Chapter 3 - Shodhganga

Chapter 3 - Shodhganga

Chapter 3 Materials & Methods 3 MATERIALS AND METHODS 3.1 Plant material Two plants of Andrographis paniculata were obtained as a gift from Forest ...

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Chapter 3

Materials & Methods

3 MATERIALS AND METHODS 3.1 Plant material Two plants of Andrographis paniculata were obtained as a gift from Forest Research Institute (FRI), Dehradun, Uttarakhand, India in the month of June 2009. Plant specimen is indexed in FRI as No. Dis. /2011-Bot-15-1(Rev.Gen.)/392.After one complete life cycle of the plants, fully matured seeds were collected and sown during last week of the month of June 2010. Crop was grown as per cultivation practices developed by Pandey and Mandal [169]. Whole plant material at different growth stages of life cycle and different parts of plant i.e. leaves; stem, flowers, root and whole plant material etc were collected, dried in shade and stored for further analysis. Whole plant material and only leaves of self grown Andrographis paniculata were also harvested at different growth stages of plant in one year life cycle. Leaves and whole plant material of Andrographis paniculata grown in natural environment (wild) from different locations were also collected, dried under shade, powdered using blender and stored in air tight bottles, for further analysis.

Loss on drying, Ash Values and evaluation of extractive value (%w/w) of all air dried samples were carried out. Extraction of all above samples was carried out using different solvents. These extracts were used for evaluation of chemical properties and spectral analysis.

3.1.1 Samples of whole plant material at different stages of life cycle Whole plant materials of Andrographis paniculata after 30, 60, 90,110and 130 days of plantation were collected and named as: Sample A-30 days. Sample B-60 days. Sample C-90 days. Sample D-110 days (after 110 days of plantation i.e., just before flowering). Sample E-130 days (At maturity of the crop i.e., bearing flowers, fully matured seed capsules etc.)

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3.1.2 Samples of different parts of plant Different parts of Andrographis paniculata were collected at 110 days and named as: Sample F - Leaves. Sample G - Whole plant material (drug). Sample H - flowers. Sample I - seed capsule. Sample J - Roots. Sample K - Stems.

3.1.3 Samples of different locations Leaves and whole plant material of Andrographis paniculata grown in natural environment (wild) from different locations were used as samples with self cultivated Andrographis paniculata. Samples of different location variation were named as – Sample LW

- Dehradun self growned whole plant material.

Sample LL

-Dehradun self growned Leaves.

Sample MW - FRI, -Dehradun (Uttarakhand) whole plant material. Sample ML

- FRI, -Dehradun (Uttarakhand) Leaves.

Sample NW

- Patanjali Ayurved, Haridwar (Uttarakhand) whole plant material.

Sample NL

- Patanjali Ayurved, Haridwar (Uttarakhand) Leaves.

Sample OW

- Selaqui Dehradun (Uttarakhand) whole plant material.

Sample OL

- Selaqui Dehradun (Uttarakhand) Leaves.

Sample PW

- Balawala (Uttarakhand) whole plant material.

Sample PL

- Balawala (Uttarakhand) Leaves.

Sample QW

- Himalaya Drug (Uttarakhand) whole plant material.

Sample QL

- Himalaya Drug (Uttarakhand) Leaves.

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240 days

Fig. 3.1: Different stages of life cycle of Andrographis paniculata

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Fig. 3.2: Different parts of plant of Andrographis paniculata

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Fig. 3.3: Andrographis paniculata grown in different locations

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3.2 Solvents used for extraction and fractionation Acetone, chloroform, ethanol, hexane, methanol, petroleum ether and water were used to prepare extracts of different samples and for fractionation. All solvents were of HPLC, HPTLC grade and all reagents were of analytical reagent grade. Water used for chromatographic separation was purified using Millipore mill purified system.

3.3 Chemicals and reagents used All reagents and chemicals used were of commercial grades. Labs of Graphic Era University, Dehradun, Uttarakhand, Himalaya Herbal Healthcare, Dehradun, Uttarakhand, and Windlass biotech Ltd, Dehradun, Uttarakhand were used for whole research work. 

Standard Andrographolide

Andrographolide standard 99.8% (Product code A009, Lot no. T11B001) was procured from Natural Remedies, Bangalore. It was used to check the purity of isolated Andrographolide and to compare the results of HPTLC and HPLC.

3.4 Study of the powder materials (drug) The whole plant materials of Andrographis paniculata were dried under shade and were powdered using a homogenizer and whole powder was considered as drug. Various chemical tests were performed according to conventional methods given in Lab manuals of Pharmacognosy by Lala, P.K., Text Book of Pharmacognosy by Wallis T.E and by Kokate[170-172].

3.4.1 Organolaptic properties Organolaptic Properties of drug were examined according to conventional methods given in Text Book of Pharmacognosy by Kokate [172]. Green powder was treated with some acidic, basic and neutral, routinely used reagents and characteristic changes were observed.

3.4.2 Fluorescence behavior The Fluorescence behavior of the powder of Andrographis paniculata as such and after treating with some chemical reagents was determined according to the methods of Chase and Pratt [173].

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The dried powder sample was treated with various chemical reagents like H2SO4, HCl, HNO3 etc. The mixture was mixed well, allowed to stand for few minutes and filtered. The filtrate was examined under both U.V. and visible light. Fluorescence characteristics of the powder were observed in day light as well as in ultraviolet radiation at 254nm and 366nm. The solvents used for the extraction procedure were Acetone, chloroform, ethanol, hexane, methanol, petroleum ether and water. After extraction all extracts were observed in day light as well as in ultraviolet (UV) light at 254nm and 366nm and colour was noted.

3.5 Preparation of extracts and fractionation Extracts of different samples were prepared according to methods developed by Wagner H et al., Khandelwal KR. and Mukherjee PK. [174]-[176].Step by step extraction using different solvents according to polarity was used for fractionation and direct extractions of different samples with different solvents were carried out to find yield difference of contents.

3.5.1 Extraction using soxhlet apparatus The solvents used for the extraction procedure were Acetone, chloroform, ethanol, hexane, methanol, petroleum ether and water. About 50gm of dried plant powder of each sample was extracted with 250 ml of the extraction solvent using soxhlet apparatus. The extracts were concentrated to dryness to yield crude residue. The extracts were autoclaved, labeled and stored at 4°c in air tight bottles. These residues were used for HPTLC, HPLC, Phytochemical, anti microbial activity against tested organisms, anti-oxidant activity and for other tests. Best yield of residue was obtained in methanol hence for fractionation methanolic extracts was used. Firstly HPTLC was used for fractionation and then HPLC. For HPTLC only methanolic extracts of different samples were used. For HPLC besides dried extracts fresh methanolic extracts of different samples were also prepared according to IP2010 Vol. 3[177]. For anti-oxidant activity the shade dried coarse powder of the leaves of Andrographis paniculata was extracted using ethanol, methanol and distilled water. Hot and Cold methanol extraction was also carried out besides extraction using soxhlet apparatus.

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Fig. 3.4: Extraction of different samples of Andrographis paniculata

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3.5.2 Hot methanol extraction The shade dried coarse powder of different samples of leaves, stems, flower, root, seed etc. and whole plant material of Andrographis paniculata (50gm each) was packed well in soxhlet apparatus separately and was subjected with methanol by continuous hot extraction for about 24hrs. The extracts were filtered through What man filter paper and concentrated on a water bath. The final concentrated extracts were stored at -180c in labeled sterile bottles.

