EXTRACTION,_PHYTOCHEMICAL_SCREENING_&_FORMULATION_OF_BOERRHAVIA.pptx

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1. THE DISSERTATION SUBMITTED TO CHHATTISGARH SWAMI VIVEKANAND TECHNICAL UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF THE DEGREE OF EXTRACTION, PHYTOCHEMICAL SCREENING & FORMULATION OF BOERRHAVIA DIFFUSA ROOT BACHELOR OF PHARMACY 8 th Semester BY ABHASH SINGH, B. Pharmacy, Roll No:- 311304119001 ANIKITA DHIWAR, B. Pharmacy, Roll No:- 311304119003 ANKIT BHINJE, B. Pharmacy, Roll No:- 311304119004 ANUSHKA BHONSLE, B. Pharmacy, Roll No:- 311304119005 ARCHISHA TIWARI, B. Pharmacy, Roll No:- 311304119006 Under the Guidance of Mr. CHANDRASHEKHAR SAHU, M. Pharm, ( Ph.D ), Associate Professor RITEE COLLEGE OF PHARMACY NH-06, Chhatauna , Mandir Hasaud , Raipur (C.G.) 492101 2019-2023

2. CONTENT INTRODUCTION LITERATURE REVIEW METHODOLOGY RESULT AND DISCUSSION CONCLUSION REFERENCES

3. INTRODUCTION The Boerrhavia diffusa is a herbal plant . The whole plant parts are used as medicinal purpose. The genus Boerhaavia consists of 40 tropical and subtropical species. Family - Nyctaginaceae Biological Source - Boerrhavia diffusa Geographical Source - India, Pakistan, China, Brazil, Australia, Africa, Middle East, Sri Lanka, Iran, USA etc. Fig :- Boerrhavia diffusa plant

4. Morphology (root) Shape - Stout, fusiform, tapering Texture - Woody Colour – light yellow, brown or brownish Size – 30 to 50cm Biological Constituents The BD plant contains a large number of compounds such as flavonoids, alkaloids,glycoside , steroids, triterpenoids , lipids, lignins , carbohydrates, proteins and glycoproteins etc. Punarnavine , boerravinone , hypoxanthine, punarnavoside are the main phytoconstituents islolated from Boerrhavia diffusa . Fig :- Boerhaavia diffusa roots

5. Uses It is used for various purposes such as : Jaundice Kidney and Liver disease Heart disease Urinary disorder, Abdominal pain, Anemia , Blood purification, Cancer, Eye problem and digestive problem

6. Ayurvedic Uses In Ayurveda this drug is known to be used as Mootrala (diuretic) Sothaghana (anti-inflammatory) Kasahara (antitussive) Jwarahara (antipyretic) Rasayana (rejuvenator)

7. Phytosome The meaning of “ phyto ” is plant and “some” is cell like . Phytosome is a novel patented technology developed to incorporate standardized plant extracts into phospholipids to give a lipid compatible molecular complexes, with improved biovalability and absorption which is called as phytosomes . Fig :- Phytosome

8. Advantages Formulation is easy as there is no problem in drug entrapment. It assures proper delivery of drug to the respective tissues. The bioavailability of drug is enhanced remarkably. Phytosome show better stability due to the formation of chemical bond between phytoconstituent and the phospahtidylcholine molecule. Phytosomes are superior to liposomes in skin care product. Disadvantages When administerd orally or topically they limit tjheir bioavalibility . Phytoconstituents are quickly eliminated from phytosome . Phytoconstituents are rapidly eliminated therefore their duration of action is short.

9. Need for study The demand for plant derived drugs seems to increase in developing countries due to their medicinal values and economic procurement. Modern herbal dosage form is greater area for concern because it offer smaller dosage size and comparatively good absorption than conventional dosage form. Modern dosage form is far more flexible in carrying and can be taken even in a busy schedule hence plays tremendous role in clinical treatment.

10. Aim and Objectives Aim : Extraction, Phytochemical Screening & Formulation of Boerrhavia diffusa root. Objectives of Study : To evaluate the solubility extractive value of the Boerrhavia diffusa extract by performing various chemical test. To check the presence of carbohydrates, glycosides, alkaloids, flavonoids, volatile oils and phenolic compounds. To improve the bioavalability of drug as compared to its statndardized form. To formulate the BD-phospholipid complex.

11. Plan of work Review Literature Solubility study of crude drug. Extractive value. Selection of better composition of solvent for extraction. Extraction of active constituent by soxhlet hot extraction method. Collection of dried extract. Phytochemical screening of extract. Preparation of phytosome .

