Dale M.M., Ritter J.M. 2003b. Pharmacology. Edinburgh: London, Churchill Livingstone, An imprint of Elsevier, 5th edition. 580.

Source of Support: Nil

Tedeschi RE, Tedeschi DH, Hitchens JT, Cook L, Mattis PA, Fellows EJ (1959). A new antitussive method involving mechanical stimulation in unanesthetized dogs. J Pharmacol Exp Ther.126: 338–344. Turner RA (1968). Screening methods in pharmacology. New York, Academic Press. 128.

Conflict of Interest: None Declared

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Original Research Article IMPACT OF AYURVEDIC SHODHANA (PURIFICATORY PROCEDURES) ON BHALLATAKA FRUITS (SEMECARPUS ANACARDIUM LINN.) BY MEASURING THE ANACARDOL CONTENT Ilanchezhian R1*, Acharya R N2, Roshy Joseph C3, Shukla V J4 1

Associate Professor, Dept. of Dravyaguna, A.L.N.Rao Memorial Ayurvedic Medical College & PG centre, Koppa – 577 126, Karnataka. 2 Associate Professor, Dept. of Dravyaguna, Institute for Postgraduate Teaching and Research in Ayurveda, Jamnagar - 361 008, Gujarat. 3 Lecturer, Dept. of Rasashastra & Bhaishajya Kalpana, Govt. Ayurveda Medical College, Nagercoil – 629 001, Tamil Nadu. 4 Head, Pharmaceutical chemistry laboratory, Institute for Postgraduate Teaching and Research in Ayurveda, Jamnagar - 361 008, Gujarat. * Corresponding Author: E-Mail: ayurilan@yahoo.com

Received: 09/06/2012; Revised: 22/06/2012; Accepted: 28/06/2012

ABSTRACT Bhallataka (Semecarpus anacardium Linn.) is reported under upavisha dravya (semi poisonous drugs), in classical Ayurvedic pharmacopoeias. It is advocated that shodhana (Purificatory procedures) of the fruits should be carried out before its internal administration. Though there are different shodhana methods mentioned in Ayurveda, Ayurvedic Pharmacopoeia of India (API) recommends only one method for the shodhana of Bhallataka fruits. In this study, cow’s urine, cow’s milk and brick powder, were used as media. The impact of shodhana was evaluated by pharamaceutical, physico-chemical and chromatographical parameters. Rf values of methanolic extract of processed bhallataka fruits shows the difference when compared to the raw bhallataka fruits, this clearly proves the chemical changes during shodhana. Increased level of anacardol was observed in Shodhita (processed) fruits in comparison to the raw fruits.

KEYWORDS:

Shodhana; Bhallataka; Pharmacopoeia of India

Semecarpus

anacardium;

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Anacardol,

Ayurvedic

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INTRODUCTION

MATERIALS AND METHODS

Bhallataka (Semecarpus anacardium Linn.; Anacardiaceae) fruit is one of the upavisha dravya (semi poisonous drugs).1 Its importance and utility is increasing day by day because of its therapeutic significance in many a diseases including cancer. Though the fruits of Bhallataka has many therapeutic values, pharmacies are scared to use this drug because of its irritant vesicating nature.2 The fruit contains tarry oil which causes contact dermatitis. Medically it is named as Urushiol Induced Contact Dermatitis because the chemical Urushiol is responsible for the dermatitis.3 If this vesicant nature is removed, the drug could be a good source for pharmaceutical industries in manufacturing many formulations containing Bhallataka as an ingredient.

Collection & Selection of drug:

Ayurveda advocates bhallataka after shodhana (purificatory procedures).4 Though there are different shodhana methods mentioned in Ayurveda,5 the shodhana method mentioned in the text Rasamrutam was adopted and quoted in API (Ayurvedic Pharmacopeia of India). The procedure is soaking the fruits in cow’s urine, cow’s milk and rubbing it in brick powder.6 Shodhana is the purificatory measure used in Ayurveda to purify toxic medicinal plants (upavisha dravyas), by various pharmaceutical procedures like soaking, rubbing and washing etc. with specific medias like gomutra (cow’s urine), godugdha (cow’s milk) etc. Physico-chemical changes and reduction of the toxic chemicals from the poisonous plants like strychnine, brucine in kupilu and scopolamine in dhattura are reported already.7 Recent studies proved the changes of Rf values in shodhita samples of bhallataka when compared to raw bhallataka.8 To prove the impact of the Ayurvedic shodhana methods, recommended by API, in this research work, an attempt has been made to analyse the raw and shodhita (purified) samples pharmaceutically and analytically including HPTLC.

Matured fruits of Semecarpus anacardium Linn., were collected from the trees growing wildly in Jalna (19°50′N 75°53′ E19.83° N 75.88°E), Maharashtra, India. The fruits were authenticated and voucher specimen was preserved in the department (Vide no. 6010/2009). The fruits were sun dried for 10 days. Then the fruits were cleaned by removing the false fruit and stored in a container. Bhallataka fruits, which sunk in water, were recommended for therapeutic purposes.9 So fruits which sunk in water were collected and dried properly and used for research purpose. The dried fruits were mixed thoroughly and sample was selected randomly. Collection of the media: Fresh cow’s urine was collected from the Goshala (cow shed) early in the morning. Amul brand cow’s milk was used for the shodhana. Ishtika choorna (brick powder) was collected from the local area. Equipments for Shodhana: Stainless steel vessel (10 litre capacity), Stainless steel spatula (30 cm long), Stainless steel filter, thick cotton cloth were used for processing of the nuts. Pharmaceutical study: 200 g of sunken Bhallataka fruits, in water, were randomly taken. The thalamus portion of the fruits was removed with the help of a steel cutter. Then it was taken in a vessel containing gomutra (cow’s urine) and kept for seven days. Every day the fruits were taken out of the media and washed with water and fresh gomutra was used. On eighth day Bhallataka was washed and shifted to the vessel containing godugdha (cow’s milk) and kept for seven days. Each day it was washed with water and fresh Godugha was added. On 15th day the samples were taken out of the media and washed with water then shifted to a bag