3.5.3 Cold methanol extraction The shade dried coarse powder of all samples of Andrographis paniculata (50gm each) was kept in stoppered flask and was macerated with 250ml of methanol for 24-48 hrs with frequent stirring. Then the extracts were filtered through what man filter paper and concentrated under air. Obtained extracts were stored at -18 °C in labeled sterile bottles. The above prepared hot and cold methanol extracts were used for HPTLC, HPLC, phytochemical and anti-microbial investigation.

3.6 Implementation of a national herbal formulary for more effective and proper use of Andrographis paniculata National herbal formulary gives a specific guideline for the chemical investigation of a medicinal plant using modern techniques available. The national drug formulary is to provide guidance for the traditional medicine practitioners and to ensure that the knowledge about the native plants and their profiles are not lost in time for the benefit of the young generation and the future generation to come. The herbal formulary comprises of the following basic structure about the medicinal plant with additional scientific data and information [178]. 

Name of plant (genus, species, authority and family).



Plant part(s) used for disease conditions.



Quality requirement (percentage of active principle, if known).



Purity (foreign matter, adulterant, other contaminant).



Physicochemical analysis:



Qualitative (including micro-chemical tests).

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Quantitative ( if major active ingredients are known, analytical procedures e.g. thin layer chromatography, high pressure liquid chromatography, chemical fingerprinting techniques should be developed to authenticate the plant species or varieties, quantify individual and total constituents.

3.7 Physico-chemical properties of the drug Physico-chemical constants like melting point, pH values, ash values, loss on drying and extractive values were determined as per method described in Indian Pharmacopoeia [177] .Three ash values including Total Ash (TA) Value, Acid Insoluble Ash (AIA) and Water Soluble Ash (WSA) was determined. Water-soluble extractives, Alcohol soluble extractives and Pet ether soluble extractives were also determined.

3.7.1 Melting point of the drug The melting point of the drug was determined by melting point apparatus .The shade dried coarse powder of the leaves and whole plant material of Andrographis paniculata was examined thrice for melting point and average values of triplicates were calculated.

3.7.2 pH values The shade dried coarse powder of the leaves and whole plant material (drug) of Andrographis paniculata was examined thrice. pH value of drug was determined by pH meter (QC/Micro/ pH 01Sr No.391505). Average value of triplicates was considered.

3.7.3 Determinations of ash values Ash is the inorganic residue left after ignition at 650 - 700 °C. The ash content is an approximate measure of the mineral content and other inorganic matter in biomass. The ash content is used in conjunction with other assays to determine the total composition of biomass samples. Methods used were according to Harborne JB., Phytochemical methods [179]. Samples of whole plant material (drug) at different stages of life cycle and samples of different location were used for the determination of ash values.

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Apparatus 

Silica crucibles



Furnace - An electric furnace was used for igniting the samples. Furnace was fitted with an indicating pyrometer, so that the desired temperature can be maintained.



Analytical balance - sensitive to 0.1 mg.



Desiccator.



Drying oven - with temperature control of 105 ± 2°C.

3.7. 3. 1 Total ash (TA) value This value was determined using a minimum of 2.0 – 3.0 g of material in a furnace heated gradually to the ignition temperature of 650 - 700 °C. Accurately 2 to3gm of air–dried samples of Andrographis paniculata were weighed in a tared silica dish and incinerate at a temperature not exceeding 700 °C until ash free from carbon is obtained. Then it was cooled and weighed. The process was repeated until at least two consecutive constant weights were obtained. The results are expressed as range or mean value ± standard deviation. The percentage of ash was calculated with reference to the air - dried drug. Same procedure was repeated for remaining samples.

3.7. 3. 2 Determination of acid insoluble ash (AIA) Above obtained ash was boiled with 25ml of 2M Hydrochloric acid for 5min, the insoluble matter was collected in a Gooch crucible or on an ash less filter paper, washed with hot water, ignited and cooled in desiccators. Then it was weighed. The percentage of acid insoluble ash was calculated with reference to the air – dried drug.

3.7.3. 3 Determination of water soluble ash (WSA) Ash was boiled for 5 min. with 25 ml of water; the insoluble matter was collected in a Gooch crucible or on an ash less filter paper, washed with hot water, and ignited for 15 min at a temperature not exceeding 450 °C .The weight of insoluble matter was subtracted from the weight of the ash, the difference in weight represent the water-soluble ash. The percentage of watersoluble ash was calculated with reference to the air-dried drug.

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Formula used for the calculation of percentage of all three types of Ash Values is Ash% = Loss in Weight x 100 W W=Weight of air – dried drug.

The results of Total Ash (TA) Value, Acid Insoluble Ash (AIA) and Water Soluble Ash (WSA) were recorded.

3.7.4 Loss on drying (LOD) Methods used were according to practical book for evaluation of Phytopharmaceuticals by Brain KR. et al., [180]. Samples of whole plant material at different stages of life cycle and samples of different location were used. Loss on drying of the air-dried samples of Andrographis paniculata was analyzed. This was carried out using a minimum of 0.5– 1.0 g of material. Accurately weighed quantity of sample was taken in a tared glass bottle and initial weight was taken. The sample was heated in a Lindberg/Blue M gravity-convention oven maintained at 105-110 °C, for 3 h, after which the sample was allowed to cool to room temperature in desiccators, and subsequently weighed. The time interval from the oven to point of weighing was usually about 30 minutes. This procedure was repeated until a constant weight was obtained. Similar process was carried out for all samples. Loss on drying (%) = loss in weight x 100/ w Where w = weight of the leaf powder in gms. The results are expressed as a range or as mean ± standard deviation.

3.7.5 Determination of extractive values Methods used were according to Kokate CK. et al., given in the Text Book of Pharmacognosy 18th ed. [172]. Whole plant material at different stages of life cycle and samples of different location were used for the determination of extractive values. Three extractive values for various solvents of air-dried sample were evaluated. i) Water-soluble extractives. ii) Alcohol soluble extractives. iii) Pet ether soluble extractives.

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3.7.5. 1 Evaluation of water extractive values About 5 g of accurately weighed coarsely powdered, air-dried sample of Andrographis paniculata was transferred into a glass-stoppered, 250-ml reflux conical flask, followed by the addition of 50 ml of boiled water. The flask was well shaken, and allowed to stand for 10 minutes. It was cooled and filtered. Filtrate was transferred to an evaporating dish, which was 7.5 cm in diameter; the solvent was evaporated on water bath, allowed to dry for 30 minutes, finally dried in an oven and residue was weighed. Percentage of water-soluble extractives was calculated with reference to the air-dried drug. Same procedure was repeated for remaining samples.

3.7. 5 .2 Evaluation of alcohol soluble extractive values 5 grams of dried samples of Andrographis paniculata were macerated with 100 ml of Alcohol in a closed flask, shaking frequently during the first 6 hours and allowed to stand for 18 hours separately. Thereafter, it was filtered rapidly taking precaution to minimize the loss of methanol. Evaporated 25ml of filtrate to dryness in a tared flat bottom shallow dish dried at 105 ºC and weighed. Percentage of Alcohol soluble extractive was calculated with reference to the air-dried samples. Similar process was carried out for other samples of Andrographis paniculata.

3.7.5.3 Evaluation of Pet. ether soluble extractive values 5 grams of dried samples

of Andrographis paniculata were macerated with 100 ml of pet ether

(40-60oC) in a conical flask, shaking frequently during the first 6 hours and allowed to stand for 18 hours separately. Thereafter, it was filtered rapidly taking precaution against loss of pet ether. Evaporated 25ml of filtrate to dryness in a tared flat bottom shallow dish dried at 105 ºC and weighed. Percentage of pet ether soluble extractive was calculated with reference to the air-dried samples. For remaining samples same procedure was repeated.

