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World Journal of Dairy & Food Sciences 8 (1): 67-73, 2013 ISSN 1817-308X © IDOSI Publications, 2013 DOI: 10.5829/idosi.wjdfs.2013.8.1.1119

Chemical Composition and Minerals Analysis of rhamnoides, Azadirachta indica, Punica granatu and Ocimum sanctum

12Muhammed Kashif and Shafqat Ullah

1Islamai College University Peshawar, KPK, Pakistan 2PCSIR Labs Complex Peshawar, KPK, Pakistan

Abstract: The chemical composition and minerals analysis of four different viz., , Azadirachta indica, Punica granatum and Ocimum sanctum leaves were studied. In the nutritional analysis the Punica granatum was found enriched source of ectin, crude fiber, sugar and as compare with other targeted plant leaves. The fat contents was also found in valuable quantity i.e., 5.50% which is relatively same as in Hippophae rhamnoides while the value of protein and nitrogen were very low in comparsion with other that is 3.53 and 0.56%. Azadirachta indica showed low level of acidity, total sugar, fiber and pectin i.e., 0.57, 10.0, 13.41 and 6.21%. The minerals analysis showed no remarkable change in the results. Although the maximum quantities of Fe2+ is found Azadirachta indica (0.0141%), Na+ in Punica granatum (0.75%), K+ in Hippophae rhamnoides (14.58%) and Ni was not found in any sample.

Key words: Plant leaves Medicinal value Chemical composition analysis

INTRODUCTION diseases such as high blood pressure and coronary heart disease. Different parts of sea-buckthorn have Sea-Bucktorn: The sea-buckthorn (Hippophae been used as traditional therapies for diseases [3]. Sea- rhamnoides) is in the buckthorn is an herbal remedy to relieve cough, aid Hippophae, family . The name sea- digestion, invigorate blood circulation and alleviate buckthorn is hyphenated here to avoid confusion with pain. Bark and leaves may be used for treating diarrhea the buckthorns (, family ). It is also and dermatological disorders. For its haemostatic and referred to as sea-buckthorn, sand-thorn or sea- [1]. anti-inflammatory effects, berry fruits are added to constituents of sea-buckthorn berries medications for pulmonary, gastrointestinal, cardiac, have potential value to affect inflammatory disorders, blood and metabolic disorders. Sea-buckthorn berry cancer or other diseases, although no specific health components have potential activity against cancer and benefits. The fruit of the plant has high vitamin C content dengue virus [4, 5]. in a range of 114 to 1550 mg/100gwith an average content (695 mg/100 g). The fruit also contains dense contents of Neem: Neem (Azadirachta indica) is a tree belongs to , , amino acids, dietary minerals, family Meliaceae. It is one of two species in the genus -sitosterol and . Flavonols were found to be Azadirachta. In 1942, for the first time, three bitter the predominating polyphenols while phenolic acids and compounds were extracted from Neem oil, which were flavan-3-ols (catechins) represent minor components. [2]. named as, Nimbin, Nimbinin and Nimbidin. The seeds Preparations of sea-buckthorn oils are recommended for contain a complex secondary metabolite i.e., Azadirachtin external use in the case of burns, bedsores and other skin [6]. complications. Internally, sea-buckthorn is used for the Neem products have been observed to be treatment of stomach and duodenal ulcers. Sea-buckthorn antimalarial, anthelmintic, antifungal, antidiaabetic, oil, juice or extracts from oil, juice, leaves and bark have antibacterial, antiviral, contraceptive and sedative been used successfully to treat high blood lipid activities. Neem products are also used in selectively symptoms, eye diseases, gingivitis and cardiovascular controlling pests in plants. It is considered a major