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containing brick powder and rubbed thoroughly. It was allowed for three days in the bag containing brick powder. On 18th day it was washed thoroughly with hot water to remove the brick powder in the sample. Later, the samples were dried properly to remove the moisture and stored in air tight glass container for further studies. The same shodhana procedure was repeated thrice to standardize the procedure pharmaceutically. Physico-chemical evaluation: Physico-chemical evaluations like moisture content, ash values, acid insoluble ash, alcohol soluble extractive and water soluble extractive values were determined. The determinations were performed in triplicate and results are expressed as mean. The percentage w/w values were calculated with reference to the air-dried drug.10,11 Preliminary phytochemical screening: The coarse powder of the fruit was subjected for extraction in methanol and water for 18 h and the extracts were evaporated to dryness. The dried extracts were weighed, and percentage yield were calculated. The extracts were used for preliminary phyto-chemical screening with a set of various chemical tests viz., Dragendorff’s, Mayer’s, Hager’s and Wagner’s tests for alkaloids; ferric chloride, lead acetate, potassium dichromate and dilute iodine tests for tannins and phenolics; foam test for saponin glycosides. These parameters were carried out by following the standard procedure.12 Equipments used for HPTLC Pre coated silica gel GF 60254 aluminium plates as 5 mm bands, 5 mm apart and 1 cm from the edge of the plates, by means of a Camag Linomate V sample applicator fitted with a 100 µL Hamilton syringe. The mobile phase used was Benzene: Ethyl acetate (6 : 1). The plates were developed in Camag twin trough chamber (20 x 10 cm2) and spots were

detected in short U.V. (254 nm), Long U.V (366 nm). Camag Scanner II (Ver. 3.14) and Cats soft ware (Ver. 3.17) were used for documentation. Preparation of raw and shodhita sample solutions: Methanol extractives were prepared by standard method. The concentrated methanol extracts were used as test solutions. 10 mg of extract was accurately weighed and dissolved in methanol in standard flask and final volumes were adjusted to 10 ml with methanol (1 µg/µl). 5 µl of each test solutions were spotted along with 5 µl standard solutions of anacardol. The plates were developed in mobile phase of Benzene : Ethyl acetate (6 : 1 v/v) and scanned at 254 nm. Application of sample: The sample solutions 5µg was applied on TLC plates (10 cm × 10 cm), precoated with silica gel as 5 mm, 5 mm apart and 1 cm from the edge of the plates by using CAMAG Linomat V sample applicator. The mobile phase used was Benzene : Ethyl acetate (6 : 1). The plates were developed in Camag twin trough chamber (20 x 10 cm2) up to a distance of 77 mm at a temperature of 30 ± 20 C. and spots were detected in short U.V. (254 nm), Long U.V (366 nm). Camag Scanner II (Ver. 3.14) and Cats soft ware (Ver. 3.17) were used for documentation. RESULTS Pharmaceutical study: 200 g of samples were taken for Shodhana. Three batches of Shodhana were carried out. The average loss of the sample after shodhana was 17.32%. (Table 1; Figure 1) Analytical study: In analytical study the parameters like physico-chemical analysis, qualitative tests of raw and shodhita Bhallataka was carried out and systematically presented in Table 2 and Table 3 respectively. Chromatography studies

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were carried out and the Rf values observed under 254 nm has been presented in Table 4.

Anacardol in raw bhallataka was 47.51% and 50.62% in processed. (Figure 2)

Table 1: Pharmaceutical study of Bhallataka fruit: Batch SBMDI – 1 SBMDI – 2 SBMDI – 3

Weight of the Bhallataka fruit (g) Initial Obtained Loss/ gain 200 155.6 44.4 200 167.7 32.3 200 172.8 27.2

% of the Bhallataka Loss /gain% Average loss % 22.2 17.32 16.2 13.6

Table 2: Showing physico-chemical parameters of raw and shodhita Bhallataka fruit: Parameters Loss on drying 1100C (% w/w) Ash Value (% w/w) Acid insoluable ash (% w/w) Methanol soluble extractive (% w/w) Water soluble extractive (% w/w)

Shodhita 8.14 5.83 3.34 28.71 4.23

Raw 6.68 2.68 − 35.40 6.99

Table 3: Showing qualitative tests of raw and shodhita bhallataka fruit: Aqueous extract Raw Shodhita + + − – − – + + + +

Parameters Tannins Alkaloids Steroid Phenolic compounds Flavonoids

Methanol extract Raw Shodhita – – – – + + – – – –

‘+’ = Presence of the compounds ‘–’ = Absence of the compounds

Table 4 - Showing Rf values of raw and shodhita Bhallataka fruit at 254 nm Sl. No. 1.

Extractive

Solvent system

Viewing reagent

Methanol

Benzene:Ethylace tate (6:1)

Iodine vapour

Raw 0.17, 0.28, 0.37, 0.42, 0.50

Rf values Shodhita 0.17, 0.30, 0.47

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Anacardol 0.17

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Fig – 1a. Thalamus removed Bhallataka fruits soaked in cow’s urine; Fig – 1b. Bhallataka fruits soaked in cow’s milk; Fig – 1c. Fruits mixed with ishtika choorna; Fig – 1d. Bhallataka tied in pottali; Fig – 1e. Fruits separated from ishtika choorna and washed with hot water; Fig – 1f. Fruits washed thrice; Fig – 1g. Drying of shodhita fruits; Fig – 1h. Dried Shodhita fruits stored in airtight glass container

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T1 – Raw Bhallataka; T2 – Shodhita Bhallataka; T3 - Anacardol Fig – 2a. Viewed under long UV; Fig – 2b. Viewed under short UV; Fig – 2c. Viewed after iodine vapour spray; Fig – 2d. Densitograph; Fig – 2e. Calibration curve

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DISCUSSION Urushiol induced contact dermatitis is a clinical condition caused by the contact of urushiol on skin. Urushiol is present in most of the species in Anacardiaceae, and one of the major chemical constituent of Bhallataka.13 Bhallataka is a drug commonly known for its blister causing nature. In Ayurvedic literature, the synonym Sopha hetu, Spota hetu, agnika are given to this drug based on its blister causing nature.14 The oil in the fruit is responsible for the irritation.15 The bhallataka fruit contains 90% Anacardic acid and 10% of Cardol. Other chemical constituents are bhilawanol (Naidu et al., 1925), 16 semecarpol17 and anacardol.18 Recent studies reported that bhilawanols are known as urushiols. Anacardic acids are closely related to urushiol. Another study reported that the corrosive juice from the pericarp of the fruit is found to contain catechol, fixed oil and anacardol (C18H13O3.COOH) to which the corrosive properties of the juice are due to two phenolic acids C16H15O3.COOH and C14H13O3.COOH.19 Three batches of bhallataka were processed by the shodhana method mentioned in API. Weight loss was observed in all the three samples (Table 1). This loss may be due to the reduction of the oil content in the fruits. The media gomutra (cow’s urine) is reported for its antimicrobial, antibacterial etc.20 Cow’s milk is recommended as one of the antidote for bhallataka blisters.21 Brick powder is having adsorbant property; by which it absorbs irritant oil in the fruit. The methanol soluble extractive was 35.40% w/w in raw Bhallataka and 28.71% w/w in shodhita bhallataka. The difference between raw and shodhita is 6.69% w/w. It reveals that after shodhana the methanol soluble extractives are reducing. The ash value of processed nut was more when compared to raw. Ash value of raw bhallataka and shodhita was 2.68% w/w and 5.83% w/w respectively. The increase in ash value may be due to the residue of the brick powder. The ash value represents the inorganic salts present in the drug. Extracts obtained by exhausting crude drugs are indicative of approximate measures of certain chemical compounds they contain,

