Extraction, isolation and identification lect 1

Extraction, isolation and identification lect 1

1. Extraction and Phytochemical A na l y s i s o f M e dic i n a l Pl an t s

2. Medicinal plants The Medicinal plants constitute an effective source of both traditional and modern medicines . H erbal medicine has been shown to have genuine utility and about 80% of rural population depends on it as primary health care. [WHO, (2005)]

3. Medicinal plants are the richest bio-resource drugs of traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates, chemical entities for synthetic drugs.

4. Evidence for Ethnopharmacology De Materia Medica, Historia Plantarum, Species Plantarum. have been variously published in attempt to provide scientific information on the medicinal uses of plants.

5. Methods in natural product chemistry

6. Bioassay guided isolation Is the physical process used to isolate biologically active chemicals from a natural source. Biomass: any sample of biological origin It does aim to find new drug leads

7. Extraction Is the separation of medicinally active portions of plant (and animal) tissues using selective solvents through standard procedures . The products so obtained from plants are relatively complex mixtures of metabolites, in liquid or semisolid state or in dry powder form (after removing the solvent ), & are intended for oral or external use.

8. Parameters for Selecting an Appropriate Extraction Method Authentication of plant material by botanist. Use the right plant part + the age of plant + the time, season & place of collection. The nature of its chemical constituents. Grinding methods & powdering techniques. Nature of constituents ( polar/nonpolar ( The quality of water / menstruum. The design & material of fabrication of the extractor. VIII.Analytical parameters of the final extract, (TL C /HPLC).

9. Selection of plant Plant based natural constituents can be derived from any part of the plant like bark, leaves, flowers, roots, fruits, seeds, etc. Plants are usually air dried to a constant weight before extraction. oven drying: every part were cut into pieces  dried in an oven @ 60 ° C for 9 hrs. & pulverized. Other method for drying the plants is the oven drying at about 40 ° C for 72 h.

10. parameters influencing the quality of an extract Plant part used as starting material Solvent used for extraction Extraction procedure

11. Steps Involved in the Extraction of Medicinal Plants Size reduction Extraction Filtration Concentration Drying

12. 1. Size Reduction Objective: To rupture plant organ, tissue & cell structures so that its medicinal ingredients are exposed to the extraction solvent. Size reduction maximizes the surface area, which in turn enhances the mass transfer of active principle from plant material to the solvent. The 30-40 mesh size is optimal. Hammer mill or a disc pulverizer which has built in sieves controlled by varying the speed of the rotor clearance b/w the hammers & the lining of the grinder.

13. 2. Extraction Medicinal plants Cold aqueous percolation Hot aqueous extraction ( decoction ( Solvent extraction (cold / hot) Aromatic plants Essential oils Concretes Bsolutes Pomades Esinoids

14. Choice of solvents Successful determination of biologically active compounds depends on the type of solvent used in the extraction procedure. The choice of solvent is influenced by what is intended with the extract.

15. Properties of a good solvent in plant extractions low toxicity, ease of evaporation at low heat, promotion of rapid physiologic absorption of the extract, preservative action, inability to cause the extract to complex or dissociate.

16. Water Water is universal solvent. used to extract plant products with antimicrobial activity. Traditional healers use primarily water & consistent antimicrobial activity is obtained. Plant extracts: organic solvents >>> water extract. Water soluble flavonoids (mostly anthocyanins) have no antimicrobial significance. only water soluble phenolics are important as antioxidant compound.

17. Acetone Dissolves many hydrophilic and lipophilic components. a very useful extractant, especially for antimicrobial studies (phenolic group extract). extraction of tannins + phenolics: aqueous acetone >>> aqueous methanol Both acetone and methanol were found to extract saponins  antimicrobial activity.

18. Alcohol The identified components from plants (antimicrobial) = aromatic or saturated organic compounds  most often obtained through initial ethanol or methanol extraction. Ethanol, found easier to penetrate the cellular membrane to extract the intracellular ingredients(polyphenols) from the plant material. Methanol is more polar than ethanol but due to its cytotoxic nature. The higher concentrations of more bioactive flavonoid compounds were detected with aqua/ ethanol , 70 % due to its higher polarity than pure ethanol.

19. Chloroform Used to obtain tannins and terpenoids. Terpenoid lactones successive extractions of dried barks with chloroform.