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3.8 Physiological variation Change in chlorophyll, carotenoids and anthocyanins content with the increasing days of plantation of plant (age related) is very important because it directly relates with effectiveness and the quality of medicinal plant. Two types of chlorophyll are found in plants - chlorophyll a and chlorophyll b. Both chlorophylls absorb light most strongly in the red and violet parts of the spectrum. Chlorophyll is responsible for photosynthesis. Carotenoids are organic pigments that are found in the chloroplasts and chromoplasts of plants. Carotenoids in general absorb blue light. Carotenoids have many physiological functions. Given their structure, carotenoids are efficient freeradical scavengers, and they enhance the vertebrate immune system. Anthocyanins are watersoluble vacuolar pigments that may appear red, purple, or blue depending on the pH. They are odorless and nearly flavorless. Anthocyanins can be used as pH indicators because their color changes with pH. Anthocyanins also act as powerful antioxidants. The experiment was conducted for the young, mature and fully mature plants leaves of selected populations of Andrographis paniculata. Young (60 days old), mature (130days old) and fully mature (240 days old) plants were selected for analyzing age-related changes in chlorophyll, carotenoid and anthocyanin content among different samples. Fresh leaves of different samples of Andrographis paniculata were used. Fig. 3.5 show variation in colour of plant leaves during life cycle.

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Fig. 3.5: Leaves of Andrographis paniculata at young and matured stages showing colour variations.

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3.8.1 Variation in chlorophyll and carotenoids Chlorophyll and carotenoids were extracted using 80% acetone as it was found best method [61,181]. 5 gm fresh leaves were homogenized in 40 ml of 80% acetone and then the extract was centrifuged at 1000 rpm for 2 min in Remi (S/N0. BCBR-2182) cooling centrifuge. The supernatant was collected and used for measuring optical density at 490, 645 and 663nm using UV10 Thermo scientific visible spectrophotometer(S/No. HEDM218008).Whole experiment was repeated thrice.

3.8.2 Variations in anthocyanin content For estimating variation in anthocyanin content of leaves, 0.5 gm leaf tissue was homogenized in 4 ml of methanol containing 1.0 M HCl and was kept at 4 °C for 4 h. The homogenate was filtered and centrifuged at 10,000 rpm for 30 min and the absorbance of the supernatant was measured at 530 nm after suitable dilution [182].

3.9 Phytochemical estimation Extracts of different samples of Andrographis paniculata were subjected to various bio-chemical tests to determine the active constituents’ present in the extracts. The various phytochemical compounds detected are known to have beneficial importance in medicinal science. Flavonoids have been referred to as “nature’s biological response modifiers”. They show anti-allergic, antiinflammatory, anti-microbial and anti-cancer activities. Phenols, the aromatic compounds with hydroxyl groups are wide spread in plant kingdom. Phenols are said to offer resistance to diseases and pest in plants. Saponins protect the plant against microbes and fungi. Alkaloids usually have marked the physiological action on human or animals. Glycosides serve as defense mechanisms against predation by many micro-organisms, insects and herbivores. Preliminary Phytochemical analysis of Andrographis paniculata was carried out for medicinally active constituents like flavonoids, phenols, alkaloid, glycosides, saponins and tannins.

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3.9.1 Preliminary phytochemical tests The following tests as described by Harborne JB., and Buerton Jean were carried out for the herb powder or extract of herb [179], [183]. (1) Tests for glycosides: Glycosides are compounds which upon hydrolysis give rise to one or more sugars (glycones) and a compound which is not a sugar (aglycone or genine). The test solution was prepared by dissolving extract in alcohol 90% or aqueous alcoholic solution. a) Baljet’s test: The test solution treated with sodium picrate gave yellow to orange colour. b) Keller-Killiani test: The test solution was treated with few drops of ferric chloride solution and mixed, and then sulphuric acid containing ferric chloride solution was added, reddish brown coloration at the junction of two layers and the bluish green color in the upper layer was observed. c) Fehling’s test for reducing sugars: To about 10 mg of the extract in test-tube was added 2 ml of water, followed by 0.2 ml of 0.1 M HCl, to effect hydrolysis of the glycosides. For the control, 0.2 ml of water was used instead of the acid. The mixture was heated in a boiling water bath to accelerate dissolution, and then left further in the bath for 5 min. Subsequently, 1 ml each of Fehling's solutions A and B were added while continuing to shake the mixture in the bath for 10 min. A brick-red precipitate indicated the presence of reducing sugars, formed from the hydrolysis of glycosides.

(2) Frothing test for saponins: A pinch of the aqueous extract was added to 5 ml of water and warmed until dissolved. The solution was subsequently shaken vigorously to generate froth, and then allowed to stand. A rich froth persisting for 10 minutes indicates the presence of saponins. (3) Ferric chloride test for phenols: Iron (III) ions form strongly coloured complexes with several organic compounds including phenol. The colour of the complexes varies from compound to compound. The reaction with iron (III) chloride solution can be used as a test for phenol. In aqueous extract of plant very dilute solution of iron (III) chloride was added. An intense violetpurple solution formed. (4) Ferric chloride test for tannins: About 10 mg of the aqueous extract was vigorously shaken with 3 ml of warm water until dissolved. This was followed by the addition of 1 ml of 15 % ferric chloride test solution. A blue-green coloration formed.

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(5)Tests for alkaloids:

Alkaloids are basic nitrogenous compounds with definite physiological

and pharmacological activity. Most alkaloids are precipitated from neutral or slightly acidic solution by Mayer’s reagent.

a) Mayer’s test: The test solution was prepared by dissolving extracts in dilute HCl acid. Test solution with Mayer’s reagent (potassium mercuric iodide) gives cream coloured precipitate.

b) Dragendorff’s test: Dragendorff’s reagent consisting of two solutions: Solution A - 1.7 g basic bismuth nitrate in 100 ml water/acetic acid (4:1), and Solution B - 40.0 g potassium iodide in 100 ml of water, was prepared. The two solutions were mixed as follows to yield 100 ml of Dragendorff’s reagent: 5 ml Solution A + 5 ml Solution B + 20 ml acetic acid + 70 ml water. The test was carried as follows: About 20 mg of the air-dried herb was extracted with 20 ml of methanol by shaking and heating over a boiling water bath. The extract was subsequently filtered and allowed to cool. Each 2 ml of the filtrate in a test-tube was treated with 2 ml of Dragendorff's reagent. The development of an orange-brown precipitate presumptively indicated the presence of alkaloids.

(6) Tests for flavonoids: The flavonoids are all structurally derived from the parent substance called flavones. Flavonoids which occur in the form of free, as well as, bound to sugars called glycosides. For this reason, when analyzing flavonoids, it is usually better to examine the flavonoids in hydrolyzed plant extracts. Preparation of test solution: To small amount of extract equal volume of 2M hydrochloric acid was added and heated the test tube for 30-40 min at 1000C.It was allowed to cool, filtered and extracted with ethyl acetate. The ethyl acetate extract was concentrated to dryness; test was followed for flavonoids to ethyl acetate fraction by dissolving the residue with ethyl acetate. a) Shinoda test: Test solution with few fragments of magnesium ribbon and conc. hydrochloric acid shows pink to magenta red colour. b) Zn/Hcl reducing test: Test solution with zinc dust and few drops of hydrochloric acid shows magenta red colour. Change in colour and physical state was observed.

