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EXTRACTION OF OLEOIDES AND ITS PERFORMANCE ALONG WITH ANTIBIOTICS FOR

ANTIMICROBIAL ACTIVITIES

BHARGAV DAVE1, PIYUSH VYAS1*, MADHU PATEL2 AND

3 Universal Impact NAINESH PATEL Factor 0.9285:2012; 1. 1.2210:2013 SHETH M.N SCIENCE COLLEGE, PATAN.

Index Copernicus 2. NAVSARI AGRICULTURAL UNIVERSITY, SURAT. ICV 2011: 5.09, 2012: 6.42, 2013: 3. PACIFIC UNIVERSITY, UDAIPUR, RAJASTHAN. 15.8, 2014:89.16 , 2015:78.30 Corresponding author’s e-mail: [email protected] NAAS Rating 2012 : 1.3; 2013-16:2.69 ABSTRACT: 2017: 3.98 Nature has given various ways to maintain people’s health. One way is to SJIF 2012: 3.947, 2013: 4.802 use herbal medicine. Herbal medicines have been used to treat various types Infobase Index 2015:4.56 of diseases for long times. The people are more attracting towards the use of Cosmos Impact Factor herbal drugs to cure various types of diseases. For treatment of several 2015: 4.366 diseases of human beings, drug ‘rasayana’ has always played a vital Received on: role. According to World health organization (WHO) more than 80% of the 6th December 2017 Revised on: world population is dependent on traditional medicine for their primary th 10 December 2017 health care needs.1 Accepted on: 12th December 2017 Herbal medicine, also called botanical medicine or phytomedicine, refers to Published on: the use of a plant’s seeds, barriers, roots, leaves, bark, or flowers for 1st January 2018 medicinal purposes. Long practiced outside of conventional medicine,

Volume No. herbalism is becoming more main stream as improvements in analysis and Online & Print quality control along with advances in clinical research show their value in 95 (2017) 2 Page No. the treatment and prevention of disease. 32 to 41 KEYWORDS: Extraction, Salvadora oleoids, Antibiotics, Antimicrobial Life Sciences Leaflets Activities. is a international open access print & e INTRODUCTION: journal, peer reviewed, In , Ayurveda medicine has used many herbs such as turmeric possibly worldwide abstract as early as 4,000 BC.3,4 Earliest Sanskrit writings such as the Rig Veda, and listed, published every month with ISSN, RNI Atharva Veda are some of the earliest available documents detailing the Free- membership, medical knowledge that formed the basis of the Ayurveda system.5 Many downloads and access. other herbs and minerals used in Ayurveda were later described by ancient http://lifesciencesleaflets.petsd.org/ PEER-REVIEWED Page | 32

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Indian herbalists such as Charaka and Sushruta during the 1st millennium BC. The Sushruta Samhita attributed to Sushruta in the 6th century BC describes 700 medicinal , 64 preparations from mineral sources, and 57 preparations based on animal sources.6 Salvadora is a small tree with drooping branches, rarely with proper bole or exceeding a height of 20 feet and a girth of 3 feet. It is very common plant in arid tracts but becomes scarce where rainfall conditions are better. It can withstand great soil salinity. It produces new leaves during April, which on maturity become thick and leathery. The tree coppices fairly well but regenerates freely by root suckers and natural layering. It is, however, very slow growing but a dense growth is often formed around the parent plant by root suckers and some natural seedlings. The plant provides a dense shade. It is often lopped for and goat fodder.

Fruit Small greenish white flowers are produced in March-April. The fruit is yellow and ripens in the months of May and June. It forms one of the main grazing sources for livestock owned by local farmers. It is often dried and preserved in large quantities. The seeds are spread by birds. The seedlings come up under the parent plant or under other bushes and are somewhat frost-tender.7

Common name: Bada Peelu • Hindi: Jal, Bada Peelu • Marathi: Diar, Pilu, Khabbar • Gujarati: Pilu Bada Peelu is a shrub or small tree, attaining 6-9 m height under favourable conditions. It has a short trunk, quite often twisted or bent, up to 2 m in diameter. Branches are numerous, drooping, stiff, often swollen at forks. Bark is gray or whitish-gray. Leaves are blue-green, linear-or ovate-lancelike, leathery and somewhat fleshy, dark greenish-yellow when young, gray when mature. Flowers are stalkless, greenish-white, minute in paniculate spikes, often clustered. Fruit is a spherical drupe, about 6 cm in diameter, usually yellow when ripe, dark brown or red when dry8 Plant taxonomy9 Kingdom: Plantae (unranked): Angiosperms (unranked): (unranked): Order: Family: Genus: Salvadora Species: S. oleiodes