20. Ether Commonly used selectively for the extraction of coumarins and fatty acids.

21. Dichloromethanol Specially used for the selective extraction of only terpenoids.

22. Flow chart of general extraction procedures

23. 3. Filtrati o n

24. 4. Co n centrati o n The enriched extract from percolators or extractors, known as miscella, is fed into a rota - evaporator where it is concentrated under vacuum to produce a thick concentrated extract. The concentrated extract is further fed into a vacuum chamber dryer to produce a solid mass free from solvent. The solvent recovered from the evaporator and vacuum chamber dryer is recycled back to the percolator or extractor for the next batch of plant material. The solid mass thus obtained is pulverized and used directly for the desired pharmaceutical formulations or further processed for isolation of its phytoconstituents.

25. 5. Drying The filtered extract is subjected to spray drying with a high pressure pump at a controlled feed rate and temperature  to get dry powder. The desired particle size of the product is obtained by controlling the inside temperature of the chamber and by varying the pressure of the pump. The dry powder is mixed with suitable diluents or excipients and blended in a double cone mixer to obtain a homogeneous powder that can be straight away used (for example, for filling in capsules or making tablets .(

26. Variation in extraction methods Length of the extraction period, Solvent used, pH of the solvent, Temperature, Particle size of the plant tissues, The solvent-to-sample ratio.

27. The general techniques of medicinal plant extraction maceration, infusion, percolation, digestion, decoction, hot continuous extraction (Soxhlet), aqueous-alcoholic extraction by fermentation, counter-current extraction, microwave-assisted extraction, ultrasound extraction (sonication), supercritical fluid extraction, phytonic extraction (with hydrofluorocarbon solvents).

28. Maceration The whole / coarsely powdered crude drug is placed in a stoppered container with the solvent. Allow to stand at room temperature for a period of at least 3 days with frequent agitation until the soluble matter gets dissolved. The mixture then is strained, the marc (the damp solid material) is pressed, The combined liquids are clarified by filtration or decantation after standing. This method is best suitable for use in case of the thermolabile drugs.

29. Infus i on Fresh infusions are prepared by macerating the crude drug for a short period of time with cold or boiling water. These are dilute solutions of the readily soluble constituents of crude drugs.

30. Digesti o n This is a form of maceration in which gentle heat is used during the process of extraction. It is used when moderately elevated temperature is not objectionable. The solvent efficiency of the menstruum is thereby increased .

31. Decoction In this process, the crude drug is boiled in a specified volume of water ( 1 : 4 ) for a defined time, Volume is reduced to 1/4 th the original It is then cooled and strained / filtered. This procedure is suitable for extracting  water- soluble, heat-stable constituents. Typically used in preparation of Ayurvedic extracts = “quath” / “kawath”

32. Percolation Used most frequently to extract active ingredients in the preparation of tinctures and fluid extracts. The solid ingredients are moistened with an appropriate amount of the specified menstruum, Allowed to stand for approximately 4 hours in a well closed container . After stand time, the tap is opened and allow to drain slowly .

33. Hot Continuous Extraction ( Soxhlet ( The finely ground crude drug is placed in a porous bag or “thimble” made of strong filter paper, which is placed in chamber of the Soxhlet apparatus. The extracting solvent in flask is heated, and its vapors condense in condenser. The condensed extractant drips into the thimble containing the crude drug & extracts it by contact.

34. Soxhlet apparatus When the level of liquid in chamber rises to the top of siphon tube, the liquid contents of chamber siphon into flask This process is continuous and is carried out until a drop of solvent from the siphon tube does not leave residue when evaporated.

35. Aqueous Alcoholic Extraction by Fermentation Some medicinal preparations of Ayurveda ( asava & arista ) adopt the technique of fermentation for extracting the active principles. The extraction procedure involves soaking the crude drug, [powder / a decoction ( kasaya )], for a specified period of time Undergoes fermentation & generates alcohol in situ. This facilitates the extraction of the active constituents contained in the plant material. The alcohol thus generated also serves as a preservative .

36. Counter-current Extraction Countercurrent   distribution is a separation process that is founded on the principles of liquid–liquid extraction where a chemical compound is distributed (partitioned) between two immiscible liquid phases (oil and water for example) according to its relative solubility in the two phases. Wet raw material is pulverized using toothed disc disintegrators to produce a fine slurry . Material to be extracted is moved in one direction generally in the form of a fine slurry within a cylindrical extractor where it comes in contact with extraction solvent .