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3.9.2 Phytochemical screening of different extracts Phytochemical screening of various extracts of Andrographis paniculata at two different stages of life cycle of crop (sample D and E) and for only leaves (sample LL) was determined. Four solvents, petroleum ether, acetone, chloroform and methanol were used to prepare extracts of sample according to method describe in 3.5.1 section of this chapter. All extracts of Andrographis paniculata were subjected to various bio-chemical tests to determine the active constituents present in the extracts. The thin layer chromatography study of various extracts was carried out to confirm the presence of phytoconstituents. A Camag HPTLC system equipped with a TLC Scanner 3, and Win cats and integration software 1.4 was used for the detection of Phytochemicals present.

3.9.3 Phytochemical screening of hot and cold methanol extracts As methanol was found to be best solvent among all tested solvents the hot and cold methanol extracts of leaves and whole plant material of Andrographis paniculata were also screened for phytochemical analysis. Extracts of sample were prepared according to method describe in 3.5.2 and 3.5.3 section. Only leaves (LL) and whole plant material (LW) of Andrographis paniculata were subjected to various bio-chemical tests and thin layer chromatography study to determine the active constituents present in the extracts.

3.9.4 Quantitative analysis of phytochemical constituents Crude extracts of whole plant (drug) and leaves of Andrographis paniculata have been investigated for quantitative analysis. Aqueous, methanolic and ethanolic extracts were tested for their tannin content, total phenolic and total flavonoids contents. These total phenolic and total flavonoids contents of leaves were also compared with total phenolic and total flavonoids contents of extracts of leaves of Tinospora cordifolia. Tinospora cordifolia, which is known by the common name Guduchi, is an herbaceous vine of the family Menispermaceae indigenous to the tropical areas of India, Myanmar and Sri Lanka. Andrographis paniculata and Tinospora cordifolia both have many common medicinal properties and anti-oxidant activity is important one of them. Anti-oxidant activity of leaves of Andrographis paniculata was compared with antioxidant activity of leaves of Tinospora cordifolia and to increase the authenticity of results total phenolic and total flavonoids contents of leaves of both plants were also compared as they affect the value of anti-oxidant activity.

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Collection of plant material: Whole plant (drug) and fresh and healthy leaves of Andrographis paniculata and Tinospora cordifolia leaves were collected at the same time and washed thoroughly with distilled water and dried in shade for seven days followed by grinding and then coarse powder stored in air tight bottles.

Preparation of extracts: The shade dried coarse powder of whole plant (drug), leaves of Andrographis paniculata and Tinospora cordifolia (50gm each) was extracted using 250ml of the extraction solvent using soxhlet apparatus. The solvents used for the extraction procedure were ethanol, methanol and distilled water. The extracts were concentrated to dryness to yield crude residues. The extracts were auto-claved and stored at 4°C, until further use.

3.9.4.1 Tannin content Quantity of tannin was determined by using the spectrophotometer method.0.5g of plant sample of Andrographis paniculata was weighed into 50ml plastic bottle. 50ml of distilled water was added and stirred for 1h.the sample was filtered into a 50ml volumetric flask and made up to mark. 5ml of the filtered sample was then pipette out into test-tube and mixed with 2ml of 0.1m FeCl 3 in 0.1m HCl and 0.008m K 4 Fe(CN) 6 .3H 2 O. The absorbance was measured with a spectrophotometer at 395nm wavelength within 10minutes.Same procedure was repeated for rest two extracts. 3.9.4.2 Total phenolic content (TPC) Total soluble phenolic content of Andrographis paniculata and Tinospora cordifolia was estimated by Folin-Ciocalteu’s reagent method using gallic acid as a standard phenolic compound [184]. Each solvent extracted solution (0.3ml in triplicate) was mixed with 1.5 ml of 10% freshly prepared Folin-Ciocalteu’s reagent and after 3 min 1.2 ml of 7.5% (W/V) sodium carbonate was added and mixed thoroughly. The tubes were placed in boiling water for one minute, cooled and the absorbance was measured at 650 nm in a spectrophotometer against a reagent blank. The concentrations of the total phenolic compounds in the extracts were obtained by extrapolating the absorbance of gallic acid on standard gallic acid graph. The experiment was repeated thrice and concentration of total phenols was expressed as mg/g of dry extract.

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3.9.4.3 Total flavonoid content The total soluble flavonoid content was estimated by aluminium chloride colorimetric method for different extracts of Andrographis paniculata and Tinospora cordifolia [185]. 0.5ml of stock solution (1g/ml) of the extract, 1.5 ml methanol, 0.1ml potassium acetate (1M) was added to reaction test tubes and volume was made up to 5 ml with distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm and compared with quercetin, a positive control. Total flavonoid content was expressed as mg/g of dry extract after aggregating the values of all three experiments.

3.10 Anti-oxidant activity The use of natural plant products with potential anti-oxidant activities are now the centre of importance because synthetic anti-oxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), as due to its instability has become the suspected carcinogenesis promoters. Extracts of many plants (for example Tinospora cordifolia) and natural products have anti-oxidant and free radical scavenging properties. Free radical oxidative stress, usually resulting from deficient natural anti-oxidant defenses [186-188]. Quantitative analysis of phytochemical constituents of drug and only leaves of Andrographis paniculata show presence of phenolic and flavonoids contents which are responsible for antioxidant activities. Therefore, it is beneficial to conduct this study on Andrographis paniculata grown in Uttarakhand. In this study, the aqueous, ethanol and methanol extracts of whole plant (drug) of Andrographis paniculata were analyzed for their potential anti-oxidant activities. Finally anti-oxidant activities of Andrographis paniculata leaves compared with leaves of Tinospora cordifolia(well known anti-oxidant plant). DPPH radical scavenging activity was compared with synthetic anti-oxidant butylated hydroxyl anisole (BHA) and total reducing power was compared with ascorbic acid. This study provides a better understanding of the anti-oxidant profile of Andrographis paniculata.

Chemicals: All chemicals used including the solvents, were of analytical grade. 1, 1-diphenyl-2picryl hydrazyl (DPPH), butylated hydroxyl toluene (BHT) and butylated hydroxyl anisole (BHA),

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gallic acid, trichlorocetic acid (TCA), Tris-HCl buffer (pH 7.4); phosphate buffer, potassium ferricyanide, ferric chloride and ascorbic acid were used.

Extracts: Samples of whole plant material (drug) of Andrographis paniculata were extracted in absolute ethanol, methanol and Milli-Q pure grade water as solvents. Only leaves were also extracted in absolute ethanol, methanol and water. The extracts were then filtered through a Whatman filter paper No. 1. The ethanol and methanol were evaporated using a rotary evaporator under vacuum and the concentrated, aqueous extracts were dried. The stock extracts were stored at -80ºC and dissolved in the respective solvents to the appropriate concentrations on the day of use. Different concentrations (0.1 - 0.9 mg/ml) of extracts were prepared from the resultant crude of aqueous, ethanol and methanol extracts of Andrographis paniculata to determine antioxidant activity. Fresh and healthy leaves of Tinospora cordifolia were also collected at the same time and washed thoroughly with distilled water and dried in shade for seven days followed by grinding. Aqueous, ethanol and methanol extracts of different concentrations (0.1 - 0.9 mg/ml) were prepared.

Fig. 3.6: Plants of Andrographis paniculata and Tinospora cordifolia growing in natural environment.

Fig. 3.7: Shaded dried leaves of Andrographis paniculata and Tinospora cordifolia.