the diversity in chemical nature and properties of the drug. Qualitative test showed no variation in raw and shodhita sample. In HPTLC study, the Rf values were observed under 254 nm. In 254 nm, raw Bhallataka showed five spots at the rf 0.17, 0.28, 0.37, 0.42, 0.50 but the shodhita bhallataka showed only three spots at the rf 0.17, 0.30, 0.47. The chemical anacardol22 was compared with both raw and shodhita sample. The anacardol was present in both the samples but the quantity was increased in shodhita sample. Research studies reported that S. anacardium fruit contains 90% of oxy acid i.e. anacardic acid and 10% of cardol. The corrosive juice from the pericarp of the fruit found to contain catechol, fixed oil and anacardol (C18H13O3.COOH) to which the corrosive properties of the juice are due to two phenolic acids C16H15O3.COOH and C14H13O3.COOH.23 In Bhallataka bhilawanols and anacardic acids are the main chemical constituent responsible for the blisters. Bhilawanol is known as Urushiol and the anacardic acids are closely related to Urushiol. Due to the decorboxylation of the oil, the anacardic acid gets converted into less toxic anacardol.24 Decorboxylation process may start right from cutting the fruit itself and will be catalyzed by giving heat/fire treatment.25 The increased level of anacardol in the shoditha bhallataka may be due to the decorboxylation of the anacardic acid in the fruits. More percentage of oil might have got reduced by soaking the fruits in the gomutra and godugdha. The brick powder is having the adsorbing nature, so some percentage of oil may be absorbed by the brick powder. There are probable chances that some chemical changes might have taken place due to the various Medias like gomutra, godugda etc used for its purification. Further studies should be carried out to find out the chemical interactions between the media and the bhallataka fruits during shodhana procedure. CONCLUSION Shodhana (purificatory procedure) increases the anacardol level in shodhita bhallataka fruit samples. More percentage of

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the anacardol was due to the conversion of toxic urushiol into Anacardol.

ACKNOWLEDGEMENT Authors are greatly thankful to the Director, I.P.G.T. & R.A., Gujarat Ayurved University, Jamnagar for the ever encouraging support and guidance to complete the work.

REFERENCES 1. Sadananda Sharma. Rasatarangini, Kasinath sastri editor. 11th ed. New Delhi: Motilal Banarasidas; 2004. p.478–479.

Physico-chemical and powder microscopic study. Journal of Herbal Medicine and Toxicology 2010; 4(2):237–247.

2. Ramasastri, B.V. and Shenolikar, I.S. 1974. Nutritive value of two unusual foods: Adda (Bauhinia vahilii) and marking nut (Semecarpus anacardium) kernels. Indian Journal of Medicinal Research 62(11): 1673–7.

9. Chunekar KC, Pandey GS. Bhavaprakasha Nighantu. 10th ed. Varanasi: Chaukhamba Bharati Academy; 1999. p.139–141.

3. http://en.wikipedia.org/wiki/Urushiolinduced_contact_dermatitis. Modified on 15 June 2011 at 12:45. 4. Sadananda Sharma. Rasatarangini, Kasinath sastri editor. 11th ed. New Delhi: Motilal Banarasidas; 2004. p.478–479. 5. Ilanchezhian R, Roshy Joseph C, Rabinarayan Acharya. Importance of media in shodhana (purification/Processing) of poisonous herbal drugs. Ancient Science of Life 2010; 30(2):27–30. 6. Yadavji Trikamji. Rasamritham. Damodar Joshi, Prabhakara Rao G editors. 1st ed. Varanasi: Chawkambha Sanskrit Bhawan; 1998. p.280. [P.147 (A.F.I., Vol.I, Part B, Appendices)] 7. Ilanchezhian R, Roshy Joseph C, Rabinarayan Acharya. Importance of media in shodhana (purification/Processing) of poisonous herbal drugs. Ancient Science of Life 2010; 30(2):27–30. 8. Venkateshwarlu G, Saraswathi P, Shantha T R, Shiddamallayya, Kishore K R, Sridhar B N. A Preliminary Study on the effect of traditional Ayurvedic purifying methods of Semecarpus anacardium Linn. Nuts – A

10. Anonymous. Ayurvedic Pharmacopoeia of India, Part-2, Vol-2, Appendices. 1st ed. New Delhi: Govt. of India, Ministry of Health of Family Welfare; 2008. p. 15–7. 11. Harborne JB. Phytochemical methods - A Guide to Modern Techniques of Plant analysis. Berlin. Springer Verlag; 2005. 12. Anonymous. Ayurvedic Pharmacopoeia of India, Part-2, Vol-2, Appendices. 1st ed. New Delhi: Govt. of India, Ministry of Health of Family Welfare; 2008. p. 15–7. 13. http://en.wikipedia.org/wiki/Urushiolinduced_contact_dermatitis 20-05-2010 14. Bapalal G Vaidya. Nighantu adarsa, Vol.1. 1st ed. Varanasi: Chaukhamba Bharati Academy; 2007.p.315. 15. Agnives CR, Unnikrishnan P, George MJ, editors. Toxicology – Ayurvedic perspective. 1st ed. Kottakkal: Department of Agadatantra, Vaidyarathnam PS Varier Ayurveda College; 2002.p.201–202. 16. Naidu, D.S. 1925. Constituents of the marking-nut: Semecarpus anacardium Linn. Journal of Indian Institute of Science 8: 129–142.

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17. Khare CP, editor. Indian Medicinal PlantsAn illustrated Dictionary. 1st ed. New Delhi: Springer (India) Private Limited; 2007.p.597. 18. Nagabhushanaa, KS, Umamaheshwari S, Tocolic FE, Prabhu SK, Green IR, Ramadoss CS. Inhibition of soybean and potato Lipoxygenases by Bhilawanols from Bhilawan (Semecarpus anacardium) nut shell liquid and some synthetic Salicylic acid analogues. Journal Enzyme Inhib. Medical Chemistry 2002; 17(4): 255–259. 19. Naidu DS. Constituents of the marking-nut: Semecarpus anacardium Linn. Journal of Indian Institute of Science 1925; 8:129– 142. 20. Anonymous, Research activities of GoVigyan Anusandhan Kendra, Go-Vigyan Anusandhan Kendra, Nagpur, 2005. 21. Sharma PV. Dravyaguna Vijnana. 1st ed. Varanasi: Chaukhambha Bharati Academy; 2005.p.169.

Source of Support: Nil

22. Nagabhushanaa, KS, Umamaheshwari S, Tocolic FE, Prabhu SK, Green IR, Ramadoss CS. Inhibition of soybean and potato Lipoxygenases by Bhilawanols from Bhilawan (Semecarpus anacardium) nut shell liquid and some synthetic Salicylic acid analogues. Journal Enzyme Inhib. Medical Chemistry 2002; 17(4): 255–259. 23. Naidu DS. Constituents of the marking-nut: Semecarpus anacardium Linn. Journal of Indian Institute of Science 1925; 8:129– 142. 24. http://www.cardochem.com/cardanol.html 25. Deborah dos Santos Garruti, Maria Elisabeth Barros de Oliveira. Effect of Cashew Nut Processing on the Burning Sensation Caused by Anacardic Acids. Proc. Interamer. Soc. Trop. Hort 2003; 47:141–142.