37. The further the starting material moves, the more concentrated the extract becomes. Complete extraction is thus possible when the quantities of solvent more than the material. Their flow rates should be optimized. sufficiently concentrated extract comes out at one end of the extractor while the marc, practically free of visible solvent falls out from the other end

38. Ultrasound Extraction ( Sonication ( The procedure involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz. This increases the permeability of cell walls & produces cavitation. Eg: extraction of rauwolfia root. Deleterious effect: Ultrasound energy (>20 kHz) on the active constituents of medicinal plants through formation of free radicals and consequently undesirable changes in the drug molecules.

39. Supercritical Fluid Extraction Cylindrical extraction vessels are used. The collection of the extracted analyte following SFE is another important step: significant analyte loss can occur during this step. CO2 as the extracting fluid. Organic solvents are frequently added to the CO2 extracting fluid to alleviate the polarity limitations The component recovery rates generally increase with increasing pressure/temperature .

40. MICROWAVE EXTRACTION Microwave-assisted extraction is a process that removes solutes from a solid matrix into a solvent. Phenomena such as electromagnetic transfer, heat transfer, mass transfer, and momentum transfer make the process complex. Accurate and controlled heat is possible because of the capacity of microwave radiation to penetrate and combine with a substrate. Therefore, the microwave procedure can be designed to transport electromagnetic energy with specific power to the location of the compounds of interest in the substrate. The energy-saving factors and short processing times lead to a reduction in manufacturing costs, and improvement of product uniformity and yields, resulting in products with high quality compared with other extraction techniques.

41. Aromatic Plant Extracts essential oils, concretes, absolutes, pomades resinoids.

42. Essential oils Used in a wide variety of consumer goods viz., detergents, soaps, toilet products, cosmetics, pharmaceuticals, perfumes, confectionery food products, soft drinks, distilled alcoholic beverages (hard drinks) and insecticides. Production technology is an essential element to improve the overall yield & quality of essential oi l.

43. Presence of Essential Oils in plant parts.

44. Hetreogenous chemical group present in essential oils

45. M ethods 1. Distillation: water distillation. water and steam distillation. direct steam distillation. Hydrolytic maceration distillation. Expression. Cold fat extraction / Enfleurage.

46. Water and steam distillation

47. Distillation methods are good for powdered almonds, rose petals and rose blossoms. Maceration is adaptable when oil yield from distillation is poor. Solvent extraction is suitable for expensive, delicate & thermally unstable materials like jasmine, tuberose, and hyacinth .

48. Concrete This is an extract of fresh flowers, herbs, leaves and the flowering tops of plants obtained by the use of a hydrocarbon solvent such as butane, pentane, hexane and petroleum ether. Concrete is rich in hydrocarbon soluble material & devoid of water-soluble components. It is generally a waxy, semisolid, dark-colored material free from the original solvent. concretes are produced in static extractors. It is a normal practice to circulate fresh solvent through a battery of extractors .

49. Absolutes To make an absolute, the concrete is mixed with absolute alcohol & agitated thoroughly in a vessel with an agitator. During agitation, the temperature is kept at 40 ° -60 ° C and the concrete is immersed in the solution. The solution is cooled down to -5 ° to -10 ° C to precipitate out the wax, since waxes are normally insoluble in alcohol below -1 ° C. The precipitated wax is removed by passing the solution through a rotary filter. The filtrate from the rotary filter is pumped into a primary evaporator, where it is concentrated to about 10% alcohol content .

50. Res i n o i d s Resinoid is an extract of naturally resinous material, made with a hydrocarbon solvent. Resinoids are usually obtained from dry materials. The extraction process is same as that of concrete production, except that perforated discs are not used for stacking the material; instead powder from dry plant material is fed into the extractor.

51. Pomades Pomades are obtained by a process known as enfleurage , which is a cold fat extraction method. The fat is spread out on glass plates contained in wooden frames, leaving a clear margin near the edges. The absorptive surface of the fat is increased by surface grooves made with a wooden spatula. Fresh flowers are spread out on the surface of the fat and the frames are stacked in piles. After the perfume oils have been absorbed from the flowers, the spent flowers are removed by hand. Fresh flowers are again spread on the fat surface. This is repeated until the fat surface is completely enriched with perfume oils. The pomade so obtained is ready for cold alcoholic extraction

52. Standardized Extraction The purpose of standardized extraction procedures for crude drugs (medicinal & aromatic plant parts) To attain the therapeutically desired portions To eliminate unwanted material by treatment with a selective solvent known as “menstrum”

53. The extract thus obtained, after standardization, may be used as medicinal agent as such in the form of tinctures fluid extracts further processed to be incorporated in any dosage form such as tablets and capsules

54. STANDARIZATION Requirement to have a minimum amount of one or several compounds or groups of compounds in the extract Applies only to extracts Standardization guarantees the content of one or more active constituents and marker compounds. The plant environment and genetic factors could significantly affect the biochemical components of the plant extract.