Corresponding Author: Shafqat ullah, PCSIR Labs Complex Peshawar, KPK, Pakistan. 67 World J. Dairy & Food Sci., 8 (1): 67-73, 2013

component in Ayurvedic and Unani medicine and is Tulsi: Tulsi (Ocimum tenuiflorum) is an aromatic plant particularly prescribed for skin disease [7]. All parts of the belongs to family Lamiaceae which is native throughout tree are said to have medicinal properties (seeds, leaves, the Old World tropics and widespread as a cultivated flowers and bark) and are used for preparing many plant and an escaped weed [15, 16]. The Tulsi also contain different medical preparations. Part of the Neem tree can sufficient quantity of antioxidants and fixed oil [17, 18]. be used as a spermicidal [8]. Neem oil has been found to Some of the main chemical constituents of Tulsi are; be an effective mosquito repellent. As neem products are Oleanolic acid, Eugenol, Carvacrol, Linalool and cheap and non-toxic to higher animals and most beneficial caryophyllene [19]. insects, they are well-suited for pest control in rural areas. Tulsi is cultivated for and medicinal purposes and Neem paste is applied to the skin to treatment and in essential/fixed oils. One study showed that Tulsi can be a similar vein is used for measles and chicken pox an effective for diabetes treatment by reducing blood sufferers. A mixture of Neem flowers are used as symbolic levels and can also reduce significantly the total of sweet and bitter. Neem is deemed very effective in the cholesterol levels [20]. Another study showed that Tulsi’s treatment of scabies. The tender shoots and flowers of the beneficial effect on blood glucose levels is due to its Neem tree are used as vegetable in some countries of the antioxidant properties [21]. Tulsi also shows some world. Neem Gum is a rich source of protein. promise for protection from radiation and cataracts [22]. The fixed oil has demonstrated anti-hyperlipidemic and Pomegranate: The pomegranate (Punica granatum), is a cardio-protective effects in rats fed a high fat diet [23]. fruit-bearing deciduous or small tree. Pomegranate Tulsi’s extracts are used for common colds, headaches, juice provides about 16% of an adult’s daily vitamin C stomach disorders, inflammation, heart disease, various requirement per 100 mL serving and is a good source of forms of poisoning and malaria. Traditionally, Tulsi is vitamin B5 (pantothenic acid), and polyphenols taken in many forms: as herbal , dried powder, such as tannins and [9]. Pomegranates are fresh leaf, or mixed with ghee. Essential oil extracted listed as high-fiber in some charts of nutritional value. from Tulsi is mostly used for medicinal purposes and in That fiber, however, is entirely contained in the edible herbal and is widely used in skin preparations seeds which also supply unsaturated oils. The most due to its anti-bacterial activity. The dried leaves of abundant polyphenols in pomegranate juice are the Tulsi have been mixed with stored grains to repel hydrolyzable tannins called ellagitannins formed when insects [24]. The other medicinal uses of Tulsi are; ellagic acid binds with a carbohydrate. Punicalagins are healing power, coughs, sore throat, respiratory disorders, tannins with free radical scavenging properties in kidney stone, heart disorder, children's ailments, stress, laboratory experiments and with potential human effects mouth infections, insect bites, skin disorders, eye [10-12]. disorders, etc. The pomegranate has been extensively used as a source of traditional remedies. The rind of the fruit and the Aim and Objectives of the Present Study: Plants or parts bark of the pomegranate tree are used as a traditional of the plants (medicinal plants) are well-known for their remedy against diarrhea, dysentery and intestinal health curing potentials, while used in its original form, parasites. The seeds and juice are considered a tonic for their crude extracts or purified chemical constituents the heart and throat and classified as a bitter-astringent isolated from them. The aim of the present study was to component under the Ayurvedic system and considered determine the chemical composition and mineral a healthful counterbalance to a diet high in sweet-fatty profile of the selected medicinal plants grown in Pakistan. components [13], leading to clinical studies of Four medicinal plants namely, Sea-buckthorn (Hippophae pomegranate juice or fruit extracts for efficacy against rhamnoides), Neem (Azadirachta indica), Pomegranate several diseases. The effects of pomegranate extracts or (Punica granatum) and Tulsi (Ocimum tenuiflorum) were juice consumption on diseases such as prostate cancer, selected. The current work will provide new reference prostatic hyperplasia, diabetes, lymphoma, rhinovirus data of the chemical constituents of the selected infection, common cold, oxidative stress, in diabetic medicinal plants, which could be used as base-line value. hemodialysis, atherosclerosis, coronary artery disease, The present study will also be helpful regarding the infant brain injury, hemodialysis for kidney disease [14]. standardization of materia medica.