Conflict of Interest: None Declared

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Original Research Article QUALITY OF LIFE CONSEQUENCES IN DIABETIC POLYNEUROPATHY Niranjan Y1*, Santwani M A2, Baghel M S3 1

Lecturer, Dept. of PG studies in Kayachikitsa, ALN Rao Memorial Ayurvedic Medical College Koppa, Chikkamagalore Dt, Karnataka, India 2 Consulting Physician, Santwani Health Center, Abhay Shopping Center, Opp. DSP Bungalow Jamnagar- 361008, Gujarat, India 3 Director, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar - 361008., Gujarat, India *Corresponding Author: E-mail- ayurniranjan@gmail.com

Received: 10/06/2012; Revised: 28/06/2012; Accepted: 05/07/2012

ABSTRACT Quality of Life (QoL) is an important outcome of healthcare measures. It is an emerging domain of interest which measures the missing dimension of heath especially in chronic disabling conditions like Diabetic Polyneuropathy. This study focuses on the QoL issues among Diabetic Polyneuropathy which form a considerable portion among diabetics. A total of 60 diabetics were surveyed; those without and with symptomatic distal sensory motor Polyneuropathy. QoL was assessed using WHO QoL (World Health Organization Quality of Life) - BREF scale to understand the consequences of Polyneuropathy on domains of QoL. The data is analyzed using student’s t test for independent samples. Patients with Polyneuropathy expressed overall lower levels of Quality of life as measured with the WHO QoL BREF scale compared with that of plain diabetic controls.

KEY WORDS: Diabetic Polyneuropathy; Quality of Life; WHO QoL BREF

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INTRODUCTION Quality of life (QoL) is defined as individual’s perceptions of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns1. QoL refers to a subjective evaluation which is embedded in a cultural, social and environmental context. Ayurveda gives immense magnitude for the improvement and assessment of QoL as an outcome of treatment. Charaka2, while describing the criteria of assessment of outcomes of therapy, has described four quartets (Chatuhshreyah) in which first is the effect of therapy on the disease but all other three i.e., Agni (Digestive power), Dehabala (Physical strength) and Satwabala (Mental strength) are predominantly concerned with the QoL. It is emerging as a new domain of interest among the recent research especially in treatment of chronic disabling conditions such as Diabetic Polyneuropathy. Charaka clearly defines the motto of treatment being restoration of equilibrium of bodily elements and there by achieving perfect quality of life in terms of alleviation of pain, accession of voice and complexion, plumpness of the body, increase of strength, desire for food, relish while eating, timely and proper digestion of the food taken, approach of the sleep at proper time, not seeing frightful dreams (that forebode disease), happy awakening, the elimination of flatus, urine, feces and semen, and freedom from impairment of any kind of the mind, the intellect, and the sense organs. The fruit of action is the attainment of happiness2 (Sukhavapti). Its characteristics are satisfaction of the mind, intellect, senses and the body. The quality of life (QoL) measurements are increasingly being used in assessing the treatment outcomes in these conditions3 as they measure the missing dimensions of healthcare. Diabetes is a metabolic illness requiring regular medications and ability on the part of patient to monitor and modify diets and lifestyle. The QoL afflictions in diabetes is well documented

in previous studies3, 4, 5 but all are broad studies involving patient oriented questionnaire based where there is a high rate of bias due to subjective perception of life and illness and also the patient awareness towards the disorder. The previous studies6 included both type I and type II diabetes mellitus whereas current research is focused on type II diabetes mellitus. Peripheral neuropathy affects about 30% of people with diabetes mellitus. Among them, 16% to 26% experience chronic pain7, 8. Chronic painful symptoms have considerable detrimental effects on individual’s life and may be associated with anxiety, dejection, depression, morbidity and dependence along with causing financial burden. Most measures of health status represent local perceptions and understanding of the disease which may not be suitable for Indian scenario. Most, measure the effect of disease or health status, mental state and not promoting holistic approach to health and health care. Hence in this study the authors have aimed at assessing the QoL using a widely accepted WHO QoL – BREF9 scale which has been extensively researched, validated9 and being quite simple for administration. This study focuses on the QoL issues among type II diabetics with and without additional neuropathy which form the majority of the population in developing countries like India. The outcomes of this study will be useful among the researchers and also the state health departments in designing or exploring future remedies and for prevention strategies along with construction of suitable rehabilitation measures. Aims and objectives: The objective of the present study was to determine the relationship between quality of life and diabetic neuropathy using WHO QoL BREF scale. MATERIALS AND METHODS Sixty patients were recruited randomly based on incidence in the study from outpatient department of Kayachikitsa, Institute for Post Graduate Teaching and Research in Ayurveda,

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Gujarat Ayurved University, Jamnagar. All were previously diagnosed with Type II diabetes mellitus and were on various oral drug regimens; among them 30 had symptomatic diabetic peripheral sensorimotor Polyneuropathy and an equal number had no additional Polyneuropathy. Diabetic Polyneuropathy is a diagnosis of exclusion10, hence presence of neuropathy was established by Neuropathy Symptom Score11 (NSS) and Neuropathy Disability Score11 (NDS) which includes evaluation of ankle reflexes, vibration perception threshold using 128 Hz tuning fork, pin prick, thermal sensitivity and 10 g monofilament testing. Additionally detailed history, clinical examinations were done to confirm the diagnosis of Diabetic Polyneuropathy. The severe form of neuropathy in stage N3 of Dyck’ staging12 were excluded from the study looking in to the fragility of the condition. International Association for the Study of Pain defines Neuropathic pain as ‘Pain caused by a lesion or disease of the somatosensory nervous system’13. In all types of neuropathic pain there is a combination of sensory loss giving rise to negative signs and pain, causing a variety of positive symptoms and signs14. All the selected subjects with neuropathy, presented with either positive or negative symptoms. Negative symptoms like numbness, loss of balance or positive ones like burning pain, prickling pain, tingling, electric shocklike, aching, tightness, hyperesthesia and allodyna often with nocturnal exacerbations were observed. Quality of Life was assessed using the WHO QoL BREF scale. WHO BREF is an international cross-culturally comparable quality of life assessment instrument which assesses the individual's perceptions in the context of their culture and value systems, and their personal goals, standards and concerns. WHO QoL BREF was administered in an interview and assistance was provided whenever necessary. The study was approved by Institutional Ethics Committee of the institute vide PGT/7/