55. Standardization involves adjusting the herbal drug preparation to a defined content of a constituent or a group of substances with known therapeutic activity Botanical extracts made directly from crude plant material show substantial (considerable) variation in composition, quality, and therapeutic effects . Standardized extracts are high-quality extracts containing consistent levels of specified compounds, and they are subjected to accurate quality controls during all phases of the growing, harvesting, and manufacturing processes.

56. No regulatory definition exists for standardization of dietary supplements. As a result, the term “standardization” may mean many different things. Some manufacturers use the term standardization incorrectly to refer to uniform manufacturing practices; following a recipe (formula) is not sufficient for a product to be called standardized. Therefore, the presence of the word “standardized” on a supplement label does not necessarily indicate product quality. When the active principles are unknown, marker substance(s) should be established for analytical purposes and standardization. Marker substances are chemically defined constituents of a herbal drug that are important for the quality of the finished product. Ideally , the chemical markers chosen would also be the compounds that are responsible for the botanical’s effects in the body.

57. Special extract: a process of defining a range for certain compounds or classes of compounds in order to enrich desired compounds and reducing the amount of undesired ones.

58. Why is standardization necessary and important? Reproducible composition and higher quality of the product Provided that the product is registered, it thus becomes a medicine that should comply with the basic standards required for all drugs Standardization allows comparison of the clinical effectiveness, pharmacological effects and side effects of a series of products Such products give patients greater security and increase their trust Ensuring the quality of the products sold is a key responsibility of the pharmacist

59. There are two types of standardization I. In the first category, “ truly” standardized extract (Type A): Extracts standardized to active constituents (single or groups) Example: belladonna leaf dry extract, standardized to 0.95-1.05% of alkaloids calculated as hyoscyamine

60. II. In the second category, “quantified ” extract (Type B1): Standardized to constituents that contribute to the activity, by blinding (randomize) different batches of herbal drug before extraction or by mixing different lots of herbal drugs preparations. Adjustment using excipients is not accepted Includes the special extracts

61. Example: Ginkgo with its 26% ginkgo flavones and 6% terpenes is a classic example. These products are highly concentrated and no longer represent the whole herb, and are now considered as phytopharmaceuticals . In many cases they are vastly (greatly) more effective than the whole herb.

62. III. In the third category, “other ” extracts (Type B2): The other type of standardization is based on manufacturers guaranteeing the presence of a certain percentage of lead compounds of unknown pharmacological relevance serve as quality marker compounds ; these are not indicators of therapeutic activity or quality of the herb. Nevertheless, the extract is pharmacologically active. Give information on the over all quality of the phytomedicine Example: Echinaceae radix

63. Phytochemical screening methods

64. Quantitative Analysis Total Phenolic Content Determined by Folin-Ciocalteau assay method (Singleton and Rossi, 1965) Instrument: UV-Vis Spectrophotometer, absorbance measured at 765 nm Expressed as Gallic acid equivalent (GAE) in milligrams per gram of fresh leaf Total Flavonoid Content Determined by Colourimetric method (Yun et al., 2009) Instrument: UV-Vis Spectrophotomer, absorbance measured at λ415 nm Expressed as mg rutin equivalent (mg RE) per gram of fresh leaf

65. Yadav and Agarwala, 2011 Assam, India Phytochemical Analysis of Some Medicinal Plants Objective To carry out qualitative and quantitative phytochemical analysis of selected medicinal plants

66. Methodology Plant sources Bryophyllum pinnatum (Leaves) Ipomea aquatica (Leaves) Oldenlandia corymbosa (Whole plant) Ricinus communis (roots) Terminalia bellerica (Leaves) Tinospora cordifolia (Leaves/Stem) Xanthium strumarium (Leaves)