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3.10.1 DPPH radical scavenging activity The hydrogen donating ability of each extract in the presence of DPPH (2, 2-diphenyl-1picrylhydrazyl) stable radical was examined according to method developed by Blois and Cao et al [189], [190]. All extracts were diluted with vehicle control to final extract concentration (0.1 to 0.9mg/ml). 0.1 ml of test sample at different concentration (0.1 - 0.9 mg/ml) was mixed with 0.9 ml of Tris-HCl buffer (pH 7.4); then 1 ml of DPPH (500 μM in ethanol) was added. The mixture was shaken vigorously and left to stand for 30 min. The absorbance of ethanolic DPPH tincture from DPPH* (violet color) to DPPH (clear) was measured at 517 nm in a spectrophotometer and compared with synthetic anti-oxidant butylated hydroxyl anisole (BHA). The experiment was repeated thrice. The percentage of DPPH scavenging was calculated using the following formula: % scavenging = [(A control - (A sample – A sample blank / A control))] x 100

3.10.2 Total reducing power The reducing power of the extracts was performed as described by Yen and Duh [191].Different concentrations of extracts of all samples were prepared ranging from 0.1 to 0.9mg/ml. An ascorbic acid stock solution was prepared from which various concentrations were prepared. Various extracts (0.1 - 0.9 mg/ml) were mixed with phosphate buffer (500 μl, 20 mM, pH 6.6) and 1% potassium ferricyanide (500 μl), and incubated at 50°C for 20 min; 500 μl of 10% trichloro acetic acid (TCA) were added, and the mixture was centrifuged at 2500 rpm for 10 min. The supernatant was mixed with distilled water (1.5 ml) and 0.1% ferric chloride (300 μl) and the absorbance were measured at 700 nm in ultra-violet visible spectrophotometer. Ascorbic acid was used as a standard solution. Phosphate buffer used as blank solution. The experiment was repeated thrice. Increase in the absorbance of the reactions mixture indicated increase in the reducing power.

3.11 Antimicrobial activities Search for new antimicrobial agents is the need of today because of the development of microbial resistance to currently available antibiotics. Natural products from plants are being investigated as they are safe and effective. Plant Andrographis paniculata has been used against different microbes from a very long time in India and other countries. Hence, it was selected for

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antimicrobial screening.

The main objective was to evaluate the antimicrobial activity of

Andrographis paniculata extracts with different solvents. The antimicrobial activity of different extracts of Andrographis paniculata was analyzed by disc diffusion susceptibility methods. Another commonly method to determine antimicrobial susceptibility is the broth dilution assay used by Hess et al., [192]. This method is difficult because the compound be tested must be diluted several times for each sample, so it is time-consuming and is more expensive than the disc diffusion test. Therefore, the disc diffusion test was chosen in the antimicrobial activity test.

Antimicrobial activity of Andrographis paniculata extracts was evaluated with different solvents. The solvents used for the extraction procedure were Acetone, chloroform, hexane, methanol, petroleum ether and water. So far antimicrobial activities of Andrographis paniculata are reported with different extracts but comparison of antimicrobial activity at different stages of life, different Parts of Plant and comparison of extracts from different geographical sites were not made yet in best of my knowledge. Comparative study also conducted with standard antibiotic Norfloxacin, Ciprofloxacin and Ofloxacin. Antimicrobial screening was done using agar well diffusion method commonly called as cup plate method [193], [194].

3.11.1Samples used for antimicrobial activities Whole Plant material at different stages of life cycle, different parts of plant and samples of different location of Andrographis paniculata were used for antimicrobial activities screening.

Samples of whole plant material at different stages of life cycle: Samples A, B, C, D and E of whole plant material (drug) were screened to observed anti-microbial activities of different stages of life cycle of Andrographis paniculata. Samples of different parts of plant of Andrographis paniculata: For comparison of anti-microbial activity of different parts of Andrographis paniculata samples F, G, H, I, J and K were screened.

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Samples of different location: To observed anti-microbial activities of Andrographis paniculata of different locations,12 samples were screened. Samples were- LW, LL, MW, ML, NW, OW, OL, PW, PL, QW and QL.

3.11.2 Target microorganism The extracts of all samples were screened against Gram-positive and Gram-negative bacteria and fungal pathogen. The pathogenic strains used for testing the antimicrobial activity of Andrographis paniculata wereGram-positive- Staphylococcus aureus (MTCC- 737), Bacillus subtilis (MTCC 441) Gram-negative- Escherichia coli (MTCC-452), Pseudomonas aeuriginosa(MTCC 1688 ). Fungal pathogens- include Aspergillus niger (MTCC 1344) and Candida albicans (MTCC 227). Pathogenic strains used were obtained from stocks of Department of microbiology of Himalaya Herbal Healthcare, Dehradun, Uttarakhand, India and Windlass biotech Ltd, Dehradun, Uttarakhand, India.

3.11.3 Culture media Nutrients Agar media (Oxoid powder CM 3) and Potato dextrose broth were used. The bacterial test organisms were sub cultured at 37°C for 24h and maintained on nutrients agar media. Similarly the fungal strains were grown in potato dextrose broth at 37°C and maintained on potato dextrose agar slants at 4°c.

3.11.4 Equipments Sterile Petri dishes with diameter of 100mm, Electronic weighing

balance(S.No.-

183680/07,QC/Micro, Citizen),Hot plate &Magnetic Stirrer(S. No.-7861-ANA-JH-002,QC/Micro, Equitron) ,Stirred water bath(S. No.-8428AJG.79,QC/Micro, Equitron) Gallenkamp plus II incubator, Becton Dickinson 2ml sterilized syringes, autoclave(S. No.-7431FA.JH.045,Media Instrument Mfg. Co.),Laminar Air Flow Chamber and Cork borer of size 6.0mm.

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The experimental conditions (temperature and aeration) were maintained constant before the antimicrobial assay was carried out. Except micro pipette other glassware used like conical flasks, Petri dishes etc. autoclaved at 120 °C for 20 minutes by steam sterilizing. Micro pipette sterilized through chemical cleaner. Acetone, chloroform, hexane, methanol, petroleum ether and hydro, extracts of all samples of Andrographis paniculata were prepared according to method describe in section 3.5.1 and screened .Comparative evolution with standard antibiotics has been also performed.

3.11.5 Anti-microbial screening All extracts of all sample of Andrographis paniculata were screened by agar well diffusion method. 25 g of the nutrient agar was weighed and was suspended in some amount of distilled water. It was heated to dissolve completely. Slowly with constant stirring 600 ml water was added. Ph was adjusted 7.3+0.2 and volume was made up to 700 ml. It was boiled on hot plate. The agar was then sterilized by autoclaving at 121°C for 15 minutes after which it was stabilized in the New Brunswick Scientific (Edison N.J. USA) reciprocal water bath shaker model R76 at 45°C for 15 minutes.

The overnight culture grown was used for inoculation. About 100 µl of standardized microbial stock suspension of bacterial strains was thoroughly mixed with nutrient agar medium and stock suspension of fungal strains with potato dextrose broth and poured into sterile Petri plates by pour plate technique. When the nutrient agar medium solidifies, in each Petri plate four 6mm wells were made using sterilized Cork borer of size 6.0 mm.Then, 0.2 ml of each extracts and standard solution were placed in each hole separately. The plates were maintained at room temperature for 2 hr to allow the diffusion of the solution into the medium. All plates were incubated at 37°±1°C for 18 hr and after incubation; the size (diameter) of the inhibition zones was measured. Triplicates were maintained for each sample of the extracts respectively. For each microbial strain control were maintained where pure solvents were used. Apart from that, the comparative study also conducted with standard antibiotic Norfloxacin, Ciprofloxacin and Ofloxacin in par with the test drug Andrographis paniculata against pathogens. All the diameters of inhibition zone were measured in mm.