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MATERIALS AND METHODS Chemical Analysis Determination of Moisture: 2 g of powder plant materials Chemicals and Reagents: Analytical grade chemicals and was taken in a pre-weighed Petri dish and was completely reagents and distilled water were used in the present dried in an oven at 100°C for 4 h. After the sample was study. n. Hexan (Purity: 96%, Scharlu Spain), Fehling’s completely dried, cooled in a desiccator and weighed Solution, Fehling A (copper sulphate solution), Fehling B again. The moisture contents (%) were determined using (alkaline tartrate solution), Methylene blue indicator, the following formula [24]. Potassium oxalate, Lead acetate, Sodium hydroxide (NaOH) (Purity: 96%, Riedel-Dehaen ), Sulphuric Weight of Sample Taken (g) −

acid (H24 SO ) (95-97% Riedel-Dehaen Germany), Asbestos, Weight of Dried Sample (g) Moisture (%) = x 100 Ethyl alcohol (Purity: 98%, Scharlu Spain), Celite (Fluka). Weight of Sample Taken (g)

Instruments and Equipments: The following instruments Determination of Ash: 1 g of the dried powder plant and equipments were used during the present work. Top materials was taken in a pre-weighed crucible and was loading balance (Model: GP 3202, Sartorius AG completely dried in an oven at 100°C for 1 h. The sample Gottingen), Electrical Oven (Ontherm Designer Series was charred on low flame and then heated at 600°C in a Oven, Hutt City, New Zealand), Barnstead/ Electrothermal muffle furnace until a white ash was obtained with (UK), pH Meter (Model: pH 3110 Set 2, WTW, Germany), constant weight. The crucible was then cooled in a Digital Refractometer (Model: RX-1000, Atago, Japan), desiccator and weighed again. The ash contents (%) were Electronic Dry Cabinet/Desiccating Cabinet (Model Dry determined using the following formula [25]. 60 todays-instrument) and atomic absorption spectrophotometer (Model: 2000, Hitachi, Tokyo-Japan). Weight of the Sample After Ashing (g) Ash (%) = ×100 Weight of Sample Taken (g) Selection and Sampling of Medicinal Plants: Four medicinal plants leaves namely, Sea-buckthorn, Neem, Determination of pH and Total Water-Soluble Solids: Pomegranate and Tulsi were selected for the present 5 g of the dried powder plant materials was added in 150ml study. This is because of their use as medicine as well as distilled water and boiled for 30 min, then cooled to room food items. Sea-buckthorn (~2 kg) and pomegranate temperature and filtered through Linen Cloth filter. The pH leaves (~1 kg) were provide by Mycotoxin Research of the filtrate was determined using pre-calibrated digital Laboratory, PCSIR Laboratories Complex, Peshawar, pH-meter and total water-soluble solid contents by digital Pakistan, which were originally collected from Skardu Refract meter [24]. District, Pakistan. The Neem leaves (~4 kg) were collected from mature Neem plants grown at the Botanical Garden, Determination of Total Acidity: 10 g of the dried Islamia College University, Peshawar, Pakistan. Similarly, powder plant materials was added to in 300ml distilled Tulsi leaves (~3.5 kg) were also collected from mature water and boiled till the volume was reduced to 250 mL. Tulsi plants grown at the Botanical Garden, Islamia The mixture was then cooled to room temperature and College University, Peshawar, Pakistan. The plants were filtered through Linen Cloth filter. 20 mL of extract was identified and authenticated by Prof. Dr. Samin Jan, poured in a titration flask, added to it few drops of Department of Botany, Islamia College University, phenolphthalein indicator and then titrated against 0.1 Peshawar, Pakistan. N NaOH solutions. The appearance of light pink coloration showed the end point. The total acidity (%) Pre-Treatment of Plant Materials: The plant materials of the sample was determined using the following formula were washed with tap water and then with the distillated [24]. water for the removal of all possible dust particles or any other external body or contaminant. The plants materials Factor × N × Titer × Dilution Total Acidity (%) = × 100 were first shad-dried at room temperature and then SWV × S grinded using grinding machine. After grinding, the power form of the plant materials were obtained, which was SWV is the weight of the sample taken (g) and S is packed in a plastic bottle, made it air tight and stored in an the volume of the sample extract (20 mL) taken for electrical desiccator till further study. analysis.