67. Hot water extraction 5gm of dried finely powdered plant material mixed with 200ml of distilled water Heated on a hot plate with continuous stirring at 30º-40ºC for 20 minutes Solvent extraction 20gm powdered plant material packed into a thimble and extracted with 250ml of solvents Extraction continues for 24 hours or till the solvent in siphon tube of an extractor become colourless filtered through filter paper kept on hot plate and heated at 30-40ºC Preparation of extracts

68. Table 1. Phytochemical constituents of medicinal plants Results and discussion L = leaves; S = stem

69. Fig 1. Total phenolic content Fig 2. Total flavonoid content

70. Conclusion Results revealed that extracts from these plants can be used as a good source for drugs Further work should be carried out to isolate, purify and characterize the active constituents responsible for the activity of these plants

71. Pranoothi et. al ., 2014 Andhra Pradesh, India Studies on Qualitative, Phytochemical Analysis and Screening of In Vitro Biological Activities of Leucas indica (L) Objective To carry out qualitative and quantitative phytochemical analysis of aerial parts of Leucas indica (L)

72. Methodology C l ea n e d , s h a d e d r i e d , m echa n i ca ll y grinded & coarsely powdered Aerial parts of Leucas indica Preparation of extracts Powdered material Subjected to Solvent extraction with hexane, acetone, methanol & water Extracts were concentrated using Rotary Evaporator Phytochemical screening

73. Table 6. Physico-chemical evaluation Solvent Initial weight of the powder (g) Final weight of the powder (g) Weight of the crude extract (g) Hexane 50 4 4 . 5 63 5.437 1 0.8 7 4 Dark brown Acetone 50 4 . 4 15 9.585 19. 1 7 Dark green Methanol 50 3 5 . 5 52 14.448 2 8.8 9 6 Dark green Water 50 3 8 . 6 21 11.379 2 2.7 5 8 Dark red Crude extract (%) Colour of the extract Figure 3. Yield of extracts Results and discussion

74. Table 7. Phytochemical analysis of whole aerial part extracts of Leucas indica (L) S l. N o . T e s t s Ex tract s 1 Alkaloids Hexane Acetone Methanol Water 2 Mayers Dragon Wagners Hagers P he n o lics FeCl 2 test - - - - - + + + + + + - + - + + + + + + 3 Flavonoids Lead acetate test - + + + 4 5 NaOH test Ethyl acetate test A nt h r a q u i n o n e s Borntrager’s test Steroids Salkowski’s test - - - + + - - + + - - + - - - +

75. 6 Tannins Hexane Acetone Methanol Water FeCl 2 test - + + - Lead acetate test - + + - Potassium dichromate test - + + - 7 Saponins Vigorous shaking test + + + + 8 Anthocyanins Ammonia-HCL test - - - - 9 L e uco- A nthocy a ni n Iso amyl alcohol test - - - - 10 Coumarins NaOH test - - - - 11 Reducing sugars Keller-Kiliani test + + + + Sl. No. T e s t s Ex tract s C o ntd. . .

76. Table 8. Total phenol content ) µg/ml ( extract extract extract 100 15.69 18.5 25.6 22.8 200 22.54 26.9 45.86 41.8 300 30.41 38.9 72.8 68.4 400 42.89 54.78 88.96 76.5 500 54.58 65.8 105.68 95.8 % of phenol content µg GAE/µg C o n c en t r ation H e xa n e A c eton e M e t h anol Water extract Figure 4. Total phenol content

77. Table 9. Total flavonoid content % of phenol content µg GAE/µg Hexane Methanol 100 00 10.28 17.25 00 200 00 15.56 26.35 00 300 00 32.47 29.12 13.20 400 00 41.58 43.52 18.25 500 00 50.24 62.34 24.69 Co n c entratio n A c etone Water Figure 5. Total flavonoid content

78. Conclusion The presence of most general phytochemicals in Leucas indica might be responsible for their therapeutic effects It further reflects a hope for the development of many more novel chemotherapeutic agents from plants which in future may serve for the production of synthetically improved therapeutic agents

79. Pandey et. al ., 2014 Uttarakhand, India Phytochemical Screening of Selected Medicinal Plant Cinnamon zeylanicum Bark Extract Objective To screen the phytochemicals present in Cinnamon zeylanicum aqueous bark extract

80. Washed, shade dried and powdered Barks of Cinnamon zeylanicum Preparation of extracts Maceration technique Powdered material mixed with 150ml distilled water for 1 hour in rotary shaker Extract was filtered using muslin cloth & Whatman filter paper Concentrated by evaporation on water bath The extract was dried & used as powder Methodology