12. LITERATURE REVIEW Vishal Gaurav et al (2021) : Studied the Phytosomes was prepared by solvent evaporation method. Applications of phytosomes like incremented bioavailability of drugs, anti-cancer and anti-oxidant agent, transdermal drug distribution, increment wound rejuvenating capacity etc. Mancini Simona et al (2018) : Studied the multi-target drugs have gained significant recognition for the treatment of multifactorial diseases such as depression. Park Ju Ho et al (2018) : Studied the centella asiatica phytosome (CA phytosome ) has potent antioxidant and anti-inflammatory properties. However, its anti-dermatitis effect has been not reported yet.

13. 4. Hanh T.H. Vu et al (2018) : Studied the phytosomes and described it as lipid nanocarriers with a complexation between the phospholipid head groups and entrapped phytochemicals. The aim of this research was to compare the structure of rutin liposomes to rutin phytosomes . FT-IR, DSC and NMR were employed to investigate the presence of any molecular interactions between the formulation components. 5. Maryana Wina et al (2016) : Studied the silymarin is a unique flavonoid complex isolated from milk thistle ( Silybum marianum ) and has been widely used as hepatoprotective agent. This study was aimed to develop silymarin -loaded phytosomes to improve silymarin bioavailability with sufficient safety and stability. 6. Bairwa Khemraj et al (2015) : To develop a phospholipid complex of rotenoid - rich fraction (RRF) and evaluate its in vivo anti-inflammatory activity and pharmacokinetic study. RRF was prepared from a 70% ethanol extract of B. diffusa roots.

14. 7 . Zhang Jifen ., et al (2013) : In this study, they developed a novel drug delivery system, curcumin - phytosome -loaded chitosan microspheres (Cur-PS-CMs) by combining polymer- and lipid-based delivery systems. They aimed to use our novel delivery system to improve the bioavailability and prolong the retention time of curcumin in the body. 8. Gupta Nishant Kumar et al (2010) : Reported the curcumin is a major constituent of rhizomes of Curcuma longa. Pharmacokinetic studies of curcumin reveal its poor absorption through intestine. Objective of the present study was to enhance bioavailability of curcumin by its complexation with phosphatidyl choline (PC). The present study clearly indicates the superiority of complex over curcumin , in terms of better absorption, enhanced bioavailability, and improved pharmacokinetics.

15. 9. Sikarwar S. Mukesh et al (2007) : Studied of formulate Marsupsin –phospholipid complex (M–P Complex) in attempt to increase the bioavailability of marsupsin and to characterize this new formulation along with its evaluation. Marsupsin –phospholipid complex was formulated by mechanical dispersion method. Hence the findings demonstrate that complexing marsupsin with phospholipids results in better oral bioavailability and improved biological response than free form of standardized marsupsin . 10. Maiti Kuntal et al (2006) : Studied the novel formulation of curcumin in combination with the phospholipids was developed to overcome the limitation of absorption and to investigate the protective effect of curcumin –phospholipid complex on carbon tetrachloride induced acute liver damage in rats. The result proved that curcumin –phospholipid complex has better hepatoprotective activity, owe to its superior antioxidant property, than free curcumin at the same dose level.

16. METHEDOLOGY Crude drug collection : The dried form of Boerrhavia diffusa root was purchased from the local market of Raipur district with proper identification. The dried form of Boerrhavia diffusa was then converted into fine powder by the help of mixer grinder. Fig :- Crude drug Boerhaavia diffusa

17. Solubility Solubility is the property of a solid, liquid, or gaseous chemical substance called  solute  to dissolve in a solid, liquid, or gaseous solvent to form a homogeneous solution of the solute in the solvent. IUPAC defines solubility as the analytical composition of a saturated solution expressed as a proportion of a designated solute in a designated solvent. Solubility may be stated in units of concentration, molality, mole fraction, mole ratio, and other units.

18. Procedure 2ml of solvent and approximately 5 mg of a sample are taken in a test tube and solubility test is performed. Solvent collection : On the basis of solubility of crude drug with different solvent, select the solvent in which crude drug is easily soluble.The BD is mostly soluble in water and ethanol. So, water and ethanol is selected for extraction process. 3 Extractive Value: This method is used to determine the amount of active constituents in a given amount of medicinal plant material when extracted with a solvent. % of extract = weight of residue/ weight of drug x 100

19. Selection of better composition of solvent for extraction Determine the fix or suitable ratio of hydro alcoholic composition for maximum yield. Different ratio of water and ethanol (80:20, 70:30, 40:60, 50:50) was determined by the extraction process. Also determine the single distilled water and ethanol extractive value. Soxhlet Hot Continuous Extraction : Crude drug placed in thimble. Soxhlet extractor is placed onto a flask containing the exytraction solvent, soxhlet is equipped with a condenser. ` The solvent is heated to reflux. The solvent vapour travels up a distillation and floods into the chamber housing the thimble of crude drug.