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Determination of Fat: 2 g of the dried powder plant water. The mixture was boiled till the volume was reduced materials was taken in a Thimble and placed Soxhlet to 140 mL and then diluted upto 250 mL with distilled extractor. A dried and pre-weighed round-bottom flask water. This mixture was then transferred to a beaker (100 mL) was connected to the Soxhlet assembly (500 mL) and few drops of methylene blue indicator were containing 80 mL n-hexane. The assembly was heated in added. The NaOH solution (1 N) was added to it a heating mental for 8 h. After extraction, the n-hexane drop-wise till the appearance of light pink colour, then was evaporated from the round-bottom flask and the 2 mL of lead acetate solution (45%) was added to the weight of the round-bottom flask along-with the extract mixture and after 10 min 2 mL of potassium oxalate was determined again and from this weight, the crude fat solution (22%) was added to the same mixture. contents (%) were obtained using the following formula The mixture was then filtered through Whatman filter [24]. paper (No. 5) and marked as Filtrate-A. The Filtrate-A was taken in a burette. Fehling-A and Fehling-B solutions W− W (each of 5 mL) were taken in two separate titration Fat (%) =12 × 100 W3 flasks and few drops of methyl blue were added as an indicator and then titrated against Filtrate-A till the

where, W1 is the weight of the round-bottom flask and appearance of dark blue color. The reducing sugar

crude extract (g), W2 is weight of the empty round-bottom contents (%) were determined using the following formula

flask (g) and W3 is the weight of the sample taken (g) for [24]; analysis. Factor × Dilution Reducing Sugar (%) = ×100 Determination of Crude Fiber: 2 g of the dried powder Titer × 1000 plant materials was taken in a Thimble and defatted using n-hexane as a solvent in a Soxhlet extractor for 8 h as where, Factor is obtained from Table. Dilution is the total mentioned in Section 2.4.6. The defatted sample was volume of the mixture (250 mL) and Titer is the volume of

boiled for 30 min in 200 mL H24 SO solution (0.255 N). the Filtrate-A used during titration (mL). After boiling, the mixture was then filtered through Linen Cloth filter and washed the residue with distilled water till Determination of Total Sugar: For the determination of obtained the acid free sample. This residue was again total sugar, 50 mL of Filtrate-A was taken in a titration boiled using 200 mL NaOH solution (0.313 N). The mixture flask (250 mL) and 5 g of citric acid and 50 mL distilled was filtered through a dried and pre-weighed Gooch water were added into it. The mixture was boiled for 10 min crucible prepared with asbestos mat. The crucible to invert the sucrose and then cooled to room along-with the samples was dried in an oven and was temperature. The mixture was then neutralized by weighed and then ignited in a muffle furnace at 600°C for drop-wise addition of 20% NaOH solution using 4 h and weighed again. The crude fiber contents (%) were phenolphthalein as an indicator till the appearance of determined using the following formula [25]. light pink color. The light pink color was disappeared by drop-wise addition of HCl solution (1 N). This colorless W− W mixture was taken in a burette and Fehling-A and Crude Fiber (%) =12 × 100 W3 Fehling-B solutions (each of 5 mL) were taken in two separate titration flasks and few drops of methylene blue where, W12 is the weight of the dried sample (g), W is the were added as an indicator and then titrated against