81. Phytoconstituents water water water Carbohydrates 15ºC + 70ºC + 45ºC + - - - Steroids + + + + + + Proteins - - - - - - Glycosides - - - - - - Alkaloids + + + + + + Flavonoids + - - + - + Saponins + + + + + + Tannins & phenol - - - + + + Table 16. Phytochemical screening of secondary metabolites of plant extracts Extracts Cold H ot W arm Et h anol M e t hanol A c etone Results and discussion

82. Conclusion Your home work

83. Singh and Bag, 2014 Manipur, India Phytochemical Analysis and Determination of Total Phenolics Content in Water Extracts of Three Species of Hedychium Objective T o id e n t i f y and c o m p a r e t he b io a c tiv e c o n s tit u e n t s pr ese n t in Hedychium species and determine total phenolic content

84. Hedychium rubrum C l eane d , s h a d e d r i e d , m e c h a n i ca ll y grinded & coarsely powdered Preparation of extracts Extracts were concentrated using Rotary Evaporator Phytochemical screening Powdered material Subjected to Solvent extraction with hexane, acetone, methanol & water Hedychium spicatum Hedychium coronarium Methodology

85. Table 17. Comparative analysis of phytochemical constituents of three different species of Genus Hedychium Phytochemical Chemical tests constituents H. spicatum Water extract H. coronarium H. rubrum Alkaloids C a r b o hyd rates (reducing sugar) Fehling’s test + + + Proteins Xanthoproteic test + + + Flavonoids Alkaline reagent test + + + Phenolic compounds Lead acetate test + + + Tannins Lead acetate test Ferric chloride test + - + + + + Steroids & terpenoids Salkowski’s test + + + Saponins Froth test + + + Cardiac glycosides Keller-killiani test + + + Oil + + + Phlobatannin - - + Hager’s test Benedict’s test - - - - - + Results and discussion

86. Table 18. Total phenolic content in the water extracts of H. Spicatum, H. Coronarium and H. rubrum Water extracts Concentration ) mg/ml ( mg of gallic acid/g of extract Mean ± ( Standard Deviation ( H. Spicatum 1 29.39 ± 0.01 H. Coronarium 1 34.93 ± 0.01 H. rubrum 1 66.48 ± 0.01 Figure 8. Callibaration curve of gallic acid

87. Conclusion Your home work

88. Vastrad et. al ., 2015 Karnataka, India Identification of Bio-active Components in Leaf Extracts of Aloe vera, Ocimum tenuiflorum (Tulasi) and Tinospora cordifolia (Amrutballi) Objective To screen various bio-active compounds present in the leaf extracts of A. vera, O. tenuiflorum and T. cordifolia and evaluate total phenolic content & total flavonoid content

89. T i no s po r a cordifolia C l eane d , s h a d e d r i e d , m e c h a n i ca ll y grinded & coarsely powdered Preparation of extracts Extracts were concentrated using Rotary Evaporator Phytochemical screening Powdered material Subjected to Solvent extraction with hexane, acetone, methanol & water Aloe vera Ocimum tenuiflorum Methodology

90. Figure 9 . Yield of extracts Results and discussion

91. Table 20. Total phenolic content (TPC) of the plant leaf extracts Total phenolic content (GAE* mg/g) Extr a ction solvent Aqueous Ethanol A. vera 94.42 ± 4.92 138.13 ± 6.63 O. tenuiflorum 80.82 ± 8.63 113.07 ± 9.81 114.34 ± 11.86 T. cordifolia 465.82 ± 23.04 264.06 ± 18.41 301.42 ± 29.69 Methanol 95.20 ± 3.23 GAE = Gallic acid equivalent Fig 10. Total phenolic content (TPC): Calibration curve

92. Table 21. Total flavonoid content (TFC) of the plant leaf extracts Figure 11. Total flavonoid content (TFC): Calibration curve Total flavonoid content (RE* mg/g) Extr a ction solvent Aqueous Ethanol M e th a n o l RE = Rutin equivalent A. vera 72.28 ± 8.70 76.50 ± 8.57 88.59 ± 8.38 O . tenuiflorum 61.84 ± 7.25 95.46 ± 4.12 96.34 ± 5.85 T. cordifolia 178.43 ± 6.61 208.36 ± 2.86 132.59 ± 7.59

93. Conclusion Your home work