Ethics/2009-2010/3494 and registered in Clinical Trial Registry of India (CTRI) under registration no. CTRI/2011/07/001885. The data obtained was analyzed statistically using Sigmastat 3.5 Software. Student’s ‘t’ test for independent samples was used to analyze the two groups of patients, viz, those without neuropathy and with neuropathy. Levels of p < 0.05 were accepted as the level of Significance. OBSERVATIONS AND RESULTS Basic demographic data of the two study groups i.e., diabetes without and with Polyneuropathy are given in Table No.1. Global perception of QoL and health along with domain scores for physical, social, psychological and environmental domains are also shown in Table no.1. The subjects studied were belonging to the age group of 35–70 years, among them 31 were males and 29 were females. Mean duration of diabetes in first group was 6.46 years and in second group, it was 9.48 years. The mean of last Fasting blood glucose levels as ascertained by the history was 186.8 mg/dl and 184.37 mg/dl. According to the drug regimen, 20 patients were using Ayurvedic drugs for diabetes and 40 were on allopathic oral hypoglycemic agents. In WHO QoL, the lower the percentage scores the lower the quality of life and vice versa. The subjects with presence of Polyneuropathy opined lower quality of life (t = 5.426; df = 58; P = < 0.001) and health (t = 5.769; df = 58; P = < 0.001). In the questions one and two assessing the global perception of one’s quality of life and health, the patients of neuropathy opined reduced scores of mean 3.33 and 2.83 respectively which was far lower than that of diabetic controls (Table No.1). The subjects with neuropathy showed statistically highly significant decrease in QoL in physical domain (Domain 1) which includes daily activity, energy and pain with mean 60.13 ± SD 6.64 (t = 8.609; df = 58; P = < 0.001). In domain 2, which includes psychological feelings and self esteem, the subjects in group 2 had statistically highly significant reduction in QoL owing to

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GJRMI, Volume 1, Issue 7, July 2012, 295–300 P = < 0.001). Financial security, health care and environment of living were included in domain 4 wherein too, people with Polyneuropathy fared statistically highly lower quality of life (t = 8.692; df = 58; P = < 0.001).

the presence of Polyneuropathy where ‘t’ value was 7.683 (df = 58; P = < 0.001). In domain three incorporating personal and social relationships and sexual activity, the people with neuropathy showed highly significant reduction in QoL (t = 11.634; df = 58;

Table No.1 Depicting demographic details and QoL of studied cases Diabetes without Neuropathy N= 30 Men= 14 ( 46.7%); Women= 16 (53.3%) Mean ± SD* Max–Min Age (in Years)

53.9 ± 8.26

70–38

Duration of Diabetes (in Years)

6.46 ± 4.17

15–0.25

Last blood sugar reading (in mg/dL) Drug regimen for Diabetes

Diabetes with Neuropathy N= 30 Men= 17 (56.7%); Women= 13 (43.3%) Mean ± SD* Max–Min 57.77 ± 7.08

68–37

9.48 ± 5.69

186.8 ± 59.12

20–0.3

184.37 ± 45.64 399–130

270–113

13 (43.3%) - Ayurveda

07 (23.3%) - Ayurveda

17 (56.7%) - Allopathic

23 (76.7%) - Allopathic

Global perception of QoL$

4.1 ± 0.40

5–3

3.33 ± 0.66

4–3

Global perception of health$$

3.6 ± 0.5

4–3

2.83 ± 0.53

4–2

Physical health#

78.17 ± 9.36

94–50

60.13 ± 6.64

75–44

Psychological##

81.5 ± 9.24

94–69

62.67± 9.75

81–44

Social relationships###

93.63 ± 7.48

100–75

67.03 ± 10.05

94–44

Environment####

86.7 ± 7.65

94–75

67.83 ± 9.1

81–44

*SD – Standard Deviation, $ t = 5.426, $$ t = 5.769, # t = 8.609, ## t = 7.683, ### t = 11.634, #### t = 8.692

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DISCUSSION: Quality of Life has been studied using a variety of measures in people with Diabetes. Benbow et al.15 opine that diabetic patients with peripheral neuropathy reported lower levels of quality of life in comparison to uncomplicated diabetes and a normal control group. But the results have been inconsistent, with several studies reporting little or no disruption, while others report a considerable impact on QoL16. The study included subjects of age group 35–70 years, mean age of diabetic neuropathy group was 57.77 years. The subjects with Polyneuropathy had diabetes for an average of 9.48 years and those with uncomplicated diabetes have given the shorter diabetes history of 6.46 years. This proves the fact17 that the duration of diabetes also plays an important role in the development of Polyneuropathy. But the last blood sugar reading ascertained by history gave contradictory finding as the readings were nearly same (186.8 and 184.37 respectively). This may be because of the fact that, the readings were based on patient’s statement and may not be the actual value. For the first question of the questionnaire, regarding their perception of global QoL, mean 3.33 and 4.1 was the score in neuropathics and non-neuropathics respectively. Majority of the subjects with neuropathy had reduced perception of health than that of those with uncomplicated diabetes (Mean 2.83 and 3.6 respectively). Since the study was conducted on symptomatic peripheral sensorimotor Polyneuropathy, the positive and negative feelings of neuropathy pose constant trouble to the daily activities including recreation and sleep. In the domains of Physical health, which includes activities of daily living, dependence on medicines, energy and fatigue, mobility, pain and discomfort, sleep and rest, work capacity the people with Polyneuropathy scored far less, mean being 60.13 compared with that of 78.17 in the diabetics. In the domain 2

comprising of psychological factors like bodily image and appearance, negative feelings, positive feelings, self-esteem, spirituality, thinking, learning, memory and concentration, the people said reduced quality of living (mean 62.67 and 81.5 respectively) due to obesity and loss of concentration in selected patients. Third domain consisted personal, sexual life wherein the reduced quality probably attributable to diminished libido and sexual performance; a well documented complication of diabetes. Fourth domain consists of financial resources freedom, physical safety and security, accessibility and quality of health and social care, home environment, opportunities for acquiring new information and skills, participation in and opportunities for recreation / leisure activities, physical environment (pollution / noise / traffic / climate) and transport. Here too the presence of Polyneuropathy caused marked decline in quality attributable to the disease. This study supports the opinion that the presence of Polyneuropathy along with diabetes significantly reduces the quality of life than that of uncomplicated diabetes. The subjective assessment of QoL becomes important especially in developing nations like India, deciding the physician visits and adherence to strict dietary, exercise and drug regimen. Diabetes being a chronic illness, the understanding of one’s illness demonstrates better coping to the disease and better outcome in terms of treatment, as the final motto of treatment is to provide a better QoL (Sukhavapti). CONCLUSION: Patients with Polyneuropathy express overall lower levels of Quality of life as measured with the WHO QoL BREF scale compared with that of plain diabetic controls. The patients also opine that their overall life is being severely affected by the presence of Polyneuropathy.

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REFERENCES: 1. Anonymous. WHO QoL BREF, Field Trial version. Geneva: World Health Organization, Geneva. 1996. 2. Mehta PM et. al. Eds. Charaka Samhita, Vimana Sthana, Rogabhishagjiteeya Vimana Adhyaya, 8/89-90, Vol. 2, First edition. Jamnagar; Shree Gulabkunverba Ayurvedic Society. 1949 : pp. 916–7 3. Rubin RR, Peyrot M. Quality of Life and Diabetes. Diabetes Metab Res Rev 1999; 15: 205–18. 4. Price P. Diabetic foot: Quality of life. Qual. Life 2004; 39 (Suppl. 2): 129–31. 5. Padua L, Saponara C, Ghirlanda G, Aprile I, Padua R, Pauri F, et al. Health related quality of life Type 1 diabetic patients and influence of peripheral nerve involvement. Neuro Sci 2001; 22: 239–45. 6. Lewko J, Polytynska B, Kochanowicz J, Zarzycki W, Okruszko A, Sierakowska M et al. Quality of life and its relationship to the degree of illness acceptance in patients with diabetes and peripheral diabetic neuropathy. Advances in Medical Sciences 2007; 52 Suppl 1: 144–6 7.