weight of the ignited sample (g) and W3 is the weight of mixture till the appearance of dark blue color. The total the sample taken (g) for analysis. sugar contents (%) were determined using the following formula [25]. Determination of Organic Nitrogen and Protein by Micro Kjeldahl’s Method: The protein and Nitrogen content in Factor × Dilution Total Sugar (%) = × 100 each sample was analyzed by Kjeldahl’s apparatus Titer × 1000 according to standard operating condition. where, Factor is obtained from Table [24], Dilution is the Determination of Reducing Sugar: About 5 g of the dried total volume of the mixture (250 mL) and Titer is the powder plant materials was mixed with 150ml distilled volume of the Filtrate-A used during titration (mL).

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Determination of Non-Reducing Sugar: The non- RESULT AND DISSCUSSION reducing sugar contents (%) were determined by subtraction of reducing sugar from the total sugar using Selection and Sampling of Medicinal Plants: Four the following formula [34]. medicinal plants namely, Sea-buckthorn (Hippophae rhamnoides), Neem (Azadirachta indica), Pomegranate Non-reducing Sugar Contents (%) = Total Sugar (%) – (Punica granatum) and Tulsi (Ocimum tenuiflorum) Reducing Sugar (%) were selected in the present study. As these plants are well-known for their medicinal values and at the same time Determination of Minerals: One gram of powdered plants they are also used as food items. The present study was materials was taken and transferred to digestion flask. therefore, conducted to determine the chemical To this about 20ml of nitric acid and perchloric acid was composition and mineral profile of the selected medicinal added. This mixture was then heated on 250°C in the plants grown in Pakistan. digestion tube. After digestion 1ml of whole mixture remained. This 1ml digested solution was diluted up to Chemical Composition: The chemical composition of the 100ml of distal water. The concentration of these solution Sea-buckthorn (Hippophae rhamnoides), Neem was reported as%( w/v) and in ppm (mg/lit) on dry weight (Azadirachta indica), Pomegranate (Punica granatum) and analyzed on Atomic Absorption Spectrophotometer. and Tulsi (Ocimum tenuiflorum) are given in Table 1.

Table 1: Proximate analysis of medicinal plants. S. No. Parameters Hippophae rhamnoides Azadirachta indica Punica granatum Ocimum tenuiflorum 1 Moisture (%) 08.10±0.28 10.30±0.28 04.32±0.57 31.35±1.04 2 Ash(%) 07.12±1.73 08.31±1.52 05.14±0.13 14.21±1.50 3 Fat(%) 05.81±1.25 03.37±0.57 05.50±0.40 03.12±0.28 4 Pectin(%) 03.54±0.87 06.21±0.54 08.90±1.10 06.30±0.76 5 Crude Fiber(%) 17.31±2.08 13.41±0.45 63.10±1.00 16.81±1.25 6 Total Sugar(%) 27.29±1.23 10.00±0.98 054.3±2.70 27.23±1.92 7 Reducing sugar(%) 17.37±1.09 03.04±0.79 051.2±2.12 26.52±1.54 8 Non reducing sugar(%) 09.92±1.32 06.96±0.76 03.16±0.21 00.79±0.08 9 Total acidity (%) 01.96±0.15 00.57±0.15 0.48±0.41 01.08±0.43 10 Vitamin-C (mg/100g) 01.12±0.15 00.31±0.04 06.10±0.74 02.41±0.91 11 Protein (%) 11.06±0.41 08.93±0.31 03.53±0.03 04.93±0.03 12 Nitrogen(%) 01.77±0.06 01.42±0.05 00.56±0.01 01.63±0.09 13 Ph of 10% Sol 05.21±0.51 05.84±0.01 03.72±0.05 05.98±0.02 14 TSS 10% Sol 01.20±0.15 00.67±0.05 00.70±1.35 00.31±0.10

