Chapter 1 INTRODUCTION INTRODUCTION Ayurveda is a Sanskrit word

Chapter 1
INTRODUCTION
INTRODUCTION
Ayurveda is a Sanskrit word, which comprises of two words ‘ayur’ and ‘veda’. “Ayur” means life and “Veda” means knowledge or science. The word “Ayurrveda” means ‘the knowledge of life’ or ‘the science of life’1.
It stems from the ancient Vedic culture and was taught for many thousands of years in an oral custom from proficient masters to their disciples. In India Ayurvedic knowledge originate more than 5,000 years ago and is also known as the “Mother of All Healing.”2
Ayurveda is an intricate medical system that originated in India thousands of years ago. The Atharvaveda contains the principles of healing on which the Ayurveda is based. ‘Ayur’ means ‘life’ in Sanskrit. Ayurveda is the most ancient science of healing which enhances longevity. 1
Ayurvedic medicine is an earliest method of health care that is native to the Indian subcontinent. In India, Nepal, Sri Lanka, China, Tibet, and Pakistan the daily use of ayurvedic medicines by millions of individuals. Now, it is also tradition for health care in Europian countries.3-4
In ancient time natural products, included plants, animals and minerals are the only basis of treatment of human diseases. But mainly plants are used as a medicine. Due to over exploitation by the human activities for their needs, this source is increasing day by day. The frequently accepted modern medicine or allopathy has developed gradually over the years by scientific anbd observational efforts of scientists. However, the development of its basis remains rooted in traditional medicines and therapies.5
1.1 Basic Principles of Ayurveda6-8
The basic principle of Ayurveda is based on the “Panchamahabhuta” theory, which says that the whole thing in this universe is made up of five basic elements namely:
Akasha (Space)
Vayu (Air)
Agni (Fire)
Jala (Water)
Prithivi(Earth)
1.2 Herbal Medicine:
In recent time, WHO has defined traditional remedy together with herbal drugs as comprising therapeutic practice that have been in continuation for hundreds of years, before the growth and increase of current medicine and are still use nowadays. Herbal medicine is still the mainstay of about 75-80% of world population. Because of this the general faith that herbal drugs be without any side effects also being inexpensive and locally available.9
Herbal products are mixtures of complex organic chemicals that might come from any raw part of a plant, including leaves, flowers, stems, seeds, and roots. Under the present law, herbs are define as dietary supplement, and manufacturer be capable of produce, sell, and market herbs devoid of first indicating safety and efficacy, while required for pharmaceutical drugs.10
Herbal medicine had develop into a well-liked form of healthcare. Still some differences survive between herbal and conventional pharmacological treatments. The herbal medicine can be experienced for efficacy using conventional trial methodology. Several specific herbal extracts have been verified to be efficient for specific conditions. Although the public is over and over again mislead to believe that all natural treatments are naturally safe. Finally, we need to know which herbal remedies do more harm than good for which condition. Because of the current popularity of herbal medicine, research in this area should be intensified.11
Plants synthesis many compounds called primary metabolites includes proteins, fats nad carbohydrates. They are mainly used for growth or reproduction. Inspite of these plants also synthesise secondary metabolites which is responsible for the therapeutic activity and thus pharmacological actions. It is a biosynthetic laboratory not only for chemical compounds like carbohydrates, proteins and lipids that are utilized as food by man, but also for a large amount of compounds like glycosides, alkaloids, volatile oils, tannins etc., that exert a physiological and therapeutic effect. Medicinal qualities of plants are due to chemicals. 12
Description of Plant (SAPINDUS MUKOROSSI: REETHA)
Sapindus mukorossi, also famous as soapnut which belong to the family Sapindaceae. It is used medicinally as an expectorant, contraceptives and for treatment of excessive salivation, epilepsy, chlorosis and migrane,9 psoriasis, eczema, for removing freckles and also have gentle insecticidal property and are habitually used for removing lice from the scalp. It is popular ingredient of ayurvedic preparation such as shampoo, cleansers 13
It is an important multipurpose tree of North India. The tree is native to China and Japan and much cultivated in North India. The tree is cultured in several parts of India as ornamental and for saponaceous fruits. In Sapindus mukorossi, the seed have mechanical dormancy due to hard seed coat.14
left259715
Figure1.1 Reetha Tree Figure 1.2 Fruit
The dried fruits of ‘Ritha’ are most valuable part of the plant. Its fleshy portion contains saponin, which is used in preparation of washing soap and as such is used for preparation of quality shampoos.13 The fruit is of considerable importance for its medical value as well. As per Ayurveda, Unani and Tibetan system of medication, it is useful in treating in number of human maladies like bad cold, facial pimples, irregularities in salivation, chlorosis, epilepsy constipation, nausea, etc. It is also used as expectorant and antihelminthic in small doses. 14
It is a deciduous tree widely grown in upper reaches of Indo-Gangetic plains, Shivaliks and Sub Himalayan tracts at altitudes from 200m to 1500m. The Sapindus mukorossi is a fairly large, deciduous tree with a straight trunk up to 12 meters in height. 15
Leaves are 30-50 cm long, alternate, paripinnate; common petiole very narrowly bordered, glabrous; leaflets 5-10 pairs, opposite or alternate, 5-18 by 2.5-5 cm, lanceolate, acuminate, entire, glabrous, petioles 2-5 m elongated.14 Flowers are about 5 mm transversely, small, incurable, polygamous, greenish white, sub sessile, numerous, mostly bisexual. Fruits are globose, fleshy, 1-seeded drupe, sometimes two drupels together, about 1.8-2.5 cm across. Seeds are 0.8-1.3 cm in diameter, globose, smooth, black and loosely placed in dry fruit.13 Bark is dark to pale yellow, fairly smooth, with many vertical lines of lenticels and fine fissures exfoliating in irregular wood scales. The blaze is 0.8-1.3 cm, hard, not fibrous, pale orange brown, brittle and granular.16
3228975100330744220100330
Figure 1.3 Leaves Figure1.4 Bark
In Japan its pericarp is called “enmei-hi”, which means “life prolonging pericarp” and in China “wu-huan-zi”, the “non-illness fruit”. The most important compounds isolated from Sapindus mukorossi are triterpenoidal saponins of mainly three oleanane, dammarane and tirucullane types. Recently many of the pharmacological actions of this plant have been explored which includes the antimicrobial, cytotoxic, molluscicidal, insecticidal, piscicidal and fungicidal activities. 14
2.1 PLANT TAXONOMY 16,17
BOTANICAL NAME Sapindus Mukorossi
FAMILY Sapindaceae
SUB-FAMILY Sapindoideae
KINGDOM Plantae
PHYLUM Spermatophyta
DIVISION Magnoliophyta (flowering plants)
CLASS Magnoliopsida (Dicotyledons)
SUB-CLASS Rosidae
ORDER Sapindales
TRIBE Andropogoneae
GENUS Sapindus (Soapberry)
SPECIES Mukorossi
POPULAR NAME
Doadni, Washnut, Soapberry, Ritha, Aritha, Dodan, Doda , Kanma & Thali, Soapnut
MORPHOLOGICAL PARTS USED Woods , Seeds , Pericarp extracts , Kernels etc
PARTS USED
Soapnut , Fruits ,Leaves , Flowers ,Soapnut Shells, Soapnut Shell Powder
2.2 VERNACULAR NAMES 13,18
SANSKRIT Hrishtah, Phenaka, Phenil, Rishtah, Rishtak, Rita, Sarishta
HINDI Phenil , Risht , Rishtak , Ritha
MANIPURI Hai kya Kekru
MARATHI Phenil
URDU Phenil , Ritha
ASSAMESE Aritha , Haithaguti
MIZO Hlingsi
NEPALI Rittha
PUNJABI Aritha , Dodan , Ritha , Thali
BENGALI Ritha
KUMAON Ritha
UNITED PROVINCES Kanmar
ITALIAN Uriya
TELUGU Kunkudu
2.3 MORPHOLOGY 13, 16, 17
LEAVES:
Leaflets 5-10 pairs, opposite or alternate, 5-18 by 2.5-5 cm, lanceolate, acuminate, entire, glabrous, often slightly falcate or oblique, petioles 2-5 m long common petiole very narrowly bordered. Leaves are 30-50 cm long, paripinnate, alternate;

Figure 1.5 Leaves of Reetha
BARK:
Bark is dark to pale yellow in colour, quite smooth and having various vertical lines of lentioels and fine fissure exfoliating in irregular wood scales.

Figure 1.6 Bark of Reetha
Flowers: The flowers are small and gresenish white, polygamous and mostly bisexual in terminal thyrses or compound cymose panicles. These are sub-sessile; numerous in number and at times occur in lose panicles at the end of branches. Pulp light brown, translucent, spontaneous rind with wrinkled surface.They are mostly bisexual, polygamous and small in terminal, compound panicles, drupes glabrous, freshly saponaceous, solitary, wrinkled or smooth, 2-2.5 cm in diameter.
Seed: It is enclosed m black, smooth, hard endocarp, 10-13 mm in diameter, containing
fleshy pericarp.

Blaze: It is 7.5 mm, hard, not fibrous pale orange- brown. Fruits are 1-3 distinct, indehiscent carpels, 1.8-2.5 cm diameter, smooth, pale-brown, globose, wrinkled before falling. Petiolules 2.5-5mm long.

Wood: It is light yellow close grained, coarse, uneven-textured, moderately hard and
heavy.

2.4 PHYTOCONSTITUENT
S. No. Chemical constituent Part of the plant
1 Triglyceride 19
Oleo-palmito-arachidin glyceride
Oleo-di-arachidin glyceride
Di-olein Seed
2 Lipid 20 Seed
3 Sesquiterpeneoidal glycosides21 Fruits
4 Flavanoids22
Quercetin, Apigenin, Kaempferol, Rutin Leaf
5 Saponin23
Triterpene24
Oleanane (sapindoside A&B)25
Dammarane(sapinmusaponin A-E)26
tricullane (sapinmusaponin F-K)27 Gall, fruit & root
fruit
gall
gall & root
2.5 PHARMACOLOGICAL ACTIVITY
S.no. Author Activity Methods used Part used
1 Ibrahim et al.28 Anti-Bacterial activity Ethanolic and chloroform extracts. Leaf
2 Garg et al.29
Rastogi et al.30 Spermicidal Activity Saponins Fruit Pericarp
3 Tiwari et al.31 Anti-Trichomonas Activity Mixing of sapindus and saponin 4 Geyter et al.32 Insecticidal Activity Ethanolic extract 5 Chakraborty et al.33 Anxiolytic Activity Metanolic extract 6 Man et al.34,35,36 Anticancer Activity Saponin from galls extracts Galls
7 Ibrahim et al.37 Hepatoprotective Activity Fruit pericarp extract Fruit
8 Upadhyay and Singh et al.38 Molluscicidal Activity Extract fruit
9 Virdi et al.39 Piscicidal Activity Fruit pericarp
10 Tsuzuki et al.40 Fungicidal Activity Crude extract Pericarp
11 Takagi et al.41 Anti-Inflammatory Activity Crude extract / isolated saponin and hederagenin Plant
12 Huang et al.26 Anti-Platelet Aggregation
Activity Isolation of compounds Gall
13 Chen et al.42 Tyrosinase Inhibition and Free Radical Scavenging Methanolic extract Seed
2.6 Uses & Benefits of Reetha
It is used as the main ingredient in soaps and shampoos for washing hair, as it is considered good for the health of hair.11
The jewellers in India use this plant to bring back the lost brightness of ornaments made of precious metals like gold, silver, etc.12
The herb is also used in the treatment of extra salivation, migraine, epilepsy and chlorosis.11
It has been placed as a popular herb in the list of herbs and minerals in Ayurveda and is used as an important ingredient in cleansers and shampoos.

It is used for the treatment of eczema, psoriasis, and for removing freckles.14
It is also used for removing lice from the scalp, as it has gentle insecticidal properties.13
The plant is also known for its antimicrobial properties which are valuable for septic systems.16
It is an important herb that is used in the treatment of contaminated soil. Moreover, it has also been used for washing and bleaching cardamoms, further helping in improving the latter’s colour and flavour.15,17

Chapter 2
REVIEW OF LITERATURE

REVIEW LITERATURE
S. No Year Author Title Abstract
1. 2016 Oguz et al.43 Extraction of Saponins from Soapnut (Sapindus Mukorossi) and Their Antimicrobial Properties Results showed that polarity of the extraction solvent affects the yield percentage of the extraction process. The extracts containing saponins were investigated for their antimicrobial activity
2. 2016 Kairon et al.44 Genetic Variation in progeny Performance Traits in soapnut (Sapindus Mukorossi Gaertn) of Himachal Pradesh All these traits showed significant variation at 3 months and 6 months of intervals among different seed sources. Variation in Sapindus mukorossi with value to the descendants performance traits could be due to the fact that the large sized seeds excelled over seed type than all the seed traits, germinability attributes, growth characters and seedling biomass
3. 2015 Mukta et.al.45 Effect of Sapindus mukorossi and Balanites aegyptiaca on colour fastness properties of Silk dyed with Butea monosperma The comparative study showed better fastness properties in Sapindus mukorossi as compared to Balanites aegyptiaca
4. 2015 Yenil et.al46 Panacea Plants for Environment and Humanity:Caper and Ritha”. It has been studied that Caper contains some biologically active compounds like glucosinolates, alkoloids, phenolics, flavonoid, tocopherol and minerals like sodium, while Ritha help to improve the quality and efficiency of the land by holding heavy metals like mercury, iron and zinc in the leaves and grabbing lead and cadmium in its fruits.

5. 2015 Sonawane et al.17 A Review of Recent and Current Research Studies on the Biological and Pharmalogical Activities of Sapindus Mukorossi The aim of the present review is to discuss the salient features of plants Sapindus mukorossi Gaertn and its different activities by fraction of crude extracts and isolated substances.
6. 2015 Londhe et al.47 Formulations of Herbal Hand Wash with Potential
Antibacterial Activity The main objective of this article is to confirm effective antimicrobial activity against skin pathogen Staphylococcus aureus, Klebsiella pneumonia, and Salmonella typhimurium.

7. 2014 Shanmugam et al.48 Phytochemical screening and antimicrobial activity of fruit
extract of Sapindus mukorossi The study revealed that the ethanolic extract of the pericarp of Sapindus mukorossi showed more prominent antibacterial activity against E.Coli, Staphylococcus aureus than aqueous extract and also showed antifungal activity against Aspergillus fumigates and Aspergillus Niger at 100% plant extract.

8. 2013 Du M et al.49 Isolation of total saponins from Sapindus mukorossi Gaertn. Studied to investigate the effect of single factors present in the pulp of Sapindus mukorossi Gaertn. It is prove that Sapindus mukorossi saponins are excellence non-ionic active agent.

9. 2013 Nimisha et al.50 Formulation and evaluation of herbal shampoo having antimicrobial potential The present investigation is done to develop a herbal shampoo for hair rising and strengthing without affecting or damaging hair.
The result found that Herbal shampoo will give hair protection and also conditioning effect, shine and manageability.

10. 2013 Sharma et.al.51 A New Triterpenoid Saponin and Antimicrobial Activity of
Ethanolic Extract from Sapindus mukorossi Gaertn. In this article the structure of the compound was determined by means of chemical and spectral analysis including advanced 2D NMR studies. And a new triterpenoid saponin has been isolated from pericarps of Sapindus mukorossi Gaertn.

11. 2013 Sharma et.al.52 Triterpenoid Saponins from the Pericarps of Sapindus mukorossi Studied a novel acetylated triterpene bisdesmoside saponin is elucidated as along with two known saponins, hederagenin from the pericarps of Sapindus mukorossi.
12. 2012 Rao et.al18 Sapindus trifoliatus: A Review The purpose of this review is to notify a small collection of Phytochemical Screening, Pharmacological activity and current uses of Sapindus trifoliatus plant. The most important phytoconstituents isolated as well as identified from different parts of this plant are saponins, sugars, fatty acids, trifoliosides, genins, phenolic acids, steroids, carbohydrates and triterpenoids.
13. 2012 Arora et.al.53 Sapindus emarginatus: Phytochemistry & Various Biological Activities The main objective of this review is to present the exploration of the uses and activities of the plant S.emarginatus with its classification, phytochemical or presence of secondary metabolites and the isolated compound.

14. 2012 Mondal et.al.15 Evaluation of Antigonorrhoeal Activity of Saponins Extract of Sapindus Mukorossi Gaertn Studied that the saponin is present in S.mukorossi Gaertn. And also revealed that saponin extract of S.mukorossi Gaertn.has useful antimicrobial properties. The ethanolic and methanolic extract of the leaves of plant was found to have antigonorrhoeal activity.

15. 2012 Verma et.al.54 Antihyperglycemic activity, antihyperlipedemic activity, haematological effects and histopathological analysis of Sapindus mukorossi Gaertn fruits in streptozotocin induced diabetic rats. Deliberate the antihyperglycemic and antihyperlipidemic properties of hydroalcoholic extract of fruits of Sapindus mukorossi Gaertn and its beneficial effect on rat and shows positive effect on blood glucose level and lipid level.

16. 2011 Sindhu et.al13 Sapindus Mukorossi (AREETHA): An Overview In this review various structure and saponins are isolated from S.mukorossi and also confirm the therapeutic value of this plant. It also provide the basic idea about most of the phytoconstituent present in S.mukorossi
17. 2011 Sati et.al.55 Chemical constituent and bio activities of genus Sapindus The main objective of this review is to present whole.research carried out with species of the genus Sapindus, in order to organize the data produced. The investigation shows that it contains numeral of bioactive novel compounds of different properties like saponins, sesquiterpene, oligoglycoside etc.

18. 2011 Ghagi et.al.56 Study of functional properties of Sapindus mukorossi as a potential bio-surfactant The main objective is to study the Functional properties of Sapindus mukorossi in aqueous solution have been investigated and hence crude ritha could be used as an economical bio-surfactant.

19.

2008 Huang et.al28 Triterpenoid saponins from die fruits and galls of Sapindus mukorossi. The main objective of this review is to study the structures of these saponins on the basis of spectroscopic analysis including 1D and 2D NMR techniques.
20. 2008 Kalola et al.57 Effect of
hydrolysis on the yield of hederagenin and High-Performance Thin-Layer
chromatography densitometric quantification of hederagenin m Fruit pericarp of
Sapindus spp. Deliberate the outcome of hydrolysis on the yield of hederagenin The yield of hederagenin ranged from 0.035 to 1.29% (w/w) with different method of hydrolysis.

21. 2008 Ibrahim et al.37 Hepatoprotective
activity of Sapindus mukorossi and Rheum emodi extracts: In vitro and in vivo
studies. Studied the Hepatoprotective activity on the extracts of the fruit pericarp of S. mukorossi (2.5 mg/mL) and rhizomes of R. emodi (3.0 mg/mL) were found to have protective properties in rats with CCI4 induced liver damage as judged from serum marker enzyme activities.
22. 2007 Tsuzuki et al.40 Antifungal activity of the extracts and saponins from Sapindus saponaria L Studied the Antifungal activity of the extracts from the dried pericarp of Sapindus saponaria L. (Sapindaceae).
23. 2007 Rahman et al.58 Investigation of Sapindus mukorossi extracts for Repellency, Insecticidal
activity and Plant growth regulatory effect. Investigated the repellency and insecticidal activity against Sitophilus oryzae and Pediculus humanus sfrom ethanolic extract of Sapindus mukorossi.
24. 2007 Huang et.al.26 Anti-platelet aggregation of Triterpene saponins from the galls of Sapindus mukorossi. Studied the Anti platelet aggregation of ethanolic extract from the galls of S. mukorossi resultant two saponins were isolated, Sapinmusaponins Q and R.

25. 2006 Sengupta et.al20 Chemical investigation of Sapindus mukorossi seed oil” Fette Seifen Anstrichmittel. The main objective is to isolate two lipid fractions ‘A’ and ‘B’ from Sapindus mukorossi seed oil by preparative TLC. Fraction ‘A’ (70.4%, Rf value 0.76) is a normal % triglyceride and its fatty acid compositions was determined by GLC( Gas Liquid Chromatography). Fraction ‘B’ (29.6%, Revalue 0.51) shows the presence of nitrogenous constituents.

26. 2006 Huang et.al.59 Sapinmusaponins F-J, bioactive tiruealiane-type saponins from the galls of
Sapindns mukorossi. Studied Tirucallane-type saponins,
sapinmusaponins were isolated from the galls of Sapindus mukorossi.

27. 2005 Yao et.al.60 New Dammarane-Type Saponins from the Galls of Sapindus
Mukorossi Gaertn. The main aim is to isolate Dammarane-type saponins, like Sapinmusaponins together with three known phenylpropanoid glycosides.

28. 2004 Teng et.al.61 New tirucallane-type
triterpenoid saponins from Sapindus mukorossi Gaetn Studied the roots of Sapindus mukorossi which contains tirucallane-type triterpenoid saponins like Sapimukoside.

29. 2004 Saxena et.al.62 Characterization of sapindosides
in Sapindus mukorossi saponin (reetha saponin) and quantitative determination of
sapindoside B. Examine six dissimilar saponins from the fruits of Sapindus mukorossi by LC-MS. They were found to be Sapindoside A, Sapindoside B,
Sapindoside C, Sapindoside D, Mukorozisaponin El and Mukorozisaponin Y1.

30. 2003 Teng et.al.63 Two New Tirucallane-Type
Triterpenoid Saponins from Sapindus mukorossi. Studied the roots of Sapindus mukorossi contains tirucallane-type triterpenoid saponins like
Sapimukoside A ; B.

31. 2003 Maikhuri et al.64 Mechanism of
action of some acrylophenones, quinolones and dithiocarbamate as potent, nondetergent
spermicidal agents. Studied another intimate use of soapnut is as a contraceptive cream. As the result shows that it is totally safe and easy to use. It is proposed for post-coital use.

32. 1994 Azhar et.al.65 Chemical constituents of Sapindus mukorossi gaertn. (Sapindaceae). Studied Fruits of Sapindus mukorossi which contain sesquiterpenoidal glycosides and six different fatty ester of tetracyclic triterpenoids.

33. 1993 Garg et al.25 Studies on contraceptive efficacy of Praneem Polyherbal Cream. Studied the Spermicidal activity on human sperm of polyherbal pessary. The mixture of
three herbal ingredients resulted in potentiation of spermicidal action by eight folds, when tested in rabbit
34. 1991 Tedlaouti et al.66 Antitrypnosomial activity of some saponins from Calendula
arvensis, Hedera helix, Sapindus mukorossi. Studied the Antitrypnosomial activity of female Anopheles in the gonotropic cycle was impair by contact to neem, reetha (S.mukorossi) and garlic.

35. 1990 Takechi et al.36 Structure activity relationships of the saponin ahederin. Study suggest that the invitro cytotoxic activity of triterpenoid saponins from Sapindus mukorossi were reported to be at least 5 times less active than the reference compound (Strychnopentamine).

36. 1990 Diwedi et.al22 Standardization of a new
spermicidal agent sapindus saponin and its estimation in its formulation. Studied a colourimetric assay method for the estimation of total saponin content of Sapindus mukorossi.

37.

1989 Dhar et al.19 Morphological changes in human spermatozoa as examined under scanning electron microscope after invitro
exposure to saponins isolated from Sapindus mukorossi. Studied the morphological changes in human ejaculated spermatozoa after contact to saponin were examined under scanning electron microscopy. These result suggest that the morphological changes observed are due to alterations in the glycoproteins associated with the lipid bilayer of plasma membrane of spermatozoa.

38. 1979 Dev et.al19 Glyceride composition of Sapindus mukorossi(Soapnut) oil The main aim is to study the seeds of Sapindus mukorossi which contain 23 % oil of which 92 % is triglycerides.

Chapter 3
AIM ; OBJECTIVES

Aim ; Objective
The chemical compounds present in the bark and leaves of Sapindus Mukorossi Gaertn., will be isolated by Column Chromatography and characterized by using different Spectroscopic method and screen these compound biologically.

The aim of present investigation is to study the phytoconstituents present in the bark and leaves of Sapindus Mukorossi Gaertn. For biological activities plant extract and compound are screen. The seeds, fruits, gall, roots of the plant contain Triterpenoids, Phytosterol, Flavonoids, Saponins, Lipids and Triglycerides which are claimed to have anti-bacterial, anti-inflammatory, anti-cancer, piscicidal, molluscicidal etc. properties. Therefore, this information encourages us to investigate more details in phytochemical and biological aspect of bark and leaves of Sapindus Mukorossi Gaertn., which have not been explored until now.

Detailed review literature suggested that the plants possess diverse pharmacological action an attempt will have been made to evaluate the biological activity of the extract of leaves and bark of Sapindus Mukorossi Gaertn.
CHAPTER 4
MATERIAL ; METHODOLOGY
Material and Methodology
4.1 Preparation of Plant Material
Collection of Plant-
The leaves and bark of Sapindus mukorossi Gaertn.was collected in month of September from Dehradun, Uttarakhand.

Washing- The collected leaves and bark were washed in tap water so that the dust can be removed and after this the leaves and bark were soaked in 1% saline water for 5 minutes to remove microbes.

Drying of material- Shade dry is recommended for the drying process for protection the rich vitamins and other photosensitive constituents. The plant material was spread on the sterile clean green net in a well-ventilated room.
Grinding- Dried plant material were crushed in small pieces and then grinded by electric grinding machine for coarse powder and the powdered was stored in an airtight container which is protected from light and humidity.

Chemical requirement and their source: And other chemical and reagents used for standardization of drug taken from our college, HIPR Dehradun.
4.2 List of chemicals used in our study
Table 8: list of chemical used in study
S.no Chemical name
1 Distilled water
2 n-hexane
3 Petroleum ether
4 Chloroform
5 Ethyl acetate
6 Methanol
7 Phenol
8 Sulphuric acid
9 Hydrochloric acid
10 Soluble starch
11 Perchloric acid
12 Folin ;cio calteu’s reagent
13 Sodium carbonate
14 Gallic acid
15 Aluminum chloride
16 Sodium nitrite
17 Sodium hydroxide
18 Rutin
19 Sodium acetate
20 Vanillin methanol solution
21 Catechin
22 Alpha amylase enzyme
23 Benedict reagents
24 Fehling solution
25 Tannic acid
26 Hager’s solution
27 Wagner reagents
28 Mayer reagents
29 Dragendroffs reagents

4.3 Physiochemical standardization:-
4.3.1 Determination of Moisture content (Loss on Drying)
Procedure: The moisture content can be done by Loss on drying method. The parameter determines the amount of moisture as well as volatile components present in a particular sample (i.e. water drying off from the drug). 2gm of powdered leaves and bark was taken in a petridish and then it was placed in hot air oven at 105 ?C. Then LOD was calculated.
4.3.2 Determination of extractive values:-
Extractive value is a measure of the content of the drug extracted by solvents. Extractive value can be water soluble, alcohol soluble and hexane soluble, ethyl acetate, chloroform.

4.3.2.1 Water extractive:
5gm of the air dried, coarsely drug powder was taken and macerated with 100ml of water in a closed flask for 24 hours, shaking had been done frequently during the first six hours and then allowed to stand for 18 hours. After that it was filtered taking precaution against loss of water, and then 20ml of filtrate evaporated to dryness in a china dish on a water bath and dried at 105 ?C and weigh. The proportion of water soluble extractive value with indication to air dried drug has to be calculated.

Formula used for calculation:
10 ml of extract solution contain = X gram extract
100 ml of extract solution contain = X100/10 =10X gram extract
2 gram powdered drug contain = 10X gram extract
100 gram powdered drug contain = 10X × 100/2 gram extract = 500X %
X = difference in pre weight and final weigh
4.3.2.2 Alcohol extractive:
5gm of the air dried, coarsely drug powder was taken and macerated with 100ml of methanol in a closed flask for 24 hours, shaking had been done frequently during the first six hours and then allowed to stand for 18 hours. Then it was filtered rapidly and 20ml of filtrate evaporated to dryness in a china dish on a water bath and dried at 105 ?C and weighed. The percentage of alcohol soluble extractive value with reference to air dried drug has to be calculated.

Formula used for calculation:
10 ml of extract solution contain = X gram extract
100 ml of extract solution contain = X 100/10 =10X gram extract
2 gram powdered drug contain = 10X gram extract
100 gram powdered drug contain = 10X × 100/2 gram extract = 500X %
X = difference in pre weight and final weight
4.3.2.3 Hexane extractive
Macerated 5gm of the air dried, coarsely powder with 100ml of n-hexane in a closed flask for 24 hours, shaking had been done frequently during the first six hours and then allowed to stand for 18 hours. Then it was filtered rapidly and 25ml of filtrate evaporated to dryness in a china dish on a water bath and dried at 105 ?C and weighed. The percentage of n-hexane soluble extractive value with reference to air dried drug has to be calculated.

Formula used for calculation:
10 ml of extract solution contain = X gram extract
100 ml of extract solution contain = X 100/10 =10X gram extract
2 gram powdered drug contain = 10X gram extract
100 gram powdered drug contain = 10X × 100/2 gram extract = 500X %
X = difference in pre weight and final weight
4.3.2.4 Ethyl acetate extractive
Macerated 5gm of the air dried, coarsely powder with 100ml of ethyl acetate in a closed flask for 24 hours, shaking had been done frequently during the first six hours and then allowed to stand for 18 hours. Then it was filtered rapidly and 25ml of filtrate evaporated to dryness in a china dish on a water bath and dried at 105 ?C and weighed. The percentage of ethyl acetate soluble extractive value with reference to air dried drug has to be calculated.

Formula used for calculation:
10 ml of extract solution contain = X gram extract
100 ml of extract solution contain = X 100/10 =10X gram extract
2 gram powdered drug contain = 10X gram extract
100 gram powdered drug contain = 10X × 100/2 gram extract = 500X %
X = difference in pre weight and final weight
4.3.2.5 Chloroform extractive
Macerated 5gm of the air dried, coarsely powder with 100ml of chloroform in a closed flask for 24 hours, shaking had been done frequently during the first six hours and then allowed to stand for 18 hours. Then it was filtered rapidly and 25ml of filtrate evaporated to dryness in a china dish on a water bath and dried at 105 ?C and weighed. The percentage of ethyl acetate soluble extractive value with reference to air dried drug has to be calculated.

Formula used for calculation:
10 ml of extract solution contain = X gram extract
100 ml of extract solution contain = X 100/10 =10X gram extract
2 gram powdered drug contain = 10X gram extract
100 gram powdered drug contain = 10X × 100/2 gram extract = 500X %
X = difference in pre weight and final weight
4.3.3 Determination of total tannins
Regents used:
A. Saturated Sodium carbonate solution: It was prepared by adding 35 g anhydrous sodium carbonate to each 100 ml distilled water, dissolved it at 70-80oc and get cool overnight, filtered through glass wool.

B. Tannic acid standard solution: (0.1 mg/ml) dissolve 10 mg tannic acid in 100 ml of distilled water.
C. Folin ; Ciocalteu’s phenol reagent.

Preparation of standard curve: Standard curve was prepared using tannic acid as standard (10 mg tannic acid in 100 ml of distilled water).

Procedure: Extracted 2 g powdered plant material with 100 ml distilled water by boiling on water bath for 6-8 hrs., filtered and made up the volume to 100 ml volumetric flask. Took 1 ml aliquot of it, added 5 ml Folin & Ciocalteu’s reagent, 10 ml saturated sodium carbonate and make up the volume up to 100 ml in volumetric flask. The instrument was calibrated through blank and took the corresponding absorbance of different samples, total tannin content calculated by using y = 0.004x + 0.002, R2 = 0.996, at 760 nm, using UV-1 Double beam spectrophotometer, where y was the absorbance and x the tannic acid equivalent (mg/ml).
4.3.4 Determination of total phenolics
Estimation of Total Phenolic content in the plant was carried out according to modified colorimetric Folin-Ciocalteu method.

Extract preparation: 1 gram air dried powdered drug percolated with pure methanol, three-time filter the extract and lyophilized to dry and was weighted.

Regents used:
7% Sodium carbonate saturated solution: It was prepared by adding 7 g anhydrous sodium carbonate dissolved in 100 ml distilled water, and get cool.

Gallic acid standard solution: (1mg/ml) dissolve 10 mg gallic acid in 10 ml of deionized water.
Folin ; Ciocalteu’s phenol reagent
Procedure: A volume of 0.5 ml of deionized water and 0.125 ml of a known dilution of the extract were added to a test tube, Folin-Ciocalteu’s reagent (0.125 ml) was added to the solution and allowed to react for 6 min. Then, 1.25 ml of 7% sodium carbonate solution was liquated into the test tubes, and the mixture was diluted to the 3 ml with deionized water. The color developed for 90 min, and the absorbance was read at 760nm, Y = 0.002x + 0.051, R2 = 0.988, using UV-1Double beam spectrophotometer. The measurement was then compared to the standard curve of prepared Gallic acid solution and expressed as milligrams of Gallic acid equivalents per 100g of the sample extract.

4.3.5 Determination of total flavonoids:
Total flavonoid contents of the extract solution based on the formation of a complex flavonoid-aluminium.

Extract preparation: 5 gram of dried powdered leaf, stem and flowers cold percolated with known volume of methanol.

Reagent used:
2% Aluminum chloride
Rutin standard: 0.1mg/ml solution
Methanol
Procedure: A volume of 0.5 ml of sample, 0.5 ml of 2% AlCl3 in methanolic solution was added. After one hour at room temperature, the absorbance was measured at 420 nm, using UV-1 Double beam spectrophotometer. Extract samples were evaluated at a final concentration of 0.01 mg/ml. All the determinations were done in triplicate. Total flavonoid content was calculated as Rutin (mg/ml) using the following equation based on the calibration curve: y = 0.003x + 0.007, R 2 = 0.993, where y was the absorbance and x was the Rutin equivalent (mg/ml).
4.3.6 Determination of total flavonols:
Extract preparation: 5 gram of dried powdered leaf, flowers and stem were percolated with known volume of methanol.

Reagent used:
2% Aluminum chloride solution.

5% Sodium acetate solution
Rutin standard: 0.1mg/ml solution
Methanol
Procedure: To 1.0 ml of sample, 2 ml of 2% AlCl3 in methanol and 3 ml 5% sodium acetate solution were added. The absorbance at 440 nm was read after 2.5 hour at 20°C using UV-1 Double beam spectrophotometer. Extract sample were evaluated at a final concentration of 0.05 mg/ml. Total flavonol content was calculated as Rutin (mg/ml) using the following equation based on the calibration curve: y = 0.005x + 0.008, R2 = 0.996, where y was the absorbance and x the Rutin equivalent (mg/ml).
4.3.7 Determination of total Proanthocyanidins:
Extract preparation: 5 gram of dried powdered leaf, stem and flowers were percolated with methanol for 48 hrs, and concentrated under vacuum using rota-vapour.

Reagent used:
4% vanillin-methanol solution.

Concentrated hydrochloric acid
Catechin standard: 0.1mg/ml solution
Methanol
Procedure: To 0.4 ml of sample with 3 ml of 4% vanillin-methanol solution and 1.5 ml concentrated hydrochloric acid were added and shake well. The absorbance at 500 nm was read after 15 min at room temperature using UV-1 Double beam spectrophotometer. Extract sample were evaluated at a final concentration of 0.01 mg/ml. Total proanthocyanidin content was calculated as Catechin (mg/ml) equivalent using the following equation based on the calibration curve: y = 0.005x +0.002, R2 = 0.997, where y was the absorbance and x the rutin equivalent (mg/ml).

4.4 Successive extraction:
In successive extraction process the powdered plant material extracted with non polar to polar solvent i.e. Hexane, chloroform, Ethyl acetate, methanol, Water so on the basis of the polarity of contain in the plant material will be extracted out in particular solvent like non polar in hexane and chloroform, intermediate polar in ethyl acetate or high polar in methanol and water. Soxhlet apparatus for (Hot percolation method) was used for successive extraction. Here continuous extraction of a drug or any other substance which is recommended in monograph is done. The procedure consists of percolating it with appropriate solvents at a temperature just about that of the boiling point of the solvent. Whichever apparatus that permit the uniform percolation of the drug and the uninterrupted flow of the vapour of the solvent around the percolator may be used.

Procedure: Assembly was arranged and thimble was prepared and place 20 gram of air dried powdered drug and was extracted with Hexane 3 days, than extract solution collected and concentrate under vacuum using Rota-vapour. Then the plant material was again collected and air dried. When completely dried placed back in thimble again. Similarly was done for chloroform, ethyl acetate, methanol, than water. Finally the dried extracts were collected in pre-weighted glass vials and post-weight for each vial was taken. Calculated % yield.

Formula used:
5 gram air dried powder contain = X gram of extract
100 gram air dried powder contain = 100X/5 = 20X
X = difference in weight of vial
4.5 Phytochemical screening:
Phytochemical screening comprise of the chemical evaluation of the plant successive extract, these are qualitative test which shows the presence or absence of different type phyto-constituent in plant from successive fraction i.e. Hexane, Chloroform, Ethyl acetate, Methanol, Water by using Soxhlet apparatus(Hot percolation method), subjected to qualitative tests for the identification of various active constituents including, Carbohydrate, Glycoside, Alkaloid, Amino acids, Flavanoids, Fixed oil, Tannins, Gum and Mucilage, Phytosterols.

4.6 Chemical requirement:
?-Naphthol, Benedict reagent, Fehling’s A and B, conc Sulfuric acid, Ferric chloride, Vanillin hydrochloride reagent, Sodium hydroxide, Copper sulphate, Millon’s reagent, Wagner’s reagent, Hager’s reagent, Ninhydrin, Dragandroff’s reagent etc.

Procedure:
Alkaloids
Preparation of test solution: The test solution was prepared by dissolving extracts in dil. hydrochloric acid solution.

Mayer’s Test: In acidic solution few drops of Mayer’s reagent (Potassium Mercuric iodide) was added to it then cream coloured ppt. was obtained.

Hager’s Test: In acidic solution few drops of Hager’s reagent (picric acid solution) was added to it then yellow coloured ppt. was obtained.

Dragendorff’s Test: In acidic solution few drops of Dragendorff’s reagent (Pot. bismuth iodide) was added to it then reddish brown ppt. was obtained.

Wagner’s Test: In extract solution few drops of Wagner’s reagent (Iodine-potassium iodide) was added to it then reddish brown ppt. was obtained.

Carbohydrates
Preparation of test solution: The test solution was prepared by dissolving extracts with water. Then it was hydrolyzed with 1 volume of 2N HCl and further subjected to following chemical tests.

Fehling’s test: Equal volume of Fehling’s A and B solutions was added to the test solution and heated for a few minutes, orange red ppt was obtained.

Molish test: The test solution was prepared and few drops of alcoholic napthol was added to it. Then along the sides of the test tube few drops of H2SO4 was added to it, purple colour was obtained at the junction.

Saponins
Foam test: Test solution was shaken with water formation of foam was observed which stable for at least 15 min confirms saponin.

Steroids and Triterpenoids
Salkowski test: To the test extract solution few drops of conc.H2SO4 was added, then it was shaken and allowed to stand, lower layer turned red indicating the presence of sterols.

Liebermann Burchard test: Chloroform solution of the extract with few drops of acetic anhydride and 1ml of conc.H2SO4 from the sides gives reddish ring at the junction of two layers.

Tannins
Ferric Chloride test: Extracts treated with 1% FeCl2 solution gives blue, green or brownish green colour.

Alkaline reagent test: Extract treated with 5% sodium hydroxide solution give yellow to red ppt within short time.

Flavonoids
Ferric Chloride test: Alcoholic solution of the extracts mixed with few drops of neutral ferric chloride solution gives green colour.

Lead Acetate test: Alcoholic solution of the extracts mixed with few drops of 10% lead acetate gives yellow precipitate.

Phenolic compounds
Ferric Chloride test: Treat the extracts with 3-4 drops ferric chloride solution gives bluish black colour.

Lead Acetate test: The extracts treated with 3ml of 10% solution of lead acetate solution give bulky white precipitate.

Glycosides
200mg of drug was extracted with 5ml of dilute sulphuric acid and warmed on a water bath. Then it was filtered. Then the acid extract was neutralized by adding 5% solution of sodium hydroxide. Then 0.1ml of Fehling’s solution A and B was added to it becomes alkaline and then heat on a water bath for 2min. Then the quantity of red ppt. was noted.

4.7 Spectroscopic Methods

4.7.1 Ultraviolet Spectroscopy

Switch on the main power and instrument. Allow to stabilize for 15 minutes.

Put the mode selector at % T position, Adjust zero percent transmittance with the help of set of zero control.

Take out the cuvette and drain out the blank solution from it. Wash it with sample and packed with sample solution.

Clean the outer surface of the reference cuvette with tissue paper and place into sample holder and cover the compartment.

Observe the wavelength of all sample.

4.8 Chromatographic parameters

4.8.1 Thin layer chromatography
The ascending TLC was run on silica gel pre-coated aluminium sheets, for checking the purity of the products as well as monitoring the progress of the sample. The final product was irradiated with UV light.

Chromatograms were eluted by the following solvent systems:
Chloroform: Methanol (5:5) (6:4) (7:3) (8:2)
Chloroform: Ethylacetate (5:5) (6:4) (7:3) (8:2)
Retention Factor = Distance travelled by solute/distance travelled by solvent

Procedure
Step1: Prepare the developing container
The developing container for TLC can be an especially intended chamber, a jar with a lid, or a beaker with a watch glass on the top. Pour solvent into the chamber to a depth of just less than 0.5cm. To support in the diffusion of the TLC chamber through solvent vapours. Cover the beaker with a watch glass, twirl it gently and allow it to stand while you prepare your TLC plate.

Figure 4.19: Preparation of developing container
Step2: Prepare the TLC plate
TLC pates used are purchased as 5cm×20cm sheets. Every large piece is cut horizontally into plates which are 5 cm tall by various widths. Measure 0.5cm from the bottom of the plate. Using a pencil, draw a line across the plate at the o.5 cm mark.

Figure 4.20: Preparation of TLC plates
Step3: Spot the TLC plate
Dissolve about 1mg of sample in 1mL of methanol. Dip the microcap into the solution and then gently touch the end of it onto the proper location on TLC plate.

Step4: Develop the plate
Place the ready TLC plate in the developing container, cover the container with the watch glass, and then leave it undisturbed on your work surface top. The solvent start rising up through TLC plate by capillary action. Make sure that the solvent does not cover the spot. Allow the plate to develop until the solvent is about half a centimetre below the top of the plate. Take away the plate from the beaker and instantly mark the solvent front with a pencil. Allow the plate to dry.

Figure 4.21: Development of plate
Step5: Visualize the spots
Hold a UV lamp over the plate and circle any spot you see.

4.8.2. Column Chromatography
Slurry Method (wet method)
Combine the silica gel with a small amount of non polar solvent in a beaker containing compound.
Carefully mix the two substances until a consistent paste is formed, but is still capable of flowing.

Transfer this homogeneous mixture into the column as carefully as possible using a spatula to scrape out the solid as you pour the liquid.
The slurry process usually gives the most excellent column packing, but is also not easy technique to master. Either the dry or slurry method is select; the most significant feature of packing the column is creating an evenly distributed and packed stationary phase.
Prevent the column from cracks, air bubbles by tapping the column to prevent the formation of voids.

Then pass the solvent n-hexane: ethylacetate (8:2) until the compound shows separation and spot is applied on TLC plates to visualize the desired compound.

Dry the compound and weighed.

4.9 Phytochemical screening of Isolated Compound

4.9.1 Steroids and Triterpenoids
Salkowski test: To the test extract solution few drops of conc.H2SO4 was added, then it was shaken and allowed to stand, lower layer turned red indicating the presence of sterols.

Liebermann Burchard test: Chloroform solution of the extract with few drops of acetic anhydride and 1ml of conc.H2SO4 from the sides gives reddish ring at the junction of two layers.

4.10 Biological Activity
4.10.1 Antifungal Activity
A Preparation of Sabourand Dextrose Agar (SDA) media
Dextrose: 40g
Peptone: 10g
Agar: 15g
Distilled water: 1L
The above constituents were weighed and dissolved in water. The mixture was warmed on water bath till agar dissolved. This was then sterilized in an autoclave at 15 lbs pressure and 121? C for fifteen minutes. The sterilized medium (20 ml) was poured in sterilized Petri dishes under aseptic condition, allowing them to solidify on a plane table.

B Preparation of Antifungal Solution

All the compounds were dissolved in dimethyl formamide (DMF). Proper drug controls were used which represent the zone of inhibition of the control and the extract. Extract was taken at concentration of 0.1mg/ml for testing antifungal activity. The extract diffused into the medium produced a concentration gradient/ After the incubation period, the zone of inhibition was measured in mm.

C Test Cultures
Following common standard strains were used for screening of antifungal activities:
Aspergillus Niger Fungus MTCC- 282
D Inoculum Preparation
The fungal strains were pre-cultured in nutrient overnight in bottle and kept it in BOD incubator at 25?C. The inoculum for determination of the sensitivity pattern considered of one loopful of an overnight grown broth culture of the test organism. The average size of the inoculum was about cells container in 2mm diameter standard loop.

E Experimental procedure
1. Agar plates are inoculated with a standardized inoculum of the test microorganism.

2. Then, filter paper discs (about 5mm in diameter) containing the test compound at a desired concentration, are placed on the agar surface.

3. The plates were placed in an incubator at 25?C.

4. Generally, antimicrobial agent diffuses into the agar and inhibits germination and growth of the test microorganism and then the diameters of inhibition growth zones are measured.

5. After days for fungal, the diameter of zone (including the diameter disc) was measured and recorded in mm. The measurements were taken with a ruler, from the bottom of the plate, without opening the lid.

Screening of antifungal activity against fungi
Screening is done with taking the different concentration of column fraction of different derivatives. Compounds of different concentration were prepared and mixed with different sterile Sabourand dextrose agar media and drug plates were prepared. For the preliminary screening, I species of gram positive fungi was taken. All the microorganisms were spot inoculated in drug plates containing different concentration of compounds. Then the plates were incubated at 22?C for 24 hrs and incubated for further 7 days for fungi. After 24 hrs of incubation, the presence or absence of fungi growth in different plates were observes. The minimum inhibitory concentration (MIC) of the extract was determined against different fungi strains as per NCCLS guidelines (National Committee for Laboratory Standards).

ANTIOXIDANT ACTIVITY
In vitro methods employed in antioxidant studies
4.10.2.1. 1, 1- Diphenyl-2-picrylhydrazyl (DPPH)
4.3 mg of DPPH (1, 1-Diphenyl –2-picrylhydrazyl) was dissolved in 30 ml methanol; it was protected from light by covering the test tubes with aluminum foil. 0.1 ml DPPH solution was added to 3ml methanol and absorbance was taken immediately at 517nm for control reading. 0.1-0.5 ml of various concentrations of isolated compounds as well as standard compound (Ascorbic acid) was taken and the volume was made uniformly using methanol. Each of the samples was then further diluted with methanol up to 3ml and to each 0.1 ml DPPH was added. Absorbance was taken after 15 min. at 517nm using methanol as blank on UV-visible spectrometer. The IC50 values for each drug compounds as well as standard preparation were calculated. The DPPH free radical scavenging activity was calculated using the following formula:
% scavenging = Absorbance of control – Absorbance of test sample/
Absorbance of control X 100
4.10.2.2. Hydrogen peroxide radical scavenging activity
1ml of (1mg/ml) test drug/standard (Ascorbic acid) was added to 0.6ml of hydrogen peroxide solution in phosphate buffer (pH?7.4). After incubating for 10 minutes at 37oC the absorbance was measured at 230nm. Corresponding blanks were taken. The absorbance of hydrogen peroxide in phosphate buffer as control was measured at 230nm. The scavenging effect (%) was measured using equation. Hydrogen peroxide produces hydroxyl radicals in cells. Scavenging of these radicals by the test drug is used as a test for antioxidant activity. The reduction of these radicals is seen by the decreased absorbance at 230nm with increasing concentration of the test drug.

% scavenging = Absorbance of control – Absorbance of test sample/
Absorbance of control X 100
CHAPTER 5
RESULT ; DISCUSSION
RESULT AND DISCUSSION
5.1 Total moisture content
Result shows that the all sample have the considerable amount of the moisture percent which are tabulated in table
Moisture percent
Parameters Moisture content %
Loss on Drying at 105°C(LOD) Leaves Bark
13.61% 12.09%
Table 9: moisture content of plant Sapindus mukorossi
5.2 Percentage Extractive value by hot percolation of Leaves (soxhlet extraction)
S.no Solvent Extractive Value
1. n-hexane 0.45
2. Chloroform 0.22
3. Ethylacetate 0.20
4. Methanol 2.07
5. Water 1.05
Table 12: % extractive value by hot percolation of leaves Sapindus mukorossi

Figure 5.22: Leaves Figure 5.22: Bark
5.3 Percentage Extractive value by hot percolation of Bark (soxhlet extraction)
S.no Solvent Extractive Value
1. n-hexane 2.00
2. Chloroform 0.13
3. Ethylacetate 0.11
4. Methanol 0.24
5. Water 0.52

Table 12: % extractive value by hot percolation of bark Sapindus mukorossi
5.4. % extractive value by cold percolation
Sample Hydro alcoholic extractive
Plant part Leaves Bark
% extractive 17.45 12.15
Table 13: % extractive value by cold percolation of plant Sapindus mukorossi
5.5 Determination of Total tannin content (Leaves)
S.no Concentration Absorbance (leaves) Absorbance (Standard drug) Statistical parameters
1. 0 0 0.03819 y= 0.097x +0.002, R2= 0.985
2. 0.1 0.01199 3. 0.2 0.02534 4. 0.3 0.03380 5. 0.4 0.04035 6. 0.5 0.04980 Table 13: Standard curve for leaves extract of Sapindus mukorossi

Standard curve for leaves extract of Sapindus mukorossi
Determination of Total tannin content (Bark)
S.no Concentration Absorbance (leaves) Absorbance (Standard drug) Statistical parameters
1. 0 0 0.02919 y= 0.097x +0.002, R2= 0.985
2. 0.1 0.01199 3. 0.2 0.02534 4. 0.3 0.03380 5. 0.4 0.04035 6. 0.5 0.04980
Table 13: Standard curve for leaves extract of Sapindus mukorossi

Standard curve for bark extract of Sapindus mukorossi
5.5 Determination of Flavonoids Content (Leaves)
S.no Concentration Absorbance (leaves) Absorbance (Standard drug) Statistical parameters
1. 0 0 0.0573 y = 0.050x – 0.047R² = 0.997
2. 0.1 0.0124 3. 0.2 0.0268 4. 0.3 0.0439 5. 0.4 0.0597 6. 0.5 0.0781
Table 13: Standard curve for leaves extract of Sapindus mukorossi

Standard curve for leaves extract of Sapindus mukorossi
Determination of Flavonoids Content (Bark)
S.no Concentration Absorbance (bark) Absorbance (Standard drug) Statistical parameters
1. 0 0 0.0488 y = 0.050x – 0.047R² = 0.997
2. 0.1 0.0124 3. 0.2 0.0268 4. 0.3 0.0439 5. 0.4 0.0597 6. 0.5 0.0781
Table 13: Standard curve for bark extract of Sapindus mukorossi

Standard curve for bark extract of Sapindus mukorossi
5.6 Determination of Total Phenolic Content (Leaves)
S.no Concentration Absorbance (leaves) Absorbance (Standard drug) Statistical parameters
1. 0 0 0.0587 y = 0.050x – 0.047R² = 0.997
2. 0.1 0.0235 3. 0.2 0.0487 4. 0.3 0.0699 5. 0.4 0.0968 6. 0.5 0.1269 Table 13: Standard curve for leaves extract of Sapindus mukorossi
Standard curve for leaves extract of Sapindus mukorossi
Determination of Total Phenolic Content (Bark)
S.no Concentration Absorbance (bark) Absorbance (Standard drug) Statistical parameters
1. 0 0 0.0875 y = 0.050x – 0.047R² = 0.997
2. 0.1 0.0235 3. 0.2 0.0487 4. 0.3 0.0699 5. 0.4 0.0968 6. 0.5 0.1269 Table 13: Standard curve for bark extract of Sapindus mukorossi

Standard curve for bark extract of Sapindus mukorossi
5.7 Determination of Total flavonols Content (Leaves)
S.no Concentration Absorbance (leaves) Absorbance (Standard drug) Statistical parameters
1. 0 0 0.1468 y = 0.050x – 0.047R² = 0.997
2. 0.1 0.0732 3. 0.2 0.1321 4. 0.3 0.1832 5. 0.4 0.2498 6. 0.5 0.2976
Table 13: Standard curve for leaves extract of Sapindus mukorossi

Standard curve for leaves extract of Sapindus mukorossi
Determination of Total flavonols Content (Bark)
S.no Concentration Absorbance (bark) Absorbance (Standard drug) Statistical parameters
1. 0 0 0.1335 y = 0.050x – 0.047R² = 0.997
2. 0.1 0.0732 3. 0.2 0.1321 4. 0.3 0.1832 5. 0.4 0.2498 6. 0.5 0.2976
Table 13: Standard curve for bark extract of Sapindus mukorossi

Standard curve for bark extract of Sapindus mukorossi
5.8 Determination of Total proanthocyanidins Content (Leaves)
S.no Concentration Absorbance (leaves) Absorbance (Standard drug) Statistical parameters
1. 0 0 0.1103 y = 0.050x – 0.047R² = 0.997
2. 0.1 0.0532 3. 0.2 0.1043 4. 0.3 0.1504 5. 0.4 0.2099 6. 0.5 0.2476 Table 13: Standard curve for leaves extract of Sapindus mukorossi

Standard curve for leaves extract of Sapindus mukorossi
Determination of Total proanthocyanidins Content (Bark)
S.no Concentration Absorbance (bark) Absorbance (Standard drug) Statistical parameters
1. 0 0 0.1185 y = 0.050x – 0.047R² = 0.997
2. 0.1 0.0532 3. 0.2 0.1043 4. 0.3 0.1504 5. 0.4 0.2099 6. 0.5 0.2476 Table 13: Standard curve for bark extract of Sapindus mukorossi

Standard curve for bark extract of Sapindus mukorossi
5.9 Preliminary phyto-chemical screening:
Phytochemical screening of successive fraction from soxhlet, (+) shows presence, (-) and show absence of content of extract.
Table 28: preliminary phytochemical screening of plant Sapindus mukorossi
S.no Plant constituent/Test/
reagent used n-hexane extract Chloroform extract Ethylacetate extract Methanol extract Water extract
1. Carbohydrates Molisch test – + + + +
Fehling – + + + +
2. Proteins Biuret Test – – – – –
Millon’s Test – – – – –
3. Phenolic compounds/ Tannins Ferric chloride solution – – – – –
Lead acetate – – – – –
4. Steroids/ Triterpenoids Salkowski reaction – – + + +
Liebermann – Burchard
Reaction – + + + +
5. Glycosides
Borntrager’s test – – – – –
Modified Borntrager’s test – – – – –
6. Flavonoids
Shinoda test – – + + –
Lead acetate solution – – + + –
7. Alkaloids Mayer’s reagent – – – – –
Dragendorff’s reagent – – – – –
Hager’s reagent – – – – –
Wagner’s reagent – – – – –
8. Saponins Foam Test + + + + +
5.10 Thin layer Chromatography
Solvent system (Chloroform : Ethylacetate = 5:5, 6:4. 7:3, 8:2)
Solvent System (Chloroform:Ethyl acetate=5:5)
Rf Value = Distance travelled by solute/ Distance travelled by solvent
Rf Value = 0.67

Figure 5.12
Solvent System (Chloroform:Ethyl acetate=6:4)
Rf Value = Distance travelled by solute/ Distance travelled by solvent
Rf Value = 0.59

Figure 5.13
Solvent System (Chloroform:Ethyl acetate=5:5)
Rf Value = Distance travelled by solute/ Distance travelled by solvent
Rf Value = 0.53

Figure 5.14
Solvent System (Chloroform:Ethyl acetate=5:5)
Rf Value = Distance travelled by solute/ Distance travelled by solvent
Rf Value = 0.45

Figure 5.15
5.11 Column Chromatography
Solvent system (chloroform: ethyl acetate =8:2)

Figure 5.16 Figure 5.17
Column chromatography of leaves Column chromatography of bark
5.12 Thin layer Chromatography of Isolated compound

Figure 5.18 Leaves Figure 5.19 Bark
S.no. Isolated Compound Rf value
1. Leaves 0.54
2. Bark 0.42
Table 5.12: Retention factor of compounds
5.13 FTIR (Fourier transform infra red spectrum)
5.13.1 Observation of chemical group from the FTIR of plant Sapindus mukorossi
Table 29: Observation of FTIR data of isolated compound of plant Sapindus mukorossi
Sample 1
Sample name Type of vibration Regions(cm-1)
Sample 1 C-H str
C=C str
CH2 str (pentane)
C-H bending
CH=CH str
O-H str (R2)
C-H str in CH3(R3)
C=O str (R4)
CH3 str(R5)
Rham (R1)
C=O vib
OH str
C-O (pri.alc)
C=C str
C-H def
C-N str
3074
1603
2925
1451
3197
2709
2854
1719
1774
3268
1019
1402
788, 747
1074
Sample 2
O-H str (R1)
O-H str
CH=CH str
C=O vib
N-O str (R4)
C-H bending (alkane)
C=C str (Aromatic)
C-N str
C-H (Alkyl halide) (R2)
C-N
CH=CH
C-H def
CH str in CH3 (R5) 3238
3275
3197
1774
1544
1454
1402
1231
1197
1074
989
672
2951
Sample 3
O-H str
C-H str (aldehyde)
Alkyne
C=O str (ketone)
N=O (nitro compound)
N-H def
N=O
O-H bending
C-Cl str
C-Cl str
3268
2933
2128
1722
1622
1510
1264
929
870
821
Interpreted structure

Figure 5.19 Interpreted Sample 1

Figure 5.20 Interpreted Sample 2

Figure 5.21 Interpreted Sample 3
Antioxidant Activity
Concentration(mg/ml) Absorbance of isolated compound and standard with DPPH
Leaf Bark Ascorbic acid
0 0 0 0
0.02 0.140 0.128 0.306
0.04 0.119 0.114 0.129
0.06 0.030 0.056 0.075
0.08 0.027 0.027 0.037
0.10 0.023 0.015 0.016
Absorbance of control =0.524
Formula= absorbance of control – absorbance of sample/absorbance of control*100
Concentration(mg/ml) % Inhibition
Leaf Bark Ascorbic acid
0 0 0 0
0.02 73.28 75.57 41.60
0.04 77.29 78.24 75.38
0.06 94.27 85.68 85.68
0.08 94.84 92.93 92.93
0.10 95.61 96.94 96.94

DPPH % free radical inhibition Vs concentration mg/ml

Bar diagram of DPPH radical scavenging activity
5.13. ANTIOXIDANT ACTIVITY OF COMPOUNDS BY USING HYDROGEN PEROXIDE METHODS
Table 5.19: Absorbance of different derivatives and standard with hydrogen peroxide
Concentration
(mg/ml) Absorbance of isolated compound and standard with H2O2
Leaf Bark Ascorbic acid
0 0 0 0
0.02 0.445 0.430 0.530
0.04 0.325 0.365 0.460
0.06 0.295 0.285 0.375
0.08 0.152 0.147 0.240
0.10 0.070 0.085 0.090
Absorbance of control =0.920
Formula= absorbance of control – absorbance of sample/absorbance of control*100
Concentration(mg/ml) % Inhibition
Leaf Bark Ascorbic acid
0 0 0 0
0.02 51.63 53.26 42.39
0.04 64.67 60.32 50.01
0.06 67.93 69.02 59.23
0.08 83.47 84.02 73.91
0.10 92.39 90.07 90.21

H2O2 % free radical inhibition Vs concentration mg/ml

Bar diagram of H2O2 radical scavenging activity
Antifungal activity
Screening results of the newly isolated compounds as antifungal activity

Figure 5.33: Preparation of agar plate

Figure 5.37: Growth of Candida albicans in SDA media

Figure 5.38 & Figure 39 : Zone of inhibition of Candida albicans

Zone of inhibition after 24hrs in concentration µg/ml (mm)
Isolated compound No. Gram +ve Fungi Standard (Itraconazole)
Aspergillus Niger 30
Sample 1 26 Sample 2 20 Sample 3 16
Discussion
From the Table.. of physiochemical parameters the Loss on Drying of Powdered leaves and bark of Sapindus mukorossi was found to be 13.61% w/w in leaves and 12.09%w/w in bark and it is an important parameter to minimize the decomposition of drug either due to chemical change or microbial contamination.

From the Table.. of extractive values by soxhlet method of the hexane soluble value indicate the presence of and the value found to be___ for leaves and ____for bark, the chloroform soluble value indicate the presence of and the value found to be___ for leaves and ____for bark, the ethyl acetate soluble value indicate the presence of and the value found to be___ for leaves and ____for bark, the methanol soluble value indicate the presence of and the value found to be___ for leaves and ____for bark, the water soluble value indicate the presence of and the value found to be___ for leaves and ____for bark.

From the Table 13 of extractive values by cold maceration process it was found that the hydro alcoholic extractive value for leaves was__ and for bark ___.

From the Table 14 of total tannin content determination, the amount of total flavonoid in the leaves and bark of Sapindus mukorossi was determined from a regression equation y=0.0045x-0.0028 R2=0.9914 and from this equation concentration of the sample was calculated.

From the Table 14 of total flavonoid content determination, the amount of total flavonoid in the leaves and bark of Sapindus mukorossi was determined from a regression equation y=0.0045x-0.0028 R2=0.9914 and from this equation concentration of the sample was calculated.

From the Table 14 of total flavonol content determination, the amount of total flavonoid in the leaves and bark of Sapindus mukorossi was determined from a regression equation y=0.0045x-0.0028 R2=0.9914 and from this equation concentration of the sample was calculated.

From the Table 14 of total phenolic content determination, the amount of total flavonoid in the leaves and bark of Sapindus mukorossi was determined from a regression equation y=0.0045x-0.0028 R2=0.9914 and from this equation concentration of the sample was calculated.

From the Table 14 of total proanthocyanidins content determination, the amount of total flavonoid in the leaves and bark of Sapindus mukorossi was determined from a regression equation y=0.0045x-0.0028 R2=0.9914 and from this equation concentration of the sample was calculated.

From the Table ___of qualitative estimation of phytoconstituents by preliminary phytochemical screening it was found that the various extracts of powdered leaves and bark of Sapindus mukorossi contains secondary metabolites of plants i.e., carbohydrates, flavonoids, saponins, steroids and triterpenoids,
Leaves and bark have been screened for in-vitro antioxidant and antifungal activities. The free radical scavenging activities of leaves and bark were done by DPPH radical scavenging method and Hydrogen peroxide radical scavenging method. Figure 5. ……… shows the DPPH radical scavenging activity and Hydrogen radical scavenging activity of leaves and bark with respect to Ascorbic acid, which were used as reference compound in the study. For DPPH radical scavenging activity, the compounds were prepared at the concentration ranges from (0.02-0.10 mg/ml). It was observed from the study that DPPH radical scavenging activity of the compounds was significantly lower than that of ascorbic acid. As the concentration of compounds increases the percentage inhibition of compounds increases. For Hydrogen peroxide radical scavenging activity, the compounds were prepared at the concentration ranges from (0.02-0.10 mg/ml). It was observed from the study that Hydrogen peroxide radical scavenging activity of the compounds was significantly lower than that of ascorbic acid. As the concentration of compounds increases the percentage inhibition of compounds increases. Compound A1 shows maximum activity against both DPPH and H2O2 radical.

The isolated compound of leaves and bark extract of Sapindus mukorossi have been screened for antifungal activity using agar disc diffusion method by measuring the zone of inhibition in mm. Itraconazole (µg/ml) was used as standard drug for antifungal activity.

The compounds were screened for antifungal activity against A.niger in subourand’s dextrose agar medium.

These sterilized agar media were spread with the help of sterilized loop. The disc of 6mm diameter was used. All the isolated compounds (?g/ml) were dipped in disc for diffusion for 1hr, DMSO was used as solvent for all compounds and as control. These plates were incubated at 28?C for 48 hr for antifungal activity respectively. The zone of inhibition were observed around the disc after incubation and measured.

CHAPTER VII
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