Extraction, Isolation and Characterization of the Antioxidants from Leaves of Tridax Plants of Melghat Region’

FINAL REPORT OF THE WORK DONE On the Major Research Project

‘Extraction, Isolation and Characterization of the Antioxidants from Leaves of Tridax Plants of Melghat Region’

Introduction: Antioxidants are substances that may protect cells against the effects of free radicals. Free radicals are molecules produced when body breaks down the food, or by environmental exposures like tobacco smoke and radiation. Free radicals can damage cells, and may play a role in heart disease, cancer and other diseases. It is therefore the demand of time to revel some new antioxidants form the nature and use them in a rational way towards the well being of mankind.

The present study is aimed towards the Extraction, Isolation and Characterization of the antioxidants form leaves of the plants of the genus Tridax of Melghat Region of Maharashtra State.

Melghat Tiger Reserve is located on southern offshoot of the Satpura Hill Range in Central India, called Gavilgarh hill in the Indian state of Maharashtra. More than 700 naturalized plant species have been enlisted in the Flora of Melghat. These species belong to about 400 genera representing as many as 97 families. There are 90 trees species, 66 shrubs species, 316 herbs species, 56 climbers, 23 sedges and 99 grass species.

Literature survey reveals that India has a rich heritage of the phytochemical analysis and herbal formulations and their curative effects on various ailments in Ayurveda. In India, Ayurvedic medicines have been using many herbs such as turmeric from ancient time. The Charaka Samhita describes the utility of medicinal plants in details. Phytochemical study has been attracting the world wide attention. Hence it is significant to identify the plants from nature for their medicinal use. Literature survey reveals that the plants of tribe Heliantheae of family Asteraceae have been extensively documented in the literature for their variety of medicinal uses since ancient times. Some

1 of the vitally important species of tribe Heliantheae viz Tridax procumbens , Zinnia peruviana, Helianthus annus, Tagetus erecta, Spilanthes fusca and Cosmos bipennatus have been reported to poses significant therapeutic activities. Tridax procumbens is reported to show antiviral, antioxidant antibiotic activities, wound healing activity, insecticidal and anti-inflammatory activity1. It has been extensively used in Indian traditional medicine as anticoagulant, antifungal, insect repellent, diarrhea and dysentery2. Moreover , it possesses wound healing activity and promotes hair growth3. Tridax procumbens is also dispensed as 'Bhringraj', which is well known Ayurvedic medicine for liver disorders4. Anti-diabetic activity of leaf extract of Tridax procumbens have also been documented 5. Oral administration of acute and sub chronic doses of T. procumbens extract showed a significant reduction in fasting blood glucose levels in diabetic rats6. In vitro studies on Tridax procumbens have been reported to have antioxidant potential of the herb with chloroform fraction of the ethanolic extract. It is also reported to possess anti-oxidant minerals such as iron, magnesium, copper and zinc7. Another plant of this tribe , Spilanthes fusca is also having wide range of therapeutic uses. The leaves and flower heads contain analgesic, antifungal, and antibacterial agents. It is also known as toothache plant and used for tooth and gum infections8. Helianthus annuus is a regular crop plant yielding oil seeds. Zinnia peruviana, Tagetus erecta, and Cosmos bipennatus are widely used as ornamental plants. These species of tribe Heliantheae of family Asteraceae are worth studying for their further medicinal uses.

Tridax procumbens

Classification

Kingdom :-Plantae (unranked) :-Angiosperms (unranked) :-Eudicots (unranked) :-Asterids Order :-Asterales Family :-Asteraceae Tribe :-Heliantheae Genus :-Tridax Species :-T .procumbens Binominal name :- Tridax procumbens

Complete plant of Tridax procumbens

Flower of Tridax procumbens

Wound healing activity The leaf extract of Tridax procumbens has been used since ancient times for healing of wounds. Tridax procumbens is known for several potential therapeutic activity , wound healing activity is also one of them9. This effect of this plant reported to promote wound healing in both normal and immune-compromised (steroid treated) rats in dead space wound model. The plant increased not only lysyl oxidase but also, protein and nucleic acid content in the granulation tissue, due to a result of increase in glycosamine glycan content10 . Traditionally in India, the fresh juice of Tridax procumbens leaves have been used since a long time as remedy for dermal wounds 11 Evolution of wound healing property of Tridax procumbens in Wister rats has been reported12. The wound healing potential of tropical formulation of leaf juice of Tridax procumbens in mice has been reported13. The wound healing activity of the ointment formulation having extract of Tridax procumbens has been evaluated experimentally upon wound in albino rats 14 and it was found that treated wound showed the faster rate of wound contraction in albino rats.

The pharmacological screening of ethanolic extract of Tridax procumbens had been carried out on the parameters like wound healing activity and leucocytes count. The extract showed significant increase in wound healing activity15.

Anti diabetic activity

The extract of leaves of Tridax have been reported to exhibit the decrease in glucose level in the blood in model of alloxan induced diabetes in rat16. Evaluation of hypoglycemic and anti hyperglycemic activity of Tridax procumbens (Linn) has been documented17. Oral administration of acute and sub chronic of Tridax procumbens extract showed a significant reduction in fasting blood glucose level in diabetic rat. The extract of Tridax procumbens has been reported to produce a significant hypoglycemic effect in rat18. Further studies has been reported to show the solvent fraction containing non polar substances would be among the active principle for lowering blood sugar level.

Pharmacognostical and pharmacological investigation on leaves of Tridax procumbens has also been carried out19.

Antioxidant activity

Tridax procumbens has been extensively documented for its antioxidant activity also. The different solvent fraction from the ethanolic extract of Tridax procumbens has been showed significant anti-oxidant activity20. Total phenolic and antioxidant activity of Tridax procumbens has been studied21. The analysis showed a very high percentage of anti oxidant activity even higher than those of Gallic acid. This shows the utility of Tridax procumbens as a source of anti-oxidant. The anti-inflammatory and anti-oxidant activity of standardized extraction of Arial parts of Tridax procumbens reported to show significant inhibition of rat paw ediema22 . The extract also exhibited anti-oxidant activity against DPPH. Effect of essential oil of Tridax procumbens on In-vivo anti-oxidant level in cancer model and in vitro free radical scavenging activity has been studied23. The DPPH essay value obtained for essential oil was found to be more than that of standard ascorbic acid. The role of Tridax procumbens in the management of oxidative stress in rats has been explored24.

Helianthus Annuus

Helianthus annuus is also another important plant of tribe Heliantheae. A tea made from the leaves is astringent, diuretic and expectorant, it is used in the treatment of high fevers25 .The crushed leaves are used on sores, swellings, snakebites and spider bites25, 26 . The leaves are harvested as the plant comes into flower and dried for later use27. A tea made from the flowers is used in the treatment of malaria and lung allments25,26 . The flowering head and seeds are nutritive and stomachic28 .the seed is also considered to be diuretic and expectorant29,30 . It has been used with success in the treatment of many Pulmonary complaints29.

Tagetus erecta

The whole herb is reported to be digestive, diuretic, sedative and stomachic31,32. It is used internally in the treatment of indigestion, colic, severe constipation32, coughs and dysentery33. Externally, it is used to treat sores, ulcers, sore eyes and rheumatism.32,33,34,35 The leaves are harvested as required for immediate use during the growing season, whilst the flowering plants can be dried and stored for later use32 .A paste of the leaves is applied externally to treat boils, carbuncles and earaches35 .The flowers are carminative and diuretic.35 A decoction is used to treat colds and mumps33 It is applied externally to treat skin diseases, conductivities and sore eyes33,35. The root is laxative.35 It is also reported to be used as a skin wash and for yellow dye.36

Scientific study shows that thiophenes, natural phytochemical that include sulfur containing rings may be the active ingredients they have been shown to kill gram negative and gram positive bacteria in vitro. This marigold may help protect certain crop plants from nematode pests when planted in field37. It is most effective against the nematodes species penetrans38. The phytochemical studies of its different parts have resulted in the isolation of various chemical constituent such flavonoids , carotenoids and triterpenoids. It has been shown to contain methyl-3,5-dihydroxy-4-methoxy benzoate, quercetin, thienyl and ethyl gallate39, 40 .

Cosmos caudatus:-

Cosmos caudatus (kunth), Asteraceae is one of Indonesian vegetable plants that is usually consumed freshly as salad or cooked by boiling with other spices. In East Java, it is also used in traditional medicine to reduce blood pressure. It is also used to improve blood circulation, as carminative, appetizer and insect repellent41.Cosmos leaves contained highest flavonoids leaves. Flavonoids have been reported to have strong antioxidant and radical scavenging activity, which is expected to have beneficial effect in the prevention of degenerative diseases42, 43.

An overview of literature among the different species of genus Tridax for their bibliographic citations has also been carried out from the important databases. The online database of Royal Botanic Garden Kew, London was searched for the plant information portal and database of Biodiversity Heritage Library was searched for bibliographic citations which are presented in table 1.

Table 1

S.N Species No. No. of No. of No. of references No. of Bibliography of references references in in micro references in entri in Kew Economic morphology Herbarium Biodiversity es in records botany bibliography catalogue Heritage IPN bibliography Library I 1 Tridax 2 0 0 0 2 7 Palmeri 2 Tridax 5 5 10 5 11 422 procumbens 3 Tridax 1 0 0 0 1 18 candidssima 4 Tridax rosea 2 0 0 0 0 2 5 Tridax 2 0 0 0 0 0 moorei 6 Tridax 2 0 0 0 0 4 purpurea 7 Tridax 2 0 0 0 0 0 tembensis 8 Tridax erecta 2 0 0 0 1 7 9 Tridax 1 0 0 0 1 7 obovata 10 Tridax 3 0 0 0 1 7 tenuifolia

From the literature survey it is obvious that the plants of tribe Heliantheae of family Asteraceae posses wide medicinal activities. The genus Tridax is one of the important genus of this tribe and the species Tridax procumbens has been extensively documented in the literature for its variety of medicinal properties over the other species of the genus.

Review of Literature

Extensive research work has been done on Tridax procumbens. Assessment and antibacterial and antifungal activities of silver nanoparticals obtained from extract of Tridax procumbens leaf have been explored by Himakshi Bhati , et al46. Chitra Pai and et al reported the antimicrobial activity of Tridax procumbens with special references to nosocomial pathogens47. Immunomodulatory effect of ethanolic extract of Tridax procumbens on Swiss Albino rats have been studied by M.K.Oladunmoye48. Tridax procumbens is reported to be useful for wound healing activities, Scabies and Dandruff49. Munjamlai Kumar and Grace studied the Hematological, Histopathological and Cytotoxicity activities of the essential oil separated from the leaves of Tridax procumbens50. Anil Kumar O and L.Mutyala Naidu studied the antibacterial activity of aqueous and solvent extract (ethanol, methanol, chloroform and petroleum ether) of different plant parts of Tridax procumbens L. against human pathogenic bacteria and found that the leaf and whole plant extracts of Tridax procumbens L. have great potential as antibacterial agents in the treatment of infectious organisms51. Priyadarshini. D. S and Priya Iyer studied antimicrobial activity of Tridax procumbens found the plant has potential source of useful drugs52.

Dhasarathan.P and et al studies the antimicrobial activities of extracts and their fraction of leaves of Tridax procumbens Linn53. Antibacterial activity of different part of Tridax procumbens against Human pathogens has studied by Veena Gayathari Krishnaswamy and Christina54. Syed Mohd.Danish Rizvi and et al used some Gram positive and Gram negative bacterial strains against different extracts (Hexan, Petroleum ether, chloroform and Methanol) of Tridax procumbens and found that the plant extract were equally susceptible to both Gram negative (Eenterobacter aerogenes) and Gram positive bacteria (Bacillus subtilis and Staphylococcus aureus)55 . A Manjamalai and et al found that the essential oil exhibited strong antioxidant activity due to its natural origin and potent free radical scavenging activity. Hence the essential oil could be used to prevent free radical Mediated diseases/disorders56. Evaluation of Analgesic and

Antipyretic activity of Tridax procumbens leaves extract have been explored by Narayan Singh Patel and et al57.

Identification of putative antitrypanosomal compounds in Tridax procumbens extract are done by A. Abubakar and et al58. Sheetal Patil and et al. found in their experimental work that the ethanolic extract of Tridax procumbens Linn. possesses potent anthelmintic activity to standard anthelmintic drug this anthelmintic activity may be due to some phytoconstituents like steroids, tannins, alkaloids, flavonoids, carbohydrates ,glycosides, found during preliminary phytochemical investigation59. J.D.Habila and et al studied the total phenolic and antioxidant activity of Tridax procumbens Linn. From their work they conclude that Tridax procumbens, has antioxidant activity which was established to correspond to the amount of total phenolic content of the plant samples60.

Aqueous extract of Tridax procumbens exhibited better antibacterial activity as compared to their petroleum ether, methanolic and ethanolic extracts 61. In vitro phytochemical screening and antibacterial activity of aqueous and methanolic leaf extracts of Tridax procumbens against Bovine Mastitis Isolated Staphylococcus aureus are studied by R.Dhanabalan and et al62. Jude Chigozie Ikewuchi found that aqueous extract of leaves of Tridax procumbens is effective agent in the treatment and prevention of carbon tetrachloride induced hepatic cytotoxicity63. Sowmya B. Jhample and et al observed that the Tridax procumbens L. extract of leaves and roots showed more significant antibacterial activity as compared to that flower extract. The acetone extracts of roots and a leaves showed superior antibacterial activity as compared to methanolic extract. Result for phytochemical screening suggests the presence of following phytochemicals such as tannins, alkaloids, saponins, flavonoids, phenols, steroids, anthocynins, proteins,amino acid and carbohydrate constituents in both acetone and methanolic leaves extract, hence it may also possess good medicinal antioxidant properties. As aqueous extract of leaves also showed enhanced blood clotting activity, it may be used as a potent haemostatic agent. Hence Tridax procumbens L. is medicinally important herb with tremendous therapeutic potential to combat bacterial infections64.

The whole plant extract of Tridax procumbens in three different solvent viz pet ether, water and ethanol have been reported to be studied for their antibacterial activity. The extract showed good antibacterial activity against the microbes vis Staphylococcus aureus, Micrococcus luteus and Proteus mirabilis65 .

Yogesh P. Talekar and Biswadeep Das studied the wound healing potential of aqueous and ethanolic extract of Tridax procumbens L. In wistar rats and reported to found good wound healing activity. Histopathological study also raveled the wound healing activity. Effect of Tridax procumbens on hydroxyproline collagen and Hexosamine content was also studied66. Antibacterial activity of Tridax procumbens against the test organisms staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhi, Escherichia coli and Bacillus tereus has been studied by V. Bharathi et al. Ethyl acetate extract of Tridax procumbens reported to show good antibacterial activity against the test organisms67 . Sunil Christuas and et al. reported that Tridax procumbens leaves can be used for treating various diseases caused by the organisms 68. N.A Maechi and V J Oye found that the extract of Tridax proumbens L. have various pharmacological effect, antimicrobial activity against both gram positive and gram negative bacteria and stimulates growth in rabbits69.

Minal Wani, Snehal Pande and Nitin More70 studied the callus induction studies in Tridax procumbens. Psychophilous and Melitophilous pollination syndrome in Tridax procumbens L.(Asteraceae) is investigated by P.Varalakshmi and A.J.solomon Raju71. Jagadesan Preveena, Subhash J Bhore worked on identification of bacterial endophytes associated with traditional medicinal plant Tridax procumbens Linn72. Demonstration of anti-inflammatory activity of alcoholic and hydroalcoholic extract of Tridax procumbens is carried out using the rat pawedema assay by R.L.Meshram and M.B.Patil73. Anti arthritic activity of ethanolic extract of Tridax procumbens (Linn) in Sprague Dawley rats was studied by R. Ramesh Petchi and et al.74 Salahdeen.H.M and et al studied the effect of aqueus leaf extract of Tridax procumbens on blood pressure and heart rate in rats75. Efficacy of antimicrobial effect of Venonia amygdalina and Tridax

12 procumbens in vitro control of tomato (Lycopersicum esculentum) post harvesting has been studied by Ijato .J. Y and et al76. Ganju Kuldeep and Pathak A.K, worked on pharmacognostic and phytochemical evaluation of Tridax procumbens.77

The effect of anti-cancer activity of Tridax procumbens flower crude aqueous and acetone extract was tested on Prostat epithelial cancerous cells pc 3 was studied by Vishnu Priya and et al78. Antiseptic and antifungal activities of wthanol-water extract of Tridax procumbens have been studied by Sutar Prashant B79.

D.S.Mohale and et al found that the methanolic extract of Tridax procumbens (L) has significant antibacterial activity against S. aureus and E.coli but Staphylococcus aureus( gram positive bacteria) are more susceptible then Escherichia coli (gram negative bacteria) 80. The activity of Tridax procumbens against the fungal mycoflora of cowpea legume was found by Umesh P. Mogle81. Rajaram .S. Sawant and Ashvin G. Godghate worked on preliminary phytochemical analysis of leaves of Tridax procumbens82. Alka Jindal and Padma Kumar have worked on Antimicrobial flavonoids from Tridax procumbens.83 R.R.Dhangar and et al. during their work found that the ethanolic extract of Tridax procumbens Linn. shows potent anthelmintic activity to standard anthelmintic drug. The drug can be further explored for the isolation and characterization of the active constituents responsible for anthelmintic activity84. K.Poornima and A.Palanisamy85 have reported the Biocontrol of mycoflora by medicinal plant extract including Tridax procumbens.

Agrawal Surendra, Mohale Deepak and Talele G.S.worked on pharmacological activity of Tridax procumbens86. Evaluation of anti-bacterial activity of local flora of Bundekhand region of Jhansi, India against plant pathogenic bacteria has been done by Sazada Siddiqui87. The effect of aqueous leaf extract of Tridax procumbens on the haematological and serum Biochemical parameters of Wistar Albino Rats has been determined by J. O. Olukunle & M.O.Abatan88. M.P.Ayyappa Das & etal. Studied phytochemical screening & antibacterial activity of Aqueous & Methanolic leaf extract of

13 two medicinal plants against Bovine Mastitis Bacterial Pathogens89. L. Isaiarasu and et al showed that both the aqueous and alcoholic extracts of Tridax procumbens are comparatively much effective against the bacterial and fungal pathogens causing diseases in the silkworm B. mori90.

Herbal formulation made from Tridax procumbens for dandruff is reported by Shalini Sharma and et al91. Influence of Tridax procumbens on lysyl oxidase activity and wound healing has been studied by S. L. Udupa and et al92. Ikram Ilahi and et al. studied In vitro antioxidant activity of four medicinal plants on the basis of DPPH free radical scavenging93. A comparative study of the antioxidant activity of methanolic leaf extracts of Various medicinal plants including Tridax procumbens L. has been documented by Melinda Krishanti P.and et al94. Aqueous extract of Tridax procumbens leaves on oxidative stress and antioxidant status in chloroquine-induced hepatotoxicity in rats have been explored by Harrison Ugo Nwanjo95. N. Savithramma and et al concluded from their work that Thespesia populnea and Tridax procumbens are widely used in traditional medicines to combat and cure various ailments thus appear to be rich in secondary metabolites. The anti-inflammatory, astringent, asthma and diarrhoea activities of these two plants can be attributed to their high flavonoids, steroids, alkaloids, tannins, terpenoids and saponins. Exploitation of these pharmacological properties involve further investigation and identification of these active ingredients by implementation of techniques like extraction, purification, separation and crystallization.96

Sutar Prashant B. has reported the use of ethanol-water extract of Tridax procumbens as antiseptic and antifungal agent97. Thus review of literature reveals that the Tridax procumbens is a vitally important plant to be explored further for its medicinal values. Hence we undertook the work on this plant under different experimental set ups which are describe in details in experimental section.

Experimental Section

A) Preliminary Phytochemical Screening 98

Tridax procumbens has been reported to exhibit various pharmacological, anti- diabetic, antiinflammatory, analgesic and marked depressant action on respiration99-104 hence phytochemical analysis of this plant is of immense importance.

Extraction Process

Sample leaves of Tridax procumbens were obtained from local market of Melghat region of Maharashtra and identified. Leaves of Tridax procumbens were collected and washed with water and then shade dried and converted in to powder.

10 g powder was taken in 25 ml of four different solvent each (Hexane, Ethyl acetate, Dichloromethane, water) and filtered. The filtrate so obtained was used for preliminary photochemical investigation.

Phytochemical Screening

Each extract of Tridax procumbens was screened for the presence of various secondary metabolites (phytochemical) such as alkaloids, saponins, tannins, steroids, flavonoids, terpenoids and phenols. The methods of analysis employed were those described by (Kokate. C.K 2000 and Harborne 1999) for the presence of various active components.

Test for alkaloid :

1 ml extract was added to one drop of Mayer’s reagent (Mixture of mercuric chloride 1.36g and of KI 5.5g in 100 ml water), a cream colored precipitate was formed which confirmed the presence of alkaloids.

Test for Saponin:

1 ml extract was added to five ml distilled water and shacked vigorously for 2 min. The appearance of foam persist for at least 15 min, confirmed the presence of saponin. Test for tannin:

2ml extract was added to few drops of 1% lead acetate, yellowish precipitate appeared confirming tannin. Test for steroid:

1 ml of extract was added to1ml acetic acid and 1 ml chloroform followed by 0.5 ml sulphuric acid. The test solution showed violet to blue green formation, which confirmed steroid Test for flavonoid:

1 ml extract was treated with 1 ml sulphuric acid resulted in orange colour confirming the presence of flavonoid. Test for terpenoid:

2ml of extract was added to 2ml of acetic acid followed by 1 ml sulphuric acid which resulted in blue green ring showed the presence of terpenoid. . Test for Total Phenol:

One ml of extract in the test tube was treated with 3% ferric chloride. The solution turned into deep blue color, it shows the presence of phenol.

Test for Glycosides:

Keller Killiani Test – Test solution was treated with few drops of glacial acetic acid and Ferric chloride solution and mixed. Concentrated sulphuric acid was added, and observed for the formation of two layers. Lower reddish brown layer and upper acetic acid layer which turns bluish green would indicate a positive test for glycosides.

RESULTS AND DISCUSSION

The tests were carried out for all four test extracts. The results obtained are reported in following table

The phytochemical analysis of the four different extract of Tridax procumbens

Table

Alkaloid Saponin Tannin Steroid flavonoid terpenoid Glycosides Phenol

Hexane + _ + _ + + _ _

Ethyl + _ + + + + + _ acetate Dichloro _ _ + + _ + + _ methane Water + + + + + + _ +

+ Present

- Absent

B) STUDY OF ANTIOXIDANT ACTIVITY OF PHYTOCONSTITUENTS

ISOLATED FROM LEAVES OF TRIDAX PROCUMBENS

A. Extraction of Non Polar flavonoids18 10 g powered and dried leaves taken as sample a) The Sample was extracted with n-hexane in soxhlet apparatus. The extract was collected and evaporated. b) The same sample was extracted with chloroform. The extract was collected and evaporated to dryness. c) Same sample was extracted with methanol; the extract was collected and evaporated to dryness. The residue a, b, c were dried and collected and used for antioxidant assay.

B. Extraction of saponin18 10 g dried leaves were added to 100 ml 20% ethanol. It was heated in water bath for 4 hours with continuous stirring at about 550C then it was filtered and re extracted the residue over water bath at 900C and transferred to 250 ml separating funnel and added 20 ml diethyl ether was added to the mixture and shacked vigorously. Aqueous layer was recovered and 60 ml n-butanol was added and combined extract was evaporated in water bath up to dryness. The residue so obtained was collected and used for antioxidant study.

C. Extraction using solvents of different polarity

10 g powered and dried leaves were subjected to soxhlet extraction separately using solvents of different polarity as water, ethanol, dichloromethane and diethyl ether. The extracts were collected and tested for

antioxidant activity using DPPH reagent. The ethanol extract showed prominent bleaching of purple color of DPPH indicating presence of antioxidants.

Antioxidant activity of non polar flavonoids TLC chromatograph of non polar flavonoids (plates 1A, 1B, 2A and 2B) were developed using dichloromethane solvent. The TLC chromatograph was sprayed with DPPH ( 2, 2 diphenyl 1-picrylhydrazyl) reagent. The spots indicated by arrow bleached the purple color of DPPH indicating presence of antioxidants. In the TLC chromatograph 1B , three spots bleached the color of DPPH and in 2B also three spots bleached the color of DPPH

TLC on DCM plate 1A After DPPH spraying plate 1 B

TLC on DCM plate 2A After DPPH spraying plate 2 B Antioxidant activity of Saponins TLC chromatograph of saponins (plate 3A and 3B) was developed using ethanol- water mixture in 6:4 proportion. The TLC chromatograph was sprayed with DPPH ( 2, 2 diphenyl 1-picrylhydrazyl) reagent. The spot indicated by arrow bleached the purple color of DPPH up to considerable extent indicating presence of antioxidants.

Plate 3A Plate 3B

Antioxidant activity of different solvent extracts

The four extracts viz water, ethanol dichloromethane and diethyl ether extracts were tested for antioxidant activity (plates 4A and 4B)

Plate 4A Plate 4B

Ethanol and water extract bleached the color of DPPH

The ethanol extract of the Tridax procumbens showed highest amount of antioxidants as it bleached the color of DPPH to a maximum extent. Water extract also showed presence of antioxidants. Saponin fraction has also bleached the color of DPPH up to considerable extent which indicated the presence of antioxidants. Non polar flavonoids showed antioxidants in traces.

C) EVALUATION OF QULITATIVE AND QUANTITATIVE ANTIOXIDANT ACTIVITY OF DIFFERENT SOLVENT EXTRACTS ISOLATED FROM THE LEAVES OF TRIDAX PROCUMBENS

MATERIALS AND METHODS Sample leaves of Tridax procumbens were obtained from local market of Melghat region of Maharashtra and identified. Extraction: leaves of Tridax procumbens were collected and washed with water and then shade dried and converted in to powder. 10 gm powder was taken in 25 ml of four different solvent each (Hexane, Diethyl ether, Dichloromethane, Dioxane) and filtered. The filtrate so obtained was used for qualitative and quantitative antioxidant evaluation. Study of qualitative antioxidant activity by DPPH After filtration the filtrate so obtained was taken into four different test tubes then one drop of each extract was taken on TLC plate and tested for antioxidant activity using DPPH reagent. The extract showed prominent bleaching of purple color of DPPH indicating presences of antioxidants. Study of quantitative antioxidant activity by DPPH The antioxidant activity of the four different extract was assessed on the basis of the radical scavenging effect of the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH). The diluted working solutions of the test extracts were prepared in four different solvent. A solution of 0.002% of DPPH was prepared in same four solvents separately and 1 ml of this solution was mixed with 1 ml of sample solution. These solution mixtures were kept in dark for 30 min and optical density was measured at 517 nm using UV visible spectrophotometer. A mixture of solvent (pure 1 ml) with DPPH solution (0.002%, 1 ml) was used as blank for each solvent. The optical density was recorded and % inhibition was calculated using the formula given below

Percent (%) inhibition of DPPH (%AA) = A – B X 100 A

RESULTS AND DISCUSSION Sample preparation : Eleven different solutions of each extract were prepared having 1 mg/ml, 1.1mg/ml, 1.2mg/ml, to 2.0 mg/ml.1ml of each of this solution was mixed with 1ml of 0.002mg/ml DPPH solution and resulting solution was used as sample. Optical density of the sample was recorded by U. V. Visible spectrophotometer and the results obtained are reported in following tables. IC50 values have been determined for each extract.

Table: 1 Optical density and percent antioxidant activity of Hexane extract

Conc.mg/ml 1mg/ml 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 O.D of 0.253 0.250 0.246 0.244 0.242 0.239 0.236 0.235 0.234 0.233 0.231 sample % AA 31.06 31.88 32.97 33.51 34.05 34.87 35.69 35.96 36.23 36.51 37.05

O.D of sample 0.255

0.25

0.245

0.24 O.D of sample 0.235

0.23

0.225 0 0.5 1 1.5 2 2.5

20 Conc.mg/ml 15 % AA

0 024681012

IC50 value calculation = MAX – 50% (MAX – MIN)

= 37.05– 50% (37.05 – 31.06)

= 34.06

= 1.5

Table 2: Optical density and percent antioxidant activity of Dichloromethane extract

Conc.mg/ml 1mg/ml 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 O.D of 0.242 0.236 0.230 0.228 0.228 0.224 0.219 0.212 0.207 0.201 0.192 sample % AA 38.70 40.25 40.51 40.51 41.81 43.11 44.93 46.23 47.79 50.12 37.14

O.D of sample 0.3

0.25

0.2

0.15 O.D of sample 0.1

0.05

0 0 0.5 1 1.5 2 2.5

% AA 60

30 % AA 20

0 0 0.5 1 1.5 2 2.5

IC50 value calculation = MAX – 50% (MAX – MIN)

= 50.12 – 50% (50.12 – 37.14)

= 43.63

= 1.6

Table 3 Optical density and percent antioxidant activity of Dioxane extract

Conc.mg/ml 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 O.D of 0.169 0.160 0.159 0.150 0.150 0.149 0.147 0.140 0.135 0.135 0.130 sample % AA 47.51 50.31 50.62 53.41 53.41 53.72 54.34 56.52 58.07 58.07 59.62

O.D of sample 0.18 0.16 0.14 0.12 0.1 0.08 O.D of sample 0.06 0.04 0.02 0 0 0.5 1 1.5 2 2.5

% AA 70

60 50

30 % AA 20 10 0 0 0.5 1 1.5 2 2.5

IC50 value calculation = MAX – 50% (MAX – MIN)

= 59.62 – 50% (59.62 – 47.51)

= 53.56

= 1.6

Table 4: Optical density and percent antioxidant activity of Diethyl ether extract

Conc.mg/ml 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 O.D of 0.215 0.213 0.210 0.209 0.208 0.206 0.205 0.203 0.201 0.200 0.199 sample % AA 51.57 52.02 52.70 52.92 53.15 53.60 53.82 54.27 54.72 54.95 55.18 O.D of sample 0.216 0.214 0.212 0.21 0.208 0.206 O.D of sample 0.204 0.202 0.2 0.198 0 0.5 1 1.5 2 2.5

% AA 55.5 55 54.5 54 53.5 53 % AA 52.5 52 51.5 51 0 0.5 1 1.5 2 2.5

IC50 value calculation = MAX – 50% (MAX – MIN)

= 55.18 – 50% (55.18 – 51.57)

= 53.37

= 1.4

Extract IC50 value Hexane 1.5mg/ml D.C.M 1.6 mg/ml Dioxane 1.6 mg/ml Diethyl Ether 1.4 mg/ml

D) EVALUATION OF ANTIOXIDANT ACTIVITY OF SAPONIN AND TANNIN FRACTIONS ISOLATED FROM THE LEAVES OF TRIDAX PROCUMBENS

Extraction of saponin10:

10gm of sample was added to 100ml 20%ethenol. It was heated in water bath for 4 hours with continuous stirring at about 550C,then it was filtrated and re extracted the residue over water bath at 900C and transferred to 250ml separating funnel then 20ml diethyl ether was added and shacked vigorously. Aqueous layer was recovered and discarded the diethyl ether layer. 60 ml of n-butanol was added and combined extract was washed twice with 10ml of 5% aqueoes sodium chloride and evaporated in water bath up to dryness and weight was measured.

Extraction of tannin10 :

10gm of sample was taken in beaker containing approximately 100ml distilled water. The flask was heated gently and boiled for 30 min. it was centrifuged at 2000 rpm and supernatant was collected. Then sample was dried and weight was measured.

Study of antioxidant activity by DPPH

The antioxidant activity of the saponin and tannin isolated was assessed on the basis of the radical scavenging effect of the stable 1, 1-diphenyl-2-picrylhydrazyl (DPPH) . The diluted working solutions of the test extracts were prepared in methanol. 0.002% of DPPH was prepared in methanol and 1 ml of this solution was mixed with 1 ml of sample solution. These solution mixtures were kept in dark for 30 min and optical density was measured at 517 nm using UV visible spectrophotometer. Methanol (1 ml) With DPPH solution (0.002%, 1 ml) was used as blank. The optical density was recorded and % inhibition was calculated using the formula given below A – B Percent (%) inhibition of DPPH activity (%AA) = ______x 100 A Where A = optical density of the blank and B = optical density of the sample.

RESULTS AND DISCUSSION

The stock solution of 1mg/ml of isolated saponin was prepared using water as solvent. The required dilutions 0.11mg/ml to 0.19 mg/ml were prepared by appropriate dilutions. The optical density and percent antioxidant activity was calculated and reported table 1

Table 1

Optical density and percent antioxidant activity for saponin

O.D. of blank DPPH = 0.565

Conc. 0.11 0.12 0.13 0.14 0.15 0.16 0.17 0.18 0.19 Mg/ml O.D of 0.483 0.449 0.421 0.412 0.400 0.390 0.384 0.370 0.362 mixture % AA 14.51 17.68 25.48 27.07 29.20 30.97 32.03 34.51 35.92

Decrease in O.D. of mixture with increase in concentration of saponin

O.D 0.6

0.5

0.4

0.3 O.D 0.2

0.1

0 0 0.05 0.1 0.15 0.2

As concentration increases the percent antioxidant activity increases.

Increase in percent antioxidant activity with increase in concentration of saponin

% AA 40 35 30 25 20 % AA 15 10 5 0 0 0.05 0.1 0.15 0.2

A graph was plotted in concentration against the %AA values and IC50 vale was calculated from graph was found to be IC50 value for saponin component = 0.13mg/ml

Table 2

Optical density and percent antioxidant activity for tannin

O.D. of blank DPPH = 0.565

Conc. 0.11 0.12 0.13 0.14 0.15 0.16 0.17 0.18 0.19 Mg/ml O.D of 0.361 0.344 0.331 0.322 0.310 0.308 0.296 0.251 0.240 mixture %AA 36.10 39.11 41.41 43.00 45.13 45.48 47.61 55.57 57.52

Decrease in O.D. of mixture with increase in concentration of tannin

O.D 0.4 0.35 0.3 0.25 0.2 O.D 0.15 0.1 0.05 0 0 0.05 0.1 0.15 0.2

Increase in percent antioxidant activity with increase in concentration of tannin

%AA 70 60

50 40

30 %AA 20

0 0 0.05 0.1 0.15 0.2

IC50 vale for tannin =0.165mg/ml

Remarkable decrease in the O.D. values of mixture for both the isolated fractions was observed indicating antioxidant activity of the fractions. Both the fraction viz saponin and tannin extracted from the leaves of Tridax procumbens showed good to moderate antioxidant activity which is evident from the graph. The IC50 values for saponin and tannin fractions were found to be 0.13mg/ml and 0.165mg/ml respectively.

E ) GCMS ANALYSIS OF VARIOUS EXTRACTS OF LEAVES OF TRIDAX PROCUMBENS

EXPERIMENTAL: - The leaves of the plant Tridax procumbens were collected from local market of hilly area nearby Melghat region of Maharashtra and identified.

Procedure -1) 10 gm dried leaves of Tridax procumbens were crushed in 100 ml distilled water and filtered. The filtrate so obtained was allowed to stand overnight. The suspended impurities so developed were filtered off. The process is repeated until no impurities are formed. The clear solution so obtained was subjected to column chromatographic separation using Ethyl acetate, ethanol 1:1 solution. The first component so obtained was collected and tested for antioxidant activity by DPPH. The fraction showed good antioxidant activity. The sample was analyzed by G.C.M.S analysis. Procedure -2)10 gm of dried coarse powder of leaves of Tridax procumbens was subjected to soxhlet extraction with n- hexane. The extract was removed and the marc was further extracted with DCM, The extract was removed and the marc so obtained was extracted with ethyl acetate and the marc obtain was again extracted with n-Butanol. The extracted sample was analyzed by G.C.M.S. Procedure -3) 10 gm of dried coarse powder of leaves of Tridax procumbens was subjected to soxhlet extraction with n- hexane. The extract so obtained was analyzed by G.C.M.S.

RESULTS AND DISCUSSION:-

1) The G.C.M.S Analysis of sample extracted by procedure (1) revealed several peaks.

33 out of these the peak of 9.2 min has been identified as 4H-pyrane-4-one,2,3-dihydro-3,5- dihydroxy-6-methyl is in 96.8% probability limit. when compared by mass M\Z(144,134,126,115,101,87,73,58,55,53,43)

O OH OH

4H-pyrane-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl

The literature survey reveals that the compound possesses antioxidant activities. Another compound identified with probability limit of 94.4% is 5-hydroxymethyl-2- furaldehyde when compared with mass spectrum M\Z(126,123,109,97,95,93,84,81,75,69,61,58,55,53,43,41)

And it is a reported antioxidant in grape and apple juice.44

H O OH O

5-hydroxymethyl-2-fururaidehyde

2) The G.C.M.S. analysis of sample extracted by procedure (2) revealed a peak at 6.9 min and identified as 1,1-dibutoxy butane with 93.5% probability. When compared with mass spectrum M\Z(159,141,129,113,103,99,89,85,79,73,67,64,57,55,53)

O O 1,1-dibutoxy butane

3) The G.C.M.S analysis of sample extracted by procedure (3) revealed several peaks out of which The peak of 19.9 minute was predominant and identified by library search as n- hexadecanoic acid with probability of 80.4%,when compared by mass spectrum M\Z(256,239,227,213,199,185,171,157,143,129,115,97,83,73,60,57). It is reported to possess some antioxidant properties45.

n-hexadecanoic acid

Another peak obtained at 28.2 min was identified as 1,2-Benzenedicarboxylic acid mono (2-ethylhexel) ester with probability limit of 50.1% when compared with mass spectrum M\Z(279,261,180,167,149,132,122,113,104,93,83,76,71,57) and known to possess some antimicrobial activity.

O OH O

1,2-Benzenedicarboxylic acid mono (2-ethylhexel) ester

CONCLUSION: - The GCMS analysis of different extracts of Tridax procumbens revealed important bioactive molecules. The structures are identified by mass spectra of library search with high probability limit.

F) ISOLATION AND IDENTIFICATION OF QUERCETIN AND RUTIN FROM LEAVES OF TRIDAX PROCUMBENS LINN. BY HPLC ANALYSIS.

Experimental:

1)Isolation of Rutin fraction : The leaves of Tridax procumbens L. were collected and washed with tap water then shade dried and after complete drying, coarse powder was prepared. Twenty grams of the powder was extracted by soxhlet apparatus with 250 ml of 80% ethanol till exhaustion. The extract was filtered and concentrated by evaporation under vacuum to about 10 ml then mixed with 25 ml distilled water and extracted with petroleum ether (50 x 3), then with chloroform (50 x 3).After extraction, the aqueous layer was collected and left to stand in a cold place for 72 hours; a yellow precipitate separated out of the solution .The precipitate was dissolved in ethanol and used for TLC, HPLC analysis and column chromatography.

TLC

A TLC was run using a pre-coated aluminum sheet with silica gel using following mobile phases

Solvent system Rf for standard rutin Rf for isolated rutin Water : AcOH (80:20) 0.52 0.56 Isopropanol : Water (60:40) 0.75 0.72

Column Chromatographic separation

A column was prepared using silica gel (100-200 mesh) in hexane. The mixture of Water- AcOH (80:20) was used as eluent. The yellow solution obtained from the column was collected and the solvent was evaporated in rotary evaporator. A yellow solid was obtained M.P. 2100C.

NMR

RTP BRUKER AVANCE II 400 NMR Spectrometer

12.6018 10.7058 9.2902 7.5603 7.5551 7.5356 6.8569 6.8365 6.3940 6.3892 6.2012 6.1964 5.3575 5.3480 5.3392 5.3056 5.1155 4.4157 4.3878 3.8389 3.7228 3.6970 3.4212 3.4019 3.3167 3.3002 3.2949 3.2710 3.2557 3.2449 3.2327 3.2157 3.1935 3.1000 3.0768 3.0537 2.5125 2.5083 2.5042 1.2256 1.0025 0.9870 SAIF Panjab University Chandigarh

Current Data Parameters NAME Mar11-2015 EXPNO 540 PROCNO 1

F2 - Acquisition Parameters Date_ 20150311 Time 17.35 INSTRUM spect PROBHD 5 mm PABBO BB- PULPROG zg30 TD 65536 SOLVENT DMSO NS 8 DS 2 SWH 12019.230 Hz FIDRES 0.183399 Hz AQ 2.7263477 sec RG 287 DW 41.600 usec DE 6.00 usec TE 295.2 K D1 1.00000000 sec TD0 1 ======CHANNEL f1 ======NUC1 1H P1 10.90 usec PL1 -3.00 dB SFO1 400.1324710 MHz F2 - Processing parameters SI 32768 SF 400.1300000 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 1.00 1.82 2.09 1.12 1.13 1.12 2.21 2.40 4.17 1.82 6.89 2.12 3.04

13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm [email protected]

RTP BRUKER AVANCE II 400 NMR Spectrometer

7.5603 7.5551 7.5356 6.8569 6.8365 6.3940 6.3892 6.2012 6.1964 SAIF Panjab University Chandigarh

Current Data Parameters NAME Mar11-2015 EXPNO 540 PROCNO 1 F2 - Acquisition Parameters Date_ 20150311 Time 17.35 INSTRUM spect PROBHD 5 mm PABBO BB- PULPROG zg30 TD 65536 SOLVENT DMSO NS 8 DS 2 SWH 12019.230 Hz FIDRES 0.183399 Hz AQ 2.7263477 sec RG 287 DW 41.600 usec DE 6.00 usec TE 295.2 K D1 1.00000000 sec TD0 1

======CHANNEL f1 ======NUC1 1H P1 10.90 usec PL1 -3.00 dB SFO1 400.1324710 MHz F2 - Processing parameters SI 32768 SF 400.1300000 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 2.09 1.12 1.13 1.12

8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 ppm [email protected]

RTP BRUKER AVANCE II 400 NMR Spectrometer

5.3575 5.3480 5.3392 5.3056 5.1155 4.4157 4.3878 3.8389 3.7228 3.6970 3.4212 3.4019 3.3167 3.3002 3.2949 3.2710 3.2557 3.2449 3.2327 3.2157 3.1935 3.1000 3.0768 3.0537 2.5125 2.5083 2.5042 1.2256 1.0025 0.9870 SAIF Panjab University Chandigarh

======CHANNEL f1 ======NUC1 1H P1 10.90 usec PL1 -3.00 dB SFO1 400.1324710 MHz F2 - Processing parameters SI 32768 SF 400.1300000 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 2.21 2.40 4.17 1.82 6.89 2.12 3.04

5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm [email protected]

2)Isolation of Quercetin fraction: The leaves of Tridax procumbens L. were collected and washed with tap water then shade dried and after complete drying coarse powder was prepared. Twenty grams of the powder was successively soxhlet extracted with petroleum ether , chloroform and methanol respectively. Each of solvent was used for a period of 24 hours. The methanolic fraction was concentrated to obtain a semisolid consistency. The same semisolid fraction was successively extracted with 50 ml of petroleum ether (fraction I) , 50 ml of ethyl acetate (fraction II) and 50 ml of ethyl acetate ( Fraction III) with the help of separating funnel. Each extraction was repeated three times to ensure complete extraction in each case .This was done for the entire methanolic extract. Fraction III was used for TLC, HPLC analysis and column chromatography.

TLC

A TLC was run using a pre-coated aluminum sheet with silica gel using following mobile phases

Solvent system Rf for standard Quercetin Rf for isolated Quercetin Toluene: ethyl acetate : 0.41 0.38 Methanol (30:60:10) Column Chromatographic separation

A column was prepared using silica gel (100-200 mesh) in hexane. The mixture Toluene: ethyl acetate: Methanol (30:60:10)was used as eluent. The yellow solution obtained from the column was collected and the solvent was evaporated in rotary evaporator. A yellow solid was obtained M.P. 2980C.

NMR

QTP BRUKER AVANCE II 400 NMR Spectrometer

12.4984 10.8023 9.6118 9.3783 9.3206 7.6804 7.6749 7.5554 7.5499 7.5342 7.5287 6.8971 6.8759 6.4126 6.4074 6.1916 6.1864 3.3633 2.5168 2.5124 2.5079 2.5034 2.4989 1.2341 SAIF Panjab University Chandigarh

Current Data Parameters NAME Mar11-2015 EXPNO 520 PROCNO 1 F2 - Acquisition Parameters Date_ 20150311 Time 17.25 INSTRUM spect PROBHD 5 mm PABBO BB- PULPROG zg30 TD 65536 SOLVENT DMSO NS 8 DS 2 SWH 12019.230 Hz FIDRES 0.183399 Hz AQ 2.7263477 sec RG 406 DW 41.600 usec DE 6.00 usec TE 295.1 K D1 1.00000000 sec TD0 1

======CHANNEL f1 ======NUC1 1H P1 10.90 usec PL1 -3.00 dB SFO1 400.1324710 MHz F2 - Processing parameters SI 32768 SF 400.1300000 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 1.03 0.99 1.07 2.02 1.03 1.00 1.05 1.01 1.02 1.37

13 12 11 10 9 8 7 6 5 4 3 2 1 0 ppm [email protected]

QTP BRUKER AVANCE II 400 NMR Spectrometer

12.4984 10.8023 9.6118 9.3783 9.3206 SAIF Panjab University Chandigarh

======CHANNEL f1 ======NUC1 1H P1 10.90 usec PL1 -3.00 dB SFO1 400.1324710 MHz F2 - Processing parameters SI 32768 SF 400.1300000 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 1.03 0.99 1.07 2.02

13.012.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 ppm [email protected]

QTP BRUKER AVANCE II 400 NMR Spectrometer

12.4984 10.8023 9.6118 9.3783 9.3206 SAIF Panjab University Chandigarh

======CHANNEL f1 ======NUC1 1H P1 10.90 usec PL1 -3.00 dB SFO1 400.1324710 MHz F2 - Processing parameters SI 32768 SF 400.1300000 MHz WDW EM SSB 0 LB 0.30 Hz GB 0 PC 1.00 1.03 0.99 1.07 2.02

13.012.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 ppm [email protected]

Result and Discussion:

The HPLC chromatogram of the sample containing rutin fraction (isolated by procedure 1) was compared with that of pure rutin, the peaks for rutin coincided indicating rutin in the extract of leaves of Tridax procumbens, analogusly the HPLC chromatogram of the sample containing quercetin fraction (isolated by procedure 2) was compared with that of pure quercetin, the peaks for quercetin coincided indicating quercetin in the extract of leaves of Tridax procumbens.

HPLC chromatogram of pure rutin HPLC chromatogram of pure quercetin

HPLC chromatogram of rutin in sample HPLC chromatogram of quercetin in sample

IR, NMR, TLC and HPLC analysis showed presence of quercetin and rutin in the leaf extract of tridax procumbens.

OH OH

OH O

OH O Quercetin

Rutin

CONCLUSION

Preliminary Phytochemical Screening of leveas of Tridax procumbens was carried out which revealed important phytoconstituents as alkaloids, saponins, tannins, steroids, flavonoids, terpenoids and phenols. Qualitative antioxiant activity of Non polar flavonoids fraction , saponin fraction and different solvent extracts has been eluciteted by using DPPH. The fractions showed moderate to good antioxidant activity. Quantitative determination of antioxidant activity of different solvent extracts of leaves of Tridax procumbens has been carried out by spectrophotometrically. IC50 values for all the solvent extracts have also been determined. Column chromatographic seperation was employed for different solvent extracts of leaves of Tridax procumbens and GCMS analysis of different frctions isolated is carried out. Further characterization has been carried out by TLC, HPLC, IR and NMR spectral analysis . Following antioxidants have been identified from the leaves of Tridax procumbens from melghat region.

S.N Name Structure Probability limit

1 Quercetin OH Confirmed OH

OH O

OH OH O 2 Rutin Confirmed

3 4H-pyrane-4- 96.8 % one,2,3-dihydro-3,5- dihydroxy-6-methyl O OH OH

4 5-hydroxymethyl-2- H 94.4% fururaidehyde O OH O

5 1,1-dibutoxy butane 93.5%

O O

6 n-hexadecanoic acid HO 80.4%

7 1,2- O OH Benzenedicarboxylic O 50.1% acid mono (2- O ethylhexel) ester

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