Table 2: Macro-elements status of four medicinal plants K Na Ca Mg ------Medicinal Plants () % ppm % ppm % ppm % ppm Hippophae rhamnoides 14.58 145800 0.09 900 1.52 15225 0.217 2175 Azadirachta indica 9.0 90000 0.15 1500 2.92 29250 0.50 5025 Punica granatum 13.65 136500 0.75 7500 0.76 7650 0.12 1200 Ocimum tenuiflorum 14.55 145500 0.45 4500 1.89 18900 0.41 4125

Table 3: Micro-elements status of four medicinal plants. Ni Cr Fe Zn Cu ------Medical Plants (Botanical Name) % ppm % ppm % ppm % ppm % ppm Hippophae rhamnoides 0.0 0.0 .0063 63 0.0224 224 0.0027 27 3.93 39375 Azadirachta indica 0.0 0.0 0.008 80 0.0927 927 0.0043 43 5.20 52050 Punica granatum 0.0 0.0 0.0244 244 0.0141 141 0.0014 14 4.33 43275 Ocimum tenuiflorum 0.0 0.0 0.0067 67 0.0546 546 0.0036 36 4.78 47850

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As can be seen, the moisture and ash contents were in 3. Rosch, D., M. Bergmann and L.W. Kroh, 2003. range of 4.3 to 31.3 and 7 to 8.3, in which Punica Structure-antioxidant efficiency relationships of granatum have the highest value of moisture and ash. phenolic compounds and their contribution to The pH and total acidity of the 10% extract of all plants theantioxidant activity of sea-uckthorn juice. J. Agric. was found same. In the determination of fiber contents the Food Chem., 51(15): 4233-4239. sea buckthorn leaves contained maximum fiber contents 4. Kallio, H., B. Yang and P. Peippo, 2002. Effects of as compare with other plants, that was 63%, while the different origins and harvesting time on vitamin C, other were in the range of 13 to 17%. The crude fat was tocopherols and tocotrienols in sea buckthorn extracted in hexane by hoxhlet apparatus in triplicate, the (Hippophaë rhamnoides) berries. J. Agric. Food mean values tabulated in Table 1. The results showed that Chem., 50(21): 6136-6142. Tulsi and sea-buckthorn contained low and high levels of 5. Jain, M., L. Ganju and R.C. Sawhney, 2008. Effect of crude fat contents, respectively, while the rest of the Hippophae rhamnoides leaf extract against Dengue plants contain intermediate values between these two. virus infection in human blood-derived macrophages. The maximum fat content was found in sea buckthorm and J. Phyto. Med., 15(10): 793-799. pomegranate, that is 5.81 and 5.50%. While 3.37 and 3.12% 6. Gunguli, S., 2002. Neem. A theraputic for all seasons. of crude fat were found in Azadirachta indica and J. Current Sci., 82(11): 1304-1306. Ocimum tenuiflorum. The pomegranate also enriched by 7. Zillur, R.S. and M.J. Shamim, 1993. Neem in Unani sugar contents. The nitrogen and protein contents were Medicine. Neem Research and Development Society determined in all the plant samples. The overall range of of Pesticide Science, India, New Delhi. J. Cur. Sci., nitrogen contents were from 0.56 to 1.63% and 3.53 to 82(11): 208-219. 8. Schubert, S.Y. and E.P. Lansky, 1999. Antioxidant and 11.06%. In which sea-buckthrom have high value of eicosanoid enzyme inhibition properties of protein and nitrogen. pomegranate seed oil and fermented juice flavonoids. J. Ethnopharmacol., 66(1): 11-17. Minerals Contents: Four macro-elements was 9. Kulkarni, A.P. and S.M. Aradhya, 2007. In vitro reported in the minerals analysis viz., K+ , Na + , Ca 2+ and studies on the binding, antioxidant and Mg2+ . The concentration reported in the minerals analysis cytotoxic actions of punicalagin. J. Agric. Food of these selected plants was in% (w/v) and ppm (mg/lit) Chem., 55(4): 1491-500. on dry weight basis in Table 2. The value of K+ , Mg 2+ and 10. Seeram, N.P., S.M Henning and Y. Zhang, 2006. Ca2+ was found high in Tulsi sample that is 14.55, 0.41 Pomegranate Juice metabolites are present in human and 1.89%. While the highest value of Na+ was found plasma and some persist in urine for up to 48 hours. in pomegrante that was recorded 0.75%. The five J. Nutr., 136(10): 2481-2485. micro- elements were reported in the minerals analysis 11. Plumb, G.W. and D.S. Pascual, 2002. Antioxidant which are Ni, Cr, Fe, Zn and Cu. The concentration properties of gallocatechin and prodelphinidins from reported in the minerals analysis of these selected plants pomegranate peel. Redox Rep., 7(41). were in% (w/v) and ppm (mg/lit) on dry weight basis in 12. Seeram, N.P., W.J Aronson and Y. Zhang, 2007. Table 3. There is not found any remarkable change in Pomegranate ellagitannin-derived metabolites micro elements of these plants while Ni was absent in all inhibit prostate cancer growth and localize to the of the tested samples. mouse prostate gland. J. Agric. Food Chem., 55(19): 7732-7737. REFERENCES 13. The Many Amazing Health Benefits of Pomegranates. Exotic Fruit for Health. 22 August 2011. Retrieved 19 1. United States Department of Agriculture. Retrieved September 2011. 2007-10-08. 14. Staples, G.E. and S.K. Michael, 1999. Ethnic Culinary 2. Teng, B.S., Z.T. Wang and D.Z. Wei, 2006. In vitro Herbs. University of Hawaii Press, pp: 73. anti-tumor activity of isorhamnetin isolated 15. Prakash, P. and N. Gupta, 2005. Therapeutic uses of from Hippophae rhamnoides L. against BEL-7402 Ocimum sanctum Linn with a note on eugenol and its cells. Pharmacol. Res. J. Agric. Food Chem., pharmacological actions: Review Article. Indian J. 54(3): 186-94. Physiol. Phamacol., 49(2): 125-131.