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Fig. 3.8: Equipments used for anti-microbial screening

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3.12 Identification of Andrographolide from Andrographis paniculata Extracts of different samples of Andrographis paniculata contain a mixture of bioactive and inactive chemical ingredients. Chemical composition of samples varies not only in different parts but also on growing region and the collection time. Therefore, efficient and effective methods of identification, extraction, standardizations, qualitative, quantitative and pharmacological testing’s are essential. To evaluate the therapeutic potential of the purified bioactive principles, method must be highly selective, in order to achieve the optimal outcomes. The main objective of this part of the study was to purify one of these major components (Andrographolide) from Andrographis paniculata, to analyze its quality and to calculate yield on weight to weight bases and exact percentage of Andrographolide. HPTLC and HPLC both are very effective methods and can be used for phytochemical profiling of whole plant, leaves and other parts of Andrographis paniculata and quantification of Andrographolide. Preliminary tests and confirmatory tests were conducted to confirm the presence of Andrographolide in Andrographis paniculata. Andrographolide standard 99.8% (Product code A009, Lot no. T11B001) was procured from Natural Remedies, Bangalore and was used as standard biomarker to confirm the results and to find correct percentage of Andrographolide.

3.12.1 Preliminary tests for Andrographis paniculata (colour Test) Preliminary Tests were carried out according to MPRI [195]. 1 g of Andrographis paniculata powder with 20 ml of ethanol was boiled in a water bath for 5 minutes. 300mg of activated charcoal was added, stirred and then filtered. The filtrate was used for the following two tests. Test (1)- The first test involved 2 drops of a 2% w/v solution of 3,5-dinitrobenzoic acid in ethanol and 2 drops of a 5.7% w/v solution of potassium hydroxide in ethanol being added to 0.5 ml of filtrate. Change in colour if occur noted. A specific change in colour indicated that the sample had active compounds. Test (2)- The second test involved 3-5 drops of the 5.7% w/v solution of potassium hydroxide in ethanol being added to 0.5 ml of filtrate until a red colour appeared. The filtrate was set aside for 10 to 15 minutes. Change in red colour if any to be noted.

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3.12.2 Confirmatory test To confirm that above obtained diterpene lactones obtained from Andrographis paniculata is Andrographolide the above filtrate was evaporated under reduced pressure until

fully dried.

About 5 mg of filtrate powder was dissolved in 5 ml of warm ethanol and 1 mg of standard Andrographolide was dissolved in 0.5 ml of warm ethanol separately. A pre coated plate of silica gel60 F254

aluminum sheets (10 x 10 cm) was used and the mobile phase used was: -

chloroform: methanol: ethyl acetate (8: 1.5: 1). 5 micro liters was used for each spot. TLC of the isolated samples was firstly detected by UV radiation (Electronic UV Trans illuminator, Quantum Scientific) and then confirmed by spraying with 2% w/v solution of 3, 5-dinitrobenzoic acid in ethanol and an excess of 5.7% w/v of potassium hydroxide in ethanol.

3.13 Isolation and purification of Andrographolide After confirmation that Andrographis paniculata contained Andrographolide, a modification of the method reported by Rajani et al [196]. was used to isolate Andrographolide from all samples of Andrographis paniculata. 200g whole dried crushed Andrographis paniculata and only leaves were extracted separately, twice with a 1:1 mixture of dichloromethane and methanol by cold maceration (one with 1 liter and then 0.2 liter of the maceration mixture) (1×1 liter and 1×0.2 liter). Each extraction was conducted for 6 hours with constant stirring. The extracts were filtered and the filtrate was concentrated at a temperature below 70°C to produce a green mass. The green mass was washed with toluene several times until most of coloured matter was removed from the residue. The toluene was completely removed from the residue by evaporation. The residue was dissolved in 60-70°C hot ethanol and filtered while hot. The filtrate was cooled in a refrigerator for crystallization. This process was repeated several times and results were noted. Further isolation of Andrographolide was confirmed by melting point, UV absorption spectrum, HPTLC and HPLC.

3.13.1 Melting point Melting point was determined using a melting point apparatus (FSE, England). Obtained melting point was compared with melting point of standard Andrographolide.

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3.13.2 UV maximum absorption spectrum UV spectrophotometry was used to determine the maximum absorption spectra within the range from 200 to 270 nm. Above purified Andrographolide was scanned in Shimadzu, UV-1700 Pharmaspec UV-vis-Spectrophotometer .The concentration of purified Andrographolide and standard Andrographolide (purchased from Natural Remedies, Bangalore, India 99.8%) was 25 μg/ml in ethanol.

3.14 Yield estimation of Andrographolide from Andrographis paniculata by high performance thin layer chromatography (HPTLC) Chromatography is a method in which the components of a mixture are separated on an adsorbent column in a flowing system which distributed between two phases. Liquid chromatography (LC) is chromatographic procedure in which the mobile phase is a liquid. There are many types of chromatography but all are based on the principle that the mobile phase carries the compounds to be separated and a stationary phase binds these compounds through intermolecular forces. Thin layer chromatography is employed to get TLC profile of various extracts and can be used in standardizing the raw drugs as well as the herbal formulations [197]. Ordinary TLC plates do not give better resolution particularly, when the herb contains many components similar to each other. Quantification of markers by conventional solvent extraction followed by calorimetric is laborious and expensive. HPTLC offers better resolution and estimation of active principles with reasonable accuracy in a shorter time [198].

Among the complex mixture of biologically active compound in Andrographis paniculata, Andrographolide can be used as an analytical marker compound to determine the quality of plant material of different sources. During crop improvement and drug analysis, a sensitive and accurate analytical method is required for the quantitation of important compound like Andrographolide, which is present in the plant. Although few methods such as gravimetric, colorimetric, spectrophotometric methods have been reported for the quantitative estimation of Andrographolide [199] but many of these methods are time consuming, not very precise and required multiple step

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extraction and purification [200].The present HPTLC method offers a sensitive, accurate and reliable method for routine detection and quantification of Andrographolide present in Andrographis paniculata [201]. High-performance thin-layer chromatographic (HPTLC) method has been developed for the simultaneous quantitative estimation of the biologically active diterpenoid Andrographolide in Andrographis paniculata. The present work focuses on development and validation of high performance thin layer chromatographic method for the determination of isolated major constituent in Andrographis paniculata extracts with an advantage of sensitive method with lower

limit of detection (LOD),as well as precise even at lower

concentration of isolated major compound in various samples of Andrographis paniculata.

Methanol was found to be the most appropriate solvent for the exhaustive extraction of Andrographolide derivatives [196]. It was confirmed in the lab during experiments hence methanol was used as a solvent. Sample Applicator System was used to apply accurate volume of samples in the form of band through micro syringe on the HPTLC plates. Accurate band size and gap between the bands can be maintained by using Sample Applicator System. Each developed band in the chromatogram can be scanned at single wave length and multiwave length in range of 200nm800nm by the help of TLC scanner. In this chromatogram scanned at 250 nm. Scanned chromatogram is obtained in the form of peaks i.e. each band has its own Rf value (retardation factor), area and height. Spectral pattern of peaks and chromatogram is used for identification and quantification of samples. The simplest method of identification of a chromatographic peak is comparison of its retention factor with that of an authentic sample of the suspected compound. Retention depends on a sample molecule's escaping into the mobile phase versus its solubility in the stationary phase.