Shaw JE, Zimmet PZ. The epidemiology of diabetic neuropathy. Diabetes Rev 1999; 7: 245–52.

8. Ziegler D, Rathmann W, Haastert B et al. Prevalence of Polyneuropathy in impaired glucose tolerance and diabetes. The MONICA/KORA Augsburg Surveys and Myocardial Infarction Registry (KORA-A Study). Diabetologia 2005;48 Suppl 1: A364–5 Source of Support: Nil

9. Anonymous. WHO QoL BREF, Field Trial version. Geneva: World Health Organization, Geneva. 1996. 10. Anonymous. Definition, diagnosis and classification of diabetes mellitus and its complications. Report of a WHO Consultation. Part 1: diagnosis and classification of diabetes mellitus. Geneva: World Health Organization; 1999. (WHO/NCD/NCS/99.2). 11. Ziegler D. Diabetic peripheral neuropathy and sexual dysfunction. In. Goldstein BJ, Muller-Wieland D editors. Type 2 Diabetes Principles and Practice. 2nd edition. New York: Informa healthcare; 2008. pp. 277– 312. 12. Dyck PJ. Detection, characterization and staging of Polyneuropathy: assessed in diabetics. Muscle Nerve 1988; 11: 21–32. 13. http://www.iasppain.org/AM/Template.cfm?Section=PainD efi...isplay.cfm&ContentID = 1728#Neuropathic pain accessed on 29/05/2011 14. Bhadada SK, Sahay RK, Jyotsna VP, Agrawal JK. Journal of Indian Academy of Clinical Medicine 2001; 2(4): pp 305–18. 15. Benbow SJ, Walleymahmed ME, Macfarlane IA. Diabetic peripheral neuropathy and quality of life. Q J Med 1998; 91: 733–7. 16. Price P. Diabetic foot: Quality of life. Qual. Life, 2004; 39 (suppl 2):129–31. 17. Joshi SR. Diabetic Neuropathy. Technical monogram. Mumbai; National book depot. 2003: pp.01 Conflict of Interest: None Declared

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Original Research Article MANAGEMENT OF NON HEALING WOUND WITH NYAGRODHADI KWATHA AND JATYADI GHRUTA (CLASSICAL AYURVEDIC PREPARATIONS) Lad Meenal D1*, Bandgar Satish B2 1

Prof & HOD Dept of Dravyaguna, PDEA’s College of Ayurved & Research Centre, Akurdi, Secter No.25 Pradhikaran, Nigadi, Pune-44, State Maharashtra, India. 2 ‘Shankar Nivas’, Plot No-18, Eng.Coloney, Near Datta Mandir, Ausa Road, Latur, Dist: Latur, State Maharashtra. Pin- 413512 India. * Corresponding Author: Email – murkute.mona@yahoo.com

Received: 10/06/2012; Revised: 29/06/2012; Accepted: 03/07/2012

ABSTRACT Chronic wound infections not responding to conventional treatment modality are the important cause of disability and mortality. Infection is responsible for delayed wound healing. In the present study, an attempt was made to develop simple and effective treatment modality for wounds that are not responding to conventional treatment modalities by using Nyogrodhadi Kwatha {Decoction of drugs starting with Ficus benghalensis L.} and Jatyadi Ghruta {Medicated ghee prepared with drugs starting with Jasminum grandiflorum}as external wound dressing material. All wounds were first washed with Nyogrodhadi Kwatha and also soaked with it for five minutes. Then these wounds were dried and Jatyadi Ghruta was applied to it. Nyogrodhadi Kwatha contains Vata {Ficus benghalensis L.}, Udumbara {Ficus racemosa L.}, Ashwatha {Ficus religiosa L.}, Triphala (Fruits of Terminalia chebula Retz., Terminalia belerica Roxb. and Phyllanthus emblica L.), Haridra {Curcuma longa L.}, Daruharidra {Berberis aristata DC}, Nimba {Azadirachta indica A.Juss} and Yastimadhu {Glycyrrhiza glabra L.}. All these plant drugs help to clean (debridement) the chronic wounds. All the chronic wounds are considered as Dushta Vrana (non healing wound) and Jatyadi Ghruta acts as wound healing enhancer. So the combination of these two medications probably act as Vrana Shodhaka (wound cleansing) and Vranaropaka (wound healing). In this study among 28 cases 23 (82.14 %) cases have got the wound completely healed. And 03 (10.71%) cases shown the signs of healing, one case (3.17 %) underwent minor amputation, and one case (3.17 %) left the treatment. In local wound care treatment, it was found most effective and economical for the successful treatment. In the management of chronic wounds Ayurvedic drugs are also potent when compared to conventional Antibiotics and local wound care treatment. KEY WORDS: Chronic wound, wound healing, Nyogrodhadi Kwatha, Jatyadi Ghruta.

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INTRODUCTION Non-healing wounds are significantly Problematic to all the health-care systems worldwide. In the industrialized world, almost 1–1.5% of the population will have a problem of wound at any point of time. Furthermore, wound management is expensive for instance, in Europe the average cost per episode is 36,650 pounds for leg wounds and 310,000 pounds for foot wounds, which accounts for 2– 4% of health-care budgets (1). In a study to determine prevalence of diabetic foot in India, the prevalence of infection noted was 6–11% and prevalence of amputation was 3% in type 2 diabetic patients. In another Indian study, the prevalence of diabetic foot ulcers in the clinic population was 3.6 % (2, 3). This figure can be expected to rise with an increasingly elderly and diabetic population. There is an urgent need to review wound strategies and treatments in order to reduce the burden of care in an efficient and cost-effective way. If patients at risk are identified sooner and aggressive interventions are taken before the wound deteriorates and complications occur, both patient morbidity and health-care costs can be significantly reduced.(4) The question is which interventions, technologies and dressing materials are the best from those available? (4) Ongoing controversy surrounds the value of various approaches to wound management and care. There is a need to consider alternative ways of achieving the highest level of evidence required for these patient groups. Drugs treating infections like the Penicillin’s, Cephalosporin’s, Fluoroquinolone’s, Tetracycline’s, Iodine, Hydrogen peroxide, Eusol solution etc are the common Antibacterial agents used in the treatment of various chronic wounds. Drug resistant bacteria are primarily a problem and help to spread infections. Sometimes the multiple drug resistant bacteria can become a therapeutic challenge. Attention should be directed towards discovering an agent, which will accelerate wound healing(4). Chronic non healing wounds continue to pose a challenge to physician (5). As