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16. Sethi, S.S. and T. Anjana, 2004. Evaluation of 20. Jyoti, S.S. and T.T Anjana, 2004. Evaluation of Hypoglycemic and Antioxidant Effect of Hypoglycemic and Antioxidant Effect of Ocimum Ocimum Sanctum, Jyoti. Indian J. Clinical Biochem., Sanctum Indian, J. Clin. Biochem., 19(2): 152-155. 19(2): 152-155. 21. Sharma, P. and S.S. Kulshreshtha, 1998. Anti-cataract 17. Suanarunsawat, T., T. Boonnak and W.D. Ayutthaya, activity of Ocimum sanctum on experimental cataract. 2010. Anti-hyperlipidemic and cardioprotective Indian J. Pharmacol., 30(1): 16-20. effects of Ocimum sanctum L. fixed oil in rats fed a 22. Suanarunsawat, T. and W.D Ayutthaya, 2010. high fat diet. J. Basic Clin. Physiol. Pharmacol., Anti-hyperlipidemic and cardioprotective effects of 21(4): 387-400. Ocimum sanctum L. fixed oil in rats fed a high fat diet. 18. Kuhn, M.D. and W. David, 2007. Winston & Kuhn's J. Basic Clin. Physiol. Pharmacol., 21(4): 387-400. Herbal Therapy & Supplements: A. Lippincott 23. Biswas, N.P. and A.K. Biswas, 2005. Evaluation of Williams & Wilkins, J. Scie Trad., pp: 260. some leaf dusts as grain protectant against rice 19. Rai, R.V., U.V. Mani and U.M Iyer, 1997. Effect of weevil Sitophilus oryzae, J. Envi Ecol., 23(3): 485-488. Ocimum sanctum Leaf Powder on Blood 24. Method of analysis AOAC Washington 2002. Lipoproteins, Glycated Proteins and Total Amino Acids in Patients with Non-insulin-dependent Diabetes Mellitus, J. Nut & Environ. Med., 7(1): 113-118.

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