20. Selection of better composition of solvent for extraction 5. Crude drug in chamber slowly fills warm solvent. 6. Desired compounds dissolves in the warm solvent. 7. Then the soxhlet chamber is almost full, the chamber is empties by the siphon. The solvent running back down the distillation flask. 8. This cycle may be allowed to repeated many times. 9. Colourless or white side cube indicates extraction complete. Fig:- Soxhlet hot continuous extraction assembly

21. (A) (B) Fig :- (A) Standardized Extract of Boerrhavia diffusa (B) Hydro-alcoholic Extract of Boerrhavia diffusa

22. Preliminary Phytochemical Screening Test for carbohydrates Molisch’s test: Take 2-3 ml aqueous extract, add few drop of alpha- napthol solution in alcohol, shake and add conc. H2SO4 from sides the test tube. Violet ring is formbed at the junction of two liquids. Selwinoff’s test: Heat 3ml selwinoff’s reagent and 1ml test solution in bearing water bath for 1-2 min. red colour is formed. TEST FOR PROTEINS Millon’s test: Mix 3ml T.S. with 5ml Millon’s reagent. White ppt. warm. Ppt. turns brick red or the ppt. dissolves giving red coloured solution. Lead acetate test : Take 3 ml T.S and add 5% lead acetate solution then it gives white colloidal ppt.

23. Ninhydrin test: Heat 3ml T.S. and 3 drops 5% Ninhydrin solution boiling water bath for 10 min. purple or bluish colour appears. TEST FOR GLYCOSIDES : Foam test: shake the drug extract or dry powder vigorously with water. Persistent stable foam observed. TEST FOR ALKALOIDS : Dragendorff’s test: Take 2-3 ml of T.S., add few drop of Dragendorff’s reagent. Orange brown ppt is formed. Mayer’s test: Take 2-3 ml of T.S., add few drop of mayer’s reagent give ppt. TEST FOR FLAVONOIDS: Sulphuric acid test: take extract and add sulphuric acid flavones and flavono gives deep yellow solution.

24. PREPARATION OF PHYTOSOME Preparation of BD-PC Complex (Anti-solvent precipitation process) BD extract and soy lecithin in molar ratio of 1:1 were taken in a round bottom flask and dissolved in 20 mL of dichloromethane. This mixture was refluxed for 2 h at room temperature on a burner at 30-50◦C. The resultant BD–PC complex was washed with n-hexane, dried under vacuum, kept in amber colored glass bottle, and stored at room temperature. Fig :- Prepration of phytosome (Reflux) Fig :- Phytosome

25. Result and discussion Solubility study of crude drug BD On the basis of solubility of drug in solvent, select the solvent for extraction of crude drug. In solubility test drug is very soluble in water and ethanol. Perform the hydro alcoholic extraction of drug in different ratio. S.No . Solvent Result 1. Ethanol Very soluble 2. Petroleum spirit Soluble 3. Acetone Not soluble 4. Chloroform Not soluble 5. Diethyl ether Soluble 6. Distilled Water Very soluble

26. Extractive value of drug S.No Alcohol : water solvent Extractive value 1. 20:80 0.16 2. 30:70 0.06 3. 40:60 0.07 4. 50:50 0.12 5. Alcohol 0.03 6. Water 0.15 On using various different solvent for the extraction of crude drug , the extractive value of drug was found to be best in alcohol : water solvent (20:80) therefore it is selected for the extraction process.

27. Solubility study of BD extract and their standard extract S. No. Solvent Extract result Standard extract result 1. Ethanol Very soluble Very soluble 2. Petroleum spirit Soluble ppt 3. Acetone Ppt ppt 4. Chloroform Partially soluble Slowly soluble 5. Diethyl ether Crystal formation ppt 6. Distilled Water Very soluble Very soluble Solubility studies states that the Boerhaavia doffusa extract and their standard extract are very soluble in ethanol and distilled water. With dissolving in acetone, both Boerrhavia diffusa extract and their standard extract gives precipitate formation. And some solvent like chloroform are partially and slowly soluble with Boerrhavia diffusa extract and their standard extract.