the last decade created tremendous interests in Ayurvedic science, the demand of herbal medicine in the world market is on a raise. Hence, it is necessary to create evidence to basic principles mentioned in Ayurvedic texts, for its worldwide acceptance. Ayurvedic texts are to be searched for appropriate medicines for a particular disease and these hypotheses and drugs are to be evaluated with modern research methodology. With this point of view a study was undertaken to evaluate the wound healing property of Nyagrodhadi kwatha and Jatyadi Ghruta as these formulations are mentioned in Ayurveda Samhitas (classical ayurvedic texts) as Vranashodhaka and Vranaropaka (1,2,3,6,9,10) which are easily available, cost effective and non-toxic in nature. Hence Nyogrodhadi Kwatha and Jatyadi Ghruta formulations were selected for this study. MATERIALS AND METHODOLOGY Nyogrodhadi Kwatha contains Vata {Ficus benghalensis L.}, Udumbara {Ficus racemosa L.}, Ashwatha {Ficus religiosa L.}, Triphala (Fruits of Terminalia chebula Retz., Terminalia belerica Roxb. and Phyllanthus emblica L.), Haridra {Curcuma longa L.}, Daruharidra {Berberis aristata DC}, Nimba {Azadirachta indica A.Juss} and Yastimadhu {Glycyrrhiza glabra L.}. The proper and potent drugs mentioned above were collected from the Botanical Garden of College of Ayurveda & Research Centre, Akurdi, Pune. Authentication and standardisation of these drugs were done at Indian Drug Research Association Laboratory, Pune, India to ensure its quality and identity. Nyogrodhadi Kwatha was prepared according to reference from Sharangdhara Samhita Di.Kh.2/1-2. Jatyadi Ghruta of Arya Vaidya Pharmacy, Coimbatore, Tamil Nadu, India was used. Wound was washed with Nyogrodhadi Kwatha, and Jatyadi ghruta was applied on the wound up to skin surface level, once in a day up to 60 days. Clinical study design: Open design.

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Simple Randomized

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GJRMI, Volume 1, Issue 7, July 2012, 301–308

Sample size: Total 28 patients of chronic Non healing wound were selected by simple randomized method irrespective of Gender, Caste, and Occupation Inclusion criteria (12) Age up to 75 yrs Gender-Both Non healing wound > 5–6 weeks Heamo-dynamicaly stable Patients having internal Allopathic treatment for Diabetes mellitus (Type-II), Hypertension Patients with I and II Grade wounds. Exclusion criteria of patients (12) Inadequate (Atherosclerosis) blood supply Poor Glycemic control Non-adherence with treatment plan End-stage renal disease Transplant recipients Differing individual goals Malnutrition Connective tissue disorders Systemic conditions such as sickle cell disease, Osteomyelitis, Immobility Heart disease, Dementia, Cancer, AIDS, Tuberculosis and advancing age > 75 yrs.

with sterile cotton gauze. Finally Jatyadi Ghruta was applied on the wound and soaked cotton gauze of Jatyadi Ghruta was dressed on the wound with a dry bandage. Patients were advised to rest. The offloading techniques were taught to the patients. (12) Period of treatment: Sixty hospitalized or OPD based patients.

days

Observation and Assessment criteria during treatment period: Daily observations of patients were done for Local as well as systemic observations and were noted and mentioned in the case sheet. Systemic examination: CVS, R/S, CNS, P/A were done daily. For wounds (12) Signs of inflammation: Temperature: Skin temperature 4 cm away from the wound area Capillary refilling time normal < 5 seconds Edema: On pitting skin, 4 cm away from wound area Presence of peripheral pulse indicator of minimum 80 mm of Hg pressure on foot. Pain scale will range from 1–10 score(13) Color Pale, Red, Ischemic

Consent & Ethical clearance: Wound Exudates: Written informed consent of each patient in his own language was taken. According to ICMR-7 Points guideline. Permission of Ethics committee was taken from Institutional Ethical committee of College Of Ayurveda & Research Centre, Akurdi, Pune, (M.S.) CARC/IEC/O.No.171/09-10} Dressing method: Wound cleaning and debridement was done regularly with the help of normal sterile saline water, sterile cotton, scoop and scalpel. Wound was let to dry with sterile cotton gauze. After which the wound was washed with Nyagrodhadi kwatha and soaked cotton gauze of Nyagrodhadi Kwatha was dressed on the wound for 5 min. Again wound was let to dry

Dry: No exudates, Moist: Small or medium, Wet: Heavy. Serous: Clear yellow fluid without blood, pus, debris, Serosanguinous: Thin, watery, pale red to pink. Sanguineous: Bloody Purulent: Thick, cloudy yellow color. Measurement of wounds for length, breadth, depth by paper tracing technique (12,14) Granulation tissue quality and Angiogenesis: Wound bed color Pink, Ischemic, Red, bleeds on touch, Black (12)

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GJRMI, Volume 1, Issue 7, July 2012, 301–308

Clinical healing outcome was considered as (15)

Healing with complete Epithilization. Partial healing without Epithilization but wound contracture.

Major Amputation Minor Amputation Death of patient Stastical analysis: It was done by using the Binomial Probability test.

OBSERVATIONS AND RESULTS Table no 1 - Details of chronic wound patients according to various characters Disease Diabetic Infected(Cellulites) Hanson’s Bed sore Total Maximum size of wound in Sq. cm. Minimum size of wound in Sq. cm. Average size of wound No. of Wounds Recurrence of wound

Male (%)

Female (%)

Total (%)

10 04 03 02 19 (67.85%) 78 Sq. cm.

05 02 0 02 09 (32.14%) _

15 (53.57%) 06 (21.42%) 03 (10.71%) 04 (14.28%) 28 _

08 Sq. cm.

35 Sq. cm.

30 First time -04

12 Second time-05

42 Recurrent-19

Table no 2- Outcome of treatment Type of Healing Complete healing with Epithelization Partial healing without Epithelization Major Amputation Minor Amputation Dead Leave treatment Total Wound healing of 26 cases in days

Number of patients 23

Percentile (%) 82.14

10.71

0 01 0 01 28 Within 20 days 06 cases

0 3.57 0 3.57 Within 40 days 15 cases

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Within 60 days 05 cases

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GJRMI, Volume 1, Issue 7, July 2012, 301–308 Table No. -3 Wound healing according to Disease

Disease Complete healing with Epithelization (23 cases) Partial healing without Epithelization (03 cases) Major Amputation Minor Amputation ( 1 case ) Dead Leave treatment (1 case) Total

Diabetes 11

Infected 06

Hanson’s 03

Bed sore 03

−

− 01

− −

−

− −

− − 15

− 01

Graph No.1 Basic Graphical representation of Disease wise dataPie chart

Diabetic

Infected(Cellulities)

Hanson's

Bed sore

11% 11% 22%

56%

From above diagram diabetic people are more when compared to people with other diseases.

Stastical analysis The Binomial Probability testDue to small sample size i.e. less than 30, we use the binomial probability test. n- Total no of persons = 27 x - Number of cured persons = 23 p – Proportion of Cured persons = x/n =0.8518 We considered here P0 = 0.8 Q0 = 1- P0 =0.2 HypothesisHo: p ≥ 0.8 vs. H1: p < 0.8

Ho: Population proportion is greater than or equal to 80% Vs H1: Population proportion is less than 80%. Test statisticsZcal = (p – P0) / (p0Q0/n) = 0.6735 P- Value = 0.25143 P-value > 0.05 therefore null hypothesis H0 is accepted. Sample size is too small so it is not possible to use more statistical tools.

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GJRMI, Volume 1, Issue 7, July 2012, 301–308

Graph No.2 multiple bar diagram showing number of persons cured, not cured versus type of disease.

Number of persons

Chart of Number of Persons vs Type of Disease 12 10 8 6 4 2 0 Cured persons Non-Cured persons

Disease

Graph No.2 shows maximum number of cured & Non-Cured persons in Diabetic disease. While in Infected and Bed Sore type cases all the patients were completely cured. DISCUSSION During the 2 month treatment period a total of 28 patients were included in study that fulfilled the inclusive criteria. When the patients were treated with the classical Ayurvedic formulations named Nyogrodhadi Kwatha and Jatyadi Ghruta, the following significant results were observed. Among the total of 28 cases, 19 (67.85%) were males and 9 (32.14%) were females. The number of male population was more in this study and it was only by chance, as the patients attending the hospital were randomly selected. The average age of included population was 57.04 years. In this population 15 (53.57%) patients had diabetic wounds which topped the cases included in the study and is because of increased prevalence rate of Diabetes mellitus in India (7). Infected Cellulitis wound were in 6 (21.42%), Hanson’s tropical wound in 3 (10.71%) and Bed sores in 4 (14.28%) patients. In these 28 patients the total numbers of wounds were 42, which was because of more than 1 or 2 wounds in a single patient. These wounds have Minimum cross sectional area of 08 Sq. cm., Maximum of 78 Sq. cm., Average was 35 Sq. cm. Among all, 6 cases had healed in a 20 day period, 15 cases had healed in 40

days and 5 cases had healed in 60 days of treatment. One case underwent for minor Amputation due to gangrene with septicemia. One case left the treatment due to some personal family problem. According to the diseases included, complete wound healing was found in 11 patients with diabetic wound, 06 patients with infected wound, 03 with Hanson’s disease wound and 03 patients having Bed sores. This variation was because of various factors such as Haemogram, LFT [Liver Function Tests], KFT [Kidney Function tests], BSL [Blood Sugar Level] level, Infection load and resistance of bacteria, use of offloading practice of patient which is subjective, level of Nutrition was also varying from patient to patient. Healing without epithelization was in 03 cases and all were Diabetic wounds. These patients had very poor control of BSL. The use of Nyogrodhadi Kwatha and Jatyadi Ghruta in the management of Chronic wound for dressing purpose was found very effective i.e. 82.14%. Due to a small sample size (less than 30 patients), Binomial Probability Test was used. (n - Total no of persons = 27); P Value = 0.25143; P value > 0.05 therefore we accept null hypothesis H0.

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GJRMI, Volume 1, Issue 7, July 2012, 301–308

After application of Kwatha and Ghruta, in the initial phases pus and debris (Vrana Shodhana) were minimized. So in the initial period of application quantity and time of Kwatha application was increased. In the later stages of wound healing rapid development of granulation and epithelial tissue (Vrana Ropana) was observed. So the time and quantity of Ghruta application was increased. During the treatment period, all observations regarding wound healing which are mentioned in Methodology are mentioned in the case paper of each patient. No any local as well as systemic side effects of the drugs were observed. Probable mode of action The drugs in Nyogrodhadi Kwatha have Kashaya, Tiktta Rasa (Astringency, Bitterness) dominancy. It has Kledaghana, Srava shoshana [drying up of exudates], Vrana sankochan [helps the wound to shrink] and krumighana [anthelmintic] actions. The Sheeta Virya [cold potency] of drugs, stop the Paka of wound. All these activities are considered as Vrana Shodhana in Ayurveda (i.e. it stops decay, reduces secretion, contracts wound, antimicrobial activity). Neem {A. indica} and

Haldi {C. longa} are already proven as antimicrobial agent by modern sciences. Yastimadhu {G. glabra} and Ghruta {Ghee} have Madhura rasa {sweetness} and Sheeta Virya {potency} which acts as Vrana ropaka (Wound healing enhancer) by increasing mamsa dhatu{muscle mass} in wound i.e. granulation tissue. CONCLUSION The use of Nyogrodhadi Kwatha and Jatyadi Ghruta in the management of Chronic wound for dressing purpose was found very effective i.e. 82.14%. Complete healing had occurred in the presence of infection. This treatment is safe, economic and very effective. Even a common man can easily use it. This will reduce the economical and physical burden over society. In Ayurveda Samhita various drugs are mentioned in the management of wounds according to its stages. In Dushta Vrana chikitsa these two formulations have been mentioned. So by this clinical trial, practical evidence has been generated on our ancient knowledge. Further detailed studies with control and experimental group are needed on large population.

REFERENCES 1. EWMA (European wound management association) journal of wound care vol19, no 6, June 2010 page no. 239 2. Vijay Viswanathan., Role of Multinational Study Group on Diabetic Foot Global prevalence of diabetes mellitus: Managing Director. V. Hospital for Diabetes and Diabetes Research Centre WHO Collaborating Centre for Research, Education, Training in Diabetes www.mydigitalfc.com/.../india’s-1stmultinational-study-group-meeting on Diabetic foot and wound management

3. www.mvdiabetes.com/Emailer Feb,2009.pdf 4. Nagoba B.S. ,Wadher B.J. and Selkar S.P. International Journal of Animal and Veterinary Advances 5. Singer A.J., Clark RAF, and Coetaneous wound healing New England Journal Of Medicine (1999) 341 PP: 739 6. Shastri Ambikadatta, Bhaishajya Ratnavali Chaukhamna Sanskrut th Sanathan 13 Ed 1997 Chapter-47, 48

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GJRMI, Volume 1, Issue 7, July 2012, 301–308

7. Shastri Ambikadatta, Charaka Samhita edited by Chakhamba bharti prakashan 22nd Ed 1997 Chikitsa sthan Chapter-25 8. Atridev Motilal, Sushruta Samhita Banarasi Publication 5th Ed Reprint 2002 Sutra sthan Chapter-37, 38 Chikitsa sthan Chapter-1 9. Chunekar K.C., Pandey G.S. Bhavprakash Nighantu Chaukhamba Bharti Academy-Reprint 2002 – Page No.12,114,119,329,513,514,516 10. Navare Krushnashatri, Sharagdhar Samhita Tukaram Book Depo Mumbai1958 Madhya Khanda 2/169,5/5,9/55– 56 Uttar Khanda 10/85–88

Ed-2002 Uttar Khanda Page No-125– 134 & 173–18 12. RNAO Guideline Development Panel, 2005 Nursing Best Practice Guideline page no-28, 37, 44, 47. 13. www.painedu.org/pain pain Assessment.

EDU

sample

14. Student British Medical Journal (BMJ)archive march 2006 14:89–132 15. Bailly and love’s 2000 23 rd Edition Short Practice Of Surgery Wound healing, Tissue repair and Scar 3/29,30 Arnold members of hodder headline group London

11. Shashtri Laxmipati, Yogratnakara Chaukhamba Sanskrita Samsthan 7th

Source of Support: Nil

Conflict of Interest: None Declared

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