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MATERIALS AND METHODS: Phytochemical analysis Phytochemistry: Fruit contains sterols, beta-sitosterol and its glucosides and stigmasterol; benzylisothiocyanate, noctacosanol and tetracosane; flavonoids including quercetin and rutin; thiourea derivatives and phospholipids10. Essential oil of leaves and stems of was extracted by hydro distillation and analyzed by gas chromatography and mass spectrometry (GC-MS). Thirty five (94.0%) and twenty five (91.1%) components were identified in the leaves and stems, respectively. The results show that leaves contain high concentration of 2-methoxy-4-vinylphenol (25.4%), cis-3-hexenyl benzoate (16.8%), phytol (13.9%), n-hexadecanoic acid (6.9%), and trans-ß-damascenone (2.1%). Stems contain high concentration of 2-methoxy4-vinylphenol (21.6%), phytol (12.9%), n- hexadecanoic acid (3.6%), octacosane (7.9%), nonacosane (7.3%), 1octadecene (5.8%), heptacosane (5.9%), hexacosane (4.5%), pentacosane (3.4%), squalene (3.9%) and transß-damascenone (2.3%)11 Medical and traditional uses This tree species is having a number of proven medicinal applications and almost all parts have been found to be pharmaceutically important. Decoction of leaves is gien o the cattles to promote the expulsion of dead fetus from the uterus. The leaves were used as a cooling agent, blood purifier, laxative, expectorant and also used as purgative in horses12,13. Salvadora oleoides is extracted by using soxhlet extraction. Here different solvents like acetone, methanol and ethanol used. These extracts are combined with different Antibiotics and their combinations were applied for zone of inhibition test against different microorganism. Antimicrobial Activity : Antimicrobial activity and antifungal activity of plant extract alone, antibiotics alone and combination of both was carried out by a well diffusion method. In vitro antibacterial activity of the crude extracts was studied by the agar well diffusion method. After getting the turbidity equal to 0.5 McFarland standards, inoculums were aseptically introduced on to the surface of sterile agar plates and sterilized cotton swabs were used for even distribution of the inoculums. Wells were prepared in the agar plates using a sterile cork borer of 10.0 mm diameter. The plant extract and antibiotic drug were dissolved in DMSO to get desired concentration. The wells were filled with plant extract 25µl, 50µl and antibiotic drug (100µl). The plates are incubated at 37℃for 48 hours and then zone of inhibition was measured. In case of combination of plant extract and antibiotic, equal volume of each was added in the well and zone of inhibition was measured. The experiment was replicated two times. (Zone of Inhibition = ±1mm) Qualitative Phytochemical Analysis Preliminary qualitative analysis of all plant extract was carried out as per standard methods as follows. http://lifesciencesleaflets.petsd.org/ PEER-REVIEWED Page | 34

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1. Test for alkaloids Extracts were dissolved in dil.HCl and filtered. A. Dragendroff’s Test: Filtrate was treated with Dragendroff’s reagent (Potassium Bismuth Iodide solution). Formation of red precipitate indicates the presence of alkaloids. B. Wagner’s Test: Filtrate was treated with Wagner’s reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate indicates the presence of alkaloids. 2. Test for flavonoids A. Lead acetate Test: Extract was treated with 10% lead acetate solution. Yellow colour precipitate formation indicates the presence of flavonoids. B. Test with Sodium hydroxide: Extract was treated with few drops of NaOH solution. The yellow colour indicates the presence of flavonoids. 3. Test for Glycosides A. Keller-Kiiani Test: Add glacial acetic acid, one drop 5% FeCl3 and conc. H2SO4 to the extract. Formation of reddish brown color at junction of the two liquid layers and bluish green at upper layer indicates the presence of glycosides. B. Legal Test: On treatment of Extracts with sodium nitropruside in pyridine and sodium hydroxide, observed pink to blood red colour that indicates the presence of cardiac glycosides. 4. Test for Phenolics Test with Ferric Chloride: Extract was treated with few drops of ferric chloride solution and formed bluish black colour indicates the presence of phenols. 5. Test for Saponins Froth Test: The extract was diluted with 20 ml of distilled water and then shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins. 6. Test for Tannins Test with Ferric Chloride: Extract was boiled with water and filtered it. Added FeCl3 to the filtrate and formation of brownish green or a blue-black coloration indicates the presence of tannins.

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7. Test for Terpenoids

Salkowski Test: To the extract, added 2ml chloroform and 3ml Conc.H2SO4 by the wall of test tube carefully. Formation reddish brown colour at interface indicates the presence of terpenoids.

RESULTS AND DISCUSSION: Table A showed phytochemical analysis of Salvadora oleoides by using acetone, methanol and ethanol extracts. Results showed that Alkaloids, Tannins are present in methanol and ethanol extract. While they were absent in acetone extract. Table 1 showed antimicrobial activity of Salvadora oleoides extract for 25µl. Results showed that at higher concentration they showed good bacterial inhibition potential. Methanol extract has little higher value of zone of inhibition compare to acetone extract. Table 2 showed antimicrobial activity of combination of Salvadora oleoides extract and amoxicillin. It is proved that combination showed good synergic effect compare to pure amoxicillin. As concentration increased, the value of zone of inhibition increased. Further all three extracts have good resistance potential against different mentioned bacterial culture Table 3 showed antimicrobial activity of combination of Salvadora oleoides extract and ciprofloxacin. The value of zone of inhibition for pure ciprofloxacin are 24mm, 29mm, 27mm, 27mm against S.aereu, B.subtilis, Pseudomonas aerugenosa and E.coli respectively. While combination shows higher zone of inhibition for all extracts. At higher concentration more powerful inhibition obtained Table 4 showed antimicrobial activity of combination of Salvadora oleoides extract and Ceftadizime. The value of zone of inhibition for combination has good resistive properties compare to pure Ceftadizime. Further methanol extract have good resistance potential compare to acetone extract. Table 5 showed antimicrobial activity of combination of Salvadora oleoides extract and Erythromycin for 25µl. pure Erythromycin have value of zone of inhibition are 16mm, 22mm, 1mm and 5mm against S.aereu, B.subtilis, Pseudomonas aerugenosa and E.coli respectively. But combination have higher zone of inhibition compare to pure antibiotic. Combination showed strong synergic effect against Pseudomonas aerugenosa. At lower concentration value of zone of inhibition slightly decreased from 1000 to 125µg Table 6 showed antifungal activity of Salvadora oleoides extract. Initially extract showed good resistance power against mentioned bacterial culture. Acetone, ethanol and methanol extract have good protective power against different bacteria. Methanol and ethanol extract showed value 15mm at 1000µg/ml against A.niger. Same way good inhibition obtained against candida albicum. Table 7 showed antifungal activity of combination of Salvadora oleoides extract with Amphoteracin B against A.niger and C. albicum. Methanol extract has little higher value of zone of inhibition against A. niger while all three extract have good synergic effect against C. albicum. http://lifesciencesleaflets.petsd.org/ PEER-REVIEWED Page | 36

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Table 8 showed antifungal activity of combination of Salvadora oleoides extract with Fluconazol. The value of zone of inhibition of fluconazol are 2mm and 0mm against A.niger and C. albicum while combination showed values between 10 to 15 against both culture from lower to higher concentration.

CONCLUSIONS: Plant extracts have showed effective antimicrobial activity against bacterial various culture. To decrease side effect of antibiotics and to increase sensitivity of plant extract, combination of Herbal extract and antibiotics used. Day by day bacteria regaining antibody against traditional antibiotics and gaining strength. To stop their regenerating power, combination of plant extracts and antibiotics provide one of the best results in such direction. If nontoxic plant extracts have been taken in suitable doses, it may prove best supplementary remedies patient. This is in vitro study and such combination must be followed by toxicity test and in vivo tests to determine its therapeutic application.

REFERENCES: Khanna, S., Gupta, S. R., & Grover, J. K. Effect of long term feeding of tulsi(Ocimum sanctum Linn) on reproductive performance of adult albino rats. Indian journal of experimental biology, 24(5), 302, 1986.

2. http://www.sunriseayurvadic.com/Herbal-Medicine.htm

3. Susan G. Wynn; Barbara Fougère . Veterinary Herbal Medicine. Elsevier Health Sciences. p. 60. ISBN 0323029981, 2007.

4. Aggarwal BB, Sundaram C, Malani N, Ichikawa H . "Curcumin: the Indian solid gold". Adv. Exp. Med. Biol. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY. 595: 1–75. doi:10.1007/978-0-387-46401-5_1. ISBN 978-0-387-46400-8. PMID 17569205, 2007.

5. Sumner, Judith . The Natural History of Medicinal Plants. Timber Press. p. 17. ISBN 0-88192- 483-0, 2000.

6. Girish Dwivedi, Shridhar Dwivedi . History of Medicine: Sushruta – the Clinician – Teacher par Excellence (PDF). National Informatics Centre. Retrieved 2008-10-08, 2007.

7. "Salvadora oleiodes Decne.". Germplasm Resources Information Network. United States Department of Agriculture. 2006-07-31. Retrieved 2010-08-21

8. www.flowersofindia.net/catalog/slides/Bada%20Peelu.html

9. www.en.wikipedia.org/wiki/Salvadora_oleiodes

10. Khare CP, Indian Medicinal Plant, An Illustrated Dictionary. Springer, New York, 574-575, 2007.

11. Samejo MQ, Memon S, Bhanger MI, Khan KM, Chemical constituents of essential oil of Salvadora oleoides, J. Pharmacy Res, 5, 2366-2367, 2012.

12. Duhan A, Chauhan B and Punia D, Nutritional value of some non-conventional plant foods of India, Plant Foods Hum Nutr, 193, 1992. http://lifesciencesleaflets.petsd.org/ PEER-REVIEWED Page | 37

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13. Anonymous, The Wealth of India – A Dictionary of Indian Raw Materials, Publications and Information Directorate, CSIR New Delhi, Vol 9 193-195, 1972.

A Qualitative phytochemical analysis of Salvadora oleoides

Name of Test Acetone Ethanol Methanol Phytochemical Extract Extract Extract Alkaloids Dragondrorff’s test + ++ ++ Wagner’s test + ++ ++

Tannins With FeCl3 + + + Saponins Froth Test + + + Glycosides Killer- kiiani Test ------Legal Test ------Flavonoids Lead acetate + + + With NaOH + ++ ++ Terpinoids Salkowski Test ------+ Phenolics With FeCl3 + --- + --- = No Presence + = Less Presence ++ = Medium Presence +++ = Complete Presence

Antimicrobial activity of Salvadora oleoides and its combination with antibiotics for 25µl.

Table 1. Shows antibacterial activity of Salvadora oleoides extract for 25 µl

Bacteria Acetone extract Ethanol extract Methanol extract

Concentration ( µg/ml )

1000 500 250 125 1000 500 250 125 1000 500 250 125 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl S. aureus 12 11 10 9 13 12 11 9 16 15 12 9 mm mm mm mm mm mm mm mm mm mm mm mm B. subtilis 10 9 9 8 12 11 10 8 15 13 11 8 mm mm mm mm mm mm mm mm mm mm mm mm Pseudo 13 13 12 11 16 15 13 11 17 16 14 11 aeruginosa mm mm mm mm mm mm mm mm mm mm mm mm E. coli 15 14 13 12 17 16 14 12 20 18 15 12 mm mm mm mm mm mm mm mm mm mm mm mm

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Table 2. Shows antibacterial activity of combination of of Salvadora oleoides extract and amoxicilline for 25 µl Bacteria Amox Acetone extract Ethanol extract Methanol extract +Amoxicilline +Amoxicilline +Amoxicilline

Concentration ( µg/ml ) 1000 500 250 125 1000 500 250 125 1000 500 250 125 100 µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl S. aureus 31 mm 43 42 41 40 42 41 40 39 43 41 39 38 mm mm mm mm mm mm mm mm mm mm mm mm B. subtilis 35 mm 40 40 39 37 41 40 39 38 42 41 39 38 mm mm mm mm mm mm mm mm mm mm mm mm Pseudo 2 mm 21 20 18 17 34 33 31 30 33 32 30 29 aeruginosa mm mm mm mm mm mm mm mm mm mm mm mm E. coli 2 mm 33 32 31 30 35 34 32 30 33 32 31 30 mm mm mm mm mm mm mm mm mm mm mm mm

Table 3. Shows antibacterial activity of combination of Salvadora oleoides extract with Ciprofloxacin for 25 µl

Bacteria Cipro Acetone extract + Ethanol extract + Methanol extract + µg/ml Cipro. Cipro. Cipro. Concentration ( µg/ml ) 1000 500 250 125 1000 500 250 125 1000 500 250 125 25 µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl S. aureus 24 39 37 36 34 38 37 36 35 40 39 38 37 mm mm mm mm mm mm mm mm mm mm mm mm mm B. subtilis 29mm 37 36 35 34 36 36 35 35 38 37 36 35 mm mm mm mm mm mm mm mm mm mm mm mm Pseudo 27 33 32 31 31 33 32 30 30 33 32 31 30 aeruginosa mm mm mm mm mm mm mm mm mm mm mm mm mm E. coli 27mm 32 31 30 30 33 32 30 30 36 35 33 32 mm mm mm mm mm mm mm mm mm mm mm mm

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Table 4. Shows antibacterial activity of combination of Salvadora oleoides extract with Ceftadizime for 25 µl Bacteria Cefta Acetone extract+Cefta Ethanol extract+Cefta Methanol extract+Cefta µg/ml Concentration ( µg/ml ) 1000 500 250 125 1000 500 250 125 1000 500 250 125 25 µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl S. aureus 0 mm 16 15 14 12 13 12 11 10 14 13 12 10 mm mm mm mm mm mm mm mm mm mm mm mm B. subtilis 0 mm 12 11 10 9 11 11 10 9 12 11 10 9 mm mm mm mm mm mm mm mm mm mm mm mm Pseudo 5 mm 10 9 9 8 11 10 9 8 11 10 9 8 aerugi mm mm mm mm mm mm mm mm mm mm mm mm E. coli 16 mm 21 20 19 18 22 21 20 19 22 21 20 19 mm mm mm mm mm mm mm mm mm mm mm mm

Table 5. Shows antibacterial activity of combination of Salvadora oleoides extract with Erythromycin for 25 µl

Bacteria Erythro Acetone extract Ethanol extract Methanol extract µg/ml + + + Erythro Erythro Erythro Concentration ( µg/ml ) 1000 500 250 125 1000 500 250 125 1000 500 250 125 25 µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl S. aureus 16 mm 36 34 33 32 37 36 34 33 35 34 32 31 mm mm mm mm mm mm mm mm mm mm mm mm B. subtilis 22 mm 34 33 31 28 35 33 30 29 31 30 29 29 mm mm mm mm mm mm mm mm mm mm mm mm Pseudo 1 mm 28 28 27 25 30 29 28 27 26 25 23 22 aeruginosa mm mm mm mm mm mm mm mm mm mm mm mm E. coli 5 mm 31 28 27 25 32 31 30 28 30 27 26 25 mm mm mm mm mm mm mm mm mm mm mm mm

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Table 6. Shows antifungal activity of Salvadora oleoides extract for 25 µl

Bacteria Acetone extract Ethanol extract Methanol extract

Concentration ( µg/ml ) 1000 500 250 125 1000 500 250 125 1000 500 250 125 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl Aspergillus 14 12 10 8 15 13 9 8 15 14 11 8 Niger mm mm mm mm mm mm mm mm mm mm mm mm Candida 10 9 8 8 12 11 10 8 16 13 11 8 Albicum mm mm mm mm mm mm mm mm mm mm mm mm

Table 7. Shows anti fungal activity of combination of Salvadora oleoides extract and Amphoteracin B for 25 µl

Bacteria Ampho Acetone extract Ethanol extract Methanol extract (µg/ml) + + + Ampho Ampho Ampho Concentration ( µg/ml ) 1000 500 250 125 1000 500 250 125 1000 500 250 125 100 µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl Aspergillus 16 mm 23 22 20 19 23 22 21 20 24 23 21 20 Niger mm mm mm mm mm mm mm mm mm mm mm mm Candida 15 mm 22 21 20 18 21 20 18 17 22 20 19 17 Albicum mm mm mm mm mm mm mm mm mm mm mm mm

Table 8. Shows antifungal activity of combination of Salvadora oleoides extract and Fluconazole for 25 µl

Bacteria Fluco Acetone extract Ethanol extract Methanol extract (µg/ml) + + + Fluco Fluco Fluco Concentration ( µg/ml ) 1000 500 250 125 1000 500 250 125 100 500 250 125 0 100 µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl 25µl Aspergillus 2 mm 15 14 13 11 16 15 14 13 17 15 14 13 Niger mm mm mm mm mm mm mm mm mm mm mm mm Candida 0 mm 15 14 12 11 16 15 13 12 17 16 14 13 Albicum mm mm mm mm mm mm mm mm mm mm mm mm

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