28. Phytochemical profile of sequential extracts of Boerrhavia diffusa S. No. Phytochemical test Test Extract Standard extract 1. Carbohydrate test Molisch’s test _ _ Selwinoff’s test _ _ 2. Protein test Millon’s test _ _ lead acetate test + _ 3. Amino acid test Ninhydrin test _ _ 6. Glycoside test Foam test _ _ 7. Flavonoid test Sulphuric acid test _ _ 9. Alkaloid test Dragendorff’s test _ + Mayer’s test _ _ Hager’s test     Wagner’s test + _ Tannic acid test + _

29. On the basis of phytochemical tests performed it can be said that: Carbohydrate, protein, volatile oil, flavonoid, steroid are present in the Boerhaavia diffusa extract as well as in their standardized extract. According to other chemical test such as tannic acid test, lead acetate test , wagner’s test and bromine water test, carbohydrate, tannic acid, phenolic compound alkaloids and steroids are also present in the Boerhaavia diffusa abstract. While secondary metabolites such as amino acids fats and oils, glycoside are not present in the Boerhaavia diffusa extract and this standardized extract.

30. CONCLUSION In the present work, phytosome of Boerrhavia diffusa has been prepared using suitable solvents such as dichloromethane to enhance the bioavailability of Boerrhavia diffusa . Hence, better absorption, enhanced bioavailability and improved pharmacokinetics is observed in phytosome of Boerrhavia diffusa . During this experiment various phytochemical test have been performed to find out the chemical constituent present in the extract of Boerhaavia diffusa . The information gathered during this experiment is going to be useful for the researchers who wish to explore a vesicular drug delivery system which can target the specific site of body part without getting metabolised and act as a potent and effective dosage form.

31. REFERENCES Vishal Gaurav, Shivangi Paliwal , Arpita Singh, Swarnima Pandey, Mohd . Aqil Siddhiqui Phytosomes : Preparation, Evaluation and Application by International Journal of Research in Engineering and Science (IJRES) 2021. Simona Mancinia,b , Luca Nardoa,b , Maria Gregoria,b , Inês Ribeiroc , Francesco Mantegazzaa,b , Cristina Delerue-Matosc , Massimo Masserinia,b , Clara Grosso Functionalized liposomes and phytosomes loading Annona muricata L. aqueous extract: Potential nanoshuttles for brain-delivery of phenolic compounds by Phytomedicine Elsevier. U Ho Park , Jun Sung Jang , Ki Chen Kim , Jin Tae Hong , Antiinflammatory effect of Centella asiatica phytosome in a mouse model of phthalic anhydride-induced atopic dermatitis, Phytomedicine 2018. H.T.H. Vu, S.M. Hook, S.D. Siqueira , A. Müllertz , T. Rades , A. McDowell, Are phytosomes a superior nanodelivery system for the antioxidant rutin ?, International Journal of Pharmaceutics 2018. Wina Maryanaa *, Heni Rachmawatib , Dicky Mudhakir Formation of Phytosome Containing Silymarin Using Thin Layer-Hydration Technique Aimed for Oral Delivery by ScienceDirect .

32. REFERENCES Khemraj Bairwa and Sanjay Madhukar Jachak Anti-inflammatory potential of a lipid-based formulation of a rotenoid -rich fraction prepared from Boerhavia diffusa by pharm Biol , 2015, 53(8), 1231–1238. Jifen Zhang∗, Qin Tang, Xiaoyu Xu , Na Li Development and evaluation of a novel phytosome -loaded chitosan microsphere system for curcumin delivery by International Journal of Pharmaceutics. Nishant kumar gupta , vinod kumar dixit Bioavailability Enhancement of Curcumin by Complexation with Phosphatidyl Choline by Department of Pharmaceutical Sciences, Dr. Hari Singh Gour Vishwavidyalaya , Sagar , Madhya Pradesh 470003, India 2010. Mukesh S. Sikarwar,1,3 Shalini Sharma,2 Amit K. Jain,1 and S. D. Parial Preparation, Characterization and Evaluation of Marsupsin –Phospholipid Complex by AAPS PharmSciTech , 2008, 9, 1. Kuntal Maiti , Kakali Mukherjee, Arunava Gantait , Bishnu Pada Saha , Pulok K. Mukherjee Curcumin –phospholipid complex: Preparation, therapeutic evaluation and pharmacokinetic study in rats by International Journal of Pharmaceutics 330, 2007, 155–163.