Quantitatively given by the partition coefficient, KD, the ratio of solubility in the two phases K D = [solute in mobile phase] [Solute in stationary phase] The quantitative analysis is for measuring the quantity of components in the mixture by peak area, Quantitative chromatography is based upon a comparison of either the height or the area of the analyze peak with that of one or more standards that the area or height of a peak is proportional to the amount of that component.

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3.14 .1 Samples used Whole plant material at different stages of life cycle from 30 days to 130 days, different parts of plant Andrographis paniculata and samples of leaves and whole plant material from different location were used for quantitative analysis by HPTLC. Isolated Andrographolide and working standard were also tested against standard biomarker of constituent.

3.14 .2 Equipments A Camag HPTLC system equipped with a sample applicator Linomat V, twin trough plate development chamber, TLC Scanner 3, Win cats and integration software 1.4 (Switzerland).

Fig. 3.9: HPTLC Equipments used for chromatographic separation.

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Stationary phase- aluminum sheets (10 x 10 cm) pre coated silica gel 60 F254 (E. Merck)

Mobile phase- A mixture of 7 volumes of chloroform and 1 volume of methanol was used.

3.14.3 Test and reference solution used 1-Test solution- 1 g of coarsely powdered samples under examination with 50 ml methanol each separately

.

2- Test solution- 5mg of isolated Andrographolide.

1-Reference solution-1g of tested Andrographis paniculata (used as working standard in Himalaya Herbal Healthcare, Dehradun, Uttarakhand, India.) 2- Reference solution-Standard Andrographolide solution.

3.14.4 Sample and standard preparation Sample preparation: (1) 1g of coarsely powdered drug sample was extracted with 50 ml absolute alcohol for 24 hours by cold extraction method. The extract was filtered by Whatmann filter paper 125mm (containing ash less circles Cat No.-1441-125) and make up to 10 ml in a volumetric flask. Same procedure was repeated for all samples. (2) 5mg of isolated Andrographolide was dissolved in 5ml of absolute alcohol and made up to 5ml in standard volumetric flask.

Reference solution: (1) 1g of coarsely powdered drug (working standard from Himalaya Drug) was extracted with 50 ml absolute alcohol for 24 hours by cold extraction method. The extract was filtered by Whatmann filter paper 125mm (containing ash less circles Cat No.-1441-125) and make up to 10 ml in a volumetric flask. Filtrate was concentrated to 2 ml and used for HPTLC.

(2) 5mg of standard Andrographolide dissolved in 5ml of absolute alcohol and made up to 5ml in standard volumetric flask.

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3.14.5 Procedure The chromatographic estimation was performed using the following conditions, stationary phase was aluminum sheets (10 x 10 cm) pre coated silica gel 60 F254 (E. Merck) plates 0.2 mm thickness and the mobile phase used was :-Chloroform: Methanol: 7:1 v/v. Camag twin trough chamber was saturated with the developing solvent. The sample (5 μL) was applied as bands on the plates using Camag linomate 5 sample applicator. The width of the applied band was 6.0 mm and the space between the bands was fixed as 13.0 mm. The speed of application was maintained at 150 μL /sec. The chamber saturation time employed was 30 minutes and the developing distance was 8.5cm. Scanning wavelength of 250nm with a slit dimension of 6.0 x 0.45mm and scanning speed of 20 mm/s were employed. When the solvent front reaches the other edge of the stationary phase, the plate was removed from the solvent reservoir. The developed plate was dried in air and examine in ultraviolet light at 250 nm using Camag TLC scanner 3 equipped with Win Cats Version 1.4 software. The constituents of extracts were identified by simultaneously running standards with the unknown. The chromatographic profiles of the test solutions were similar to that of the reference solution and standard Andrographolide The fingerprint of HPTLC profile of all extracts and isolated compound Andrographolide were taken using computer by the help of Wincats and integration software 1.4 (Switzerland) to increase the authenticity of results. Rf value of various samples was evaluated. Rf value of various samples was evaluated using following formula: Rf = Distance traveled by sample from base line Distance traveled by solvent from base line The Peak table, Peak display and Peak densitogram were noted. The amount of Andrographolide in test samples was determined.

HPTLC profile of various extract of Andrographis paniculata was compared to determine maximum yield containing stage and part of plant. Quantitative analysis of samples of different locations was also compared.

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3.15 Yield estimation of Andrographolide from Andrographis paniculata by high performance liquid chromatography (HPLC) For the broad spectrum of work HPLC techniques were also used for the estimation of Andrographolide in different samples besides HPTLC. All samples of fresh extracts and dry extracts (6 months old) were analyzed by HPLC for their pharmaceutical potential in terms of Andrographolide content present. Bio-marker of Andrographolide was used as a standard to compare all results of yield estimation of Andrographolide content by HPLC. Isolated Andrographolide was also tested for its purity and yield percentage.

3.15.1 HPLC method validation HPLC was carried out according to procedure given in IP2010 Vol. 3[33]. The development of suitable mobile phase is an important step in devising an analytical procedure. The composition of the HPLC mobile phase was optimized to achieve good resolution. The best resolution and peak shape was obtained by: A. a buffer solution prepared by dissolving 0.136 g of potassium dihydrogen orthophosphate in 500 ml of water and 0.5 ml of orthophosphoric acid, dilute to 1000 ml with water, B. acetonitrile, for dry extracts and 65Volumes of methanol and 35 Volumes of water for freshly prepared methanolic extracts of all samples. The compound with a retention time 2.871 ± 0.004min.was identified as Andrographolide. Specificity can be ascertained by comparing the standard and sample peak purity. The peak corresponding to Andrographolide in the sample was confirmed by comparing the spectrum obtained by photodiode array detector, which was completely in agreement with the standard Precision is a measure of either reproducibility or repeatability of the analytical method. Intermediate precision express the laboratory variations, by intra- and inter-day variation.

Six determinations of three concentrations of standard Andrographolide on the same day (intraday) and on different days (inter-day) were carried out and expressed as percent relative standard deviation (% RSD) or co-efficient of variation (CV). The results noted reveal that no significant intra- and inter-day variations. The accuracy of the method was determined from recovery studies.

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The pre-analyzed sample was spiked with three different concentrations of standard Andrographolide and the mixtures were analyzed by the proposed method. The accurate, simple, specific and reproducible HPLC method has been developed and validated for the determination of Andrographolide in Andrographis paniculata herb.

3.15.2 Samples used For the determination of Andrographolide in Andrographis paniculata samples at different stages of life cycle, different parts of plant and samples from different locations were used. Leaves and whole plant material samples of self cultivated Andrographis paniculata were also collected every month in one year-cycle and their extracts were tested for Andrographolide content. Fresh green and dry leaves of Andrographis paniculata were also tested for Andrographolide content. Plants of Andrographis paniculata did not died after one complete life cycle. Good growth of plants was recorded for the second year in Dehradun. Study of change in its main chemical constituent “Andrographolide” in comparison to the one year old plants was also carried out. Besides fresh extracts dry extracts of leaves and whole plant material at two different growth stages 110 days and 130 days were also tested for diterpenoid lactones Andrographolide content. Isolated Andrographolide was also examined to check the purity of isolated content.

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Fig. 3.10: Life cycle of plants of Andrographis paniculata

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Fig. 3.11: Dried samples of Andrographis paniculata

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3.15.3 HPLC instrumentation Shimadzu Model-LC2010 CHT, Serial No. C-21254505638 HPLC instrument was used for the chromatographic separation using C 18 column (250 nm x 4.6 nm). Isocratic elution was carried out with methanol at a flow rate 1.5 ml/min. The detection was performed with a D2 lamp at 223 nm wavelength. L C Solution software was used for integration and calibration. Evaluation was via peak areas with linear regression.

3.15.4 Standard Andrographolide Andrographolide standard 99.8% (Product code A009, Lot no. T11B001) was procured from Natural Remedies, Bangalore.

Fig. 3.12: Standard biomarker of Andrographolide

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3.15.5 Mobile phase Two different types of mobile Phase were used for freshly prepared extracts and for dry extract.

(1) Mobile Phase for freshly prepared extracts: Mobile Phase 65Volumes of methanol and 35 Volumes of water were used for freshly prepared methanolic extracts of all samples. (2) Mobile Phase for dry Extract: Mobile phase: A. a buffer solution prepared by dissolving 0.136 g of potassium dihydrogen orthophosphate in 500 ml of water and 0.5 ml of orthophosphoric acid, dilute to 1000 ml with water, B. acetonitrile.

3.15.6 Sample preparation for HPLC All solvents were of HPLC Grade and all reagents were of analytical reagent grade. Water used for chromatographic separation was purified using Millipore mill purified system.

3.15.6.1For freshly prepared extracts Freshly prepared extracts of dry powder of different samples were used for HPLC. About 2.5 gm of Andrographis paniculata plant powder was weighed of all samples separately into a round bottom flask. About 50 ml of methanol was added and refluxed on a water bath for 30 minutes. It was cooled and filtered. Residue was refluxed further with methanol till the last extract turns colorless, it was cooled and filtered. The same operation was repeated twice with methanol (2×30 ml) for all samples. All the methanolic extracts of sample were combined and made up to 50 ml with methanol and filtered through 0.45 μm membrane filter. Same procedure was repeated for all other samples.

3.15 .6.2 For dry extract For this dry extracts (6 months old) of different samples of Andrographis paniculata were used. About 250 mg of the dry extract (under examination) or quantity equivalent to 20 mg of Andrographolide was dissolved in methanol by gently heating. It was made up to 100.0 ml and filtered. Samples of dry extract of leaves and whole plant material at two different growth stages 110 days and 130 days were tested.

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3.15.7 Preparation of standard solution Standard working solution was prepared by dissolving 10 mg of Andrographolide [99.8%] with 100ml of methanol to give a concentration of 100μg/ml.

3.15.8 Estimation of Andrographolide Freshly prepared extracts as well as dry extracts were used for the estimation of Andrographolide in different samples of Andrographis paniculata.

3.15.8.1 Estimation of Andrographolide in freshly prepared extracts To estimate the content of Andrographis paniculata plant in all samples, 20μl aliquots of samples were injected in to HPLC. Spectrophotometer was set at 223nm. The HPLC analysis was continued for 15 min, since the retention time of the Andrographolide was 2.871 ± 0.004 min. The content of Andrographolide was calculated by linear regression and mean percentages were calculated from six replicate experiments. Since Andrographolide is freely soluble in methanol, the plant materials were extracted with methanol. Mobile phase for HPLC was methanol: water (65:35). First run was of blank to check error from mobile phase and to determine the retention time of the solvent. The chromatograms of Andrographolide from plant samples were obtained and compared with standard chromatogram of Andrographolide on the basis of their retention time and peak area. The retention time and peak area were recorded for calculating total Andrographolide content in all samples.

3.15.8.2 Estimation of Andrographolide in dry extract This test was carried out to check whether there is any difference in Andrographolide content in freshly prepared and old dry extracts of Andrographis paniculata. Dry extracts were stored at -180c (ideal environment) in labeled sterile bottles as given in IP2010 Vol. 3.This test also gives the shelves value of extracts of Andrographis paniculata. Two types of mobile phases were used. A. a buffer solution prepared by dissolving 0.136 g of potassium dihydrogen orthophosphate in 500 ml of water and 0.5 ml of orthophosphoric acid, dilute to 1000 ml with water, B. Acetonitrile. Reference solution: 0.02 per cent w/v solution of Andrographolide RS in methanol. Flow rate was 1.5 ml per minute. A linear gradient programme using the conditions given was used. Spectrophotometer was set at 223 nm and injection volume was 20μl. The test is not valid unless

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the relative standard deviation for the replicate injections is not more than 2.0 per cent. The relative retention time with respect to Andrographolide is about 1.2. First run was of blank to check error from mobile phase. Test solution and reference solution (A) were injected. The content of Andrographolide in the extracts was calculated by summing the peak areas of Andrographolide.

3.15.8.3 Checking of purity of isolated Andrographolide Purity of isolated Andrographolide was checked against standard biomarker of Andrographolide by HPLC. Solution was prepared by dissolving 10 mg of isolated Andrographolide in100ml of methanol to give a concentration of 100μg/ml. The sample was sonicated for 5 minutes to ensure complete dissolution and allowed to equilibrate at room temperature. From this, 5 ml solution was taken and was diluted up to 25 ml with methanol. 20 μl of the above sample was injected in the HPLC instrument and HPLC chromatogram was obtained.

3.15.9 Linearity The linearity of the standard curve was evaluated by injecting five standard working solutions containing 120 to280 ppm spot based on 20μL sample volume concentration of Andrographolide. Peak area and concentrations were subjected to least square linear regression analysis to calculate the calibration equation and correlation coefficient.

3.16 Fourier transforms infra-red spectroscopy (FTIR) 3.16.1 Introduction Fourier Transform Infrared Spectroscopy (FTIR) can provide very useful information about functional group. The technique can be used to analyze organic materials and some of inorganic materials. In infrared spectroscopy, IR radiation is passed through a sample. Some of the infrared radiation is absorbed by the sample and some of it is passed through (transmitted). The resulting spectrum represents the molecular absorption and transmission, creating a molecular fingerprint of the sample. Like a fingerprint no two unique molecular structures produce the same infrared spectrum. This makes infrared spectroscopy useful for several types of analysis. The term frequency is used for band/peak position throughout, and this is expressed in the commonly used

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units of wave number (cm-1). The average modern infrared instrument records spectra from an upper limit of around 4000 cm-1 (by convention) down to 400 cm-1 as defined by the optics of the instrument (commonly based on potassium bromide, KBr). For this reason, when a spectral region is quoted in the text, the higher value will be quoted first, consistent with the normal left-to-right (high to low cm-1) representation of spectra. Also, the terms infrared band, peak and absorption will be used interchangeably within the text to refer to a characteristic spectral feature. The FTIR technique is to measure the absorption of various infrared radiations by the target material, to produces an IR spectrum that can be used to identify functional groups and molecular structure in the sample. The unknown IR spectrum is compared with standard spectra in commercial libraries or a spectrum collected from a reference material to identify the analyzed material. Latest technique is comparison of IR of samples with IR of standard biomarker and that was used.

3.16.2 Procedure for FTIR study of Andrographolide IR transmission spectra were obtained using a FTIR spectrophotometer (Perkin Elmer spectrumVX (4000-400 cm-1) 5% sample w/w was mixed with dry potassium bromide (KBr) and KBr disc was prepared. The mixture was ground into a fine powder using an agate mortar before compressing into KBr disc under a hydraulic press at 10,000 psi. Each KBr disc was scanned at 4 mm/s at a resolution of 2 cm at wave number region of 400-4000 cm-1. The characteristic peaks were recorded using the FTIR spectrophotometer. The IR spectrum showed characteristic peak positions of active ingredient- Andrographolide. Peaks of Andrographolide standard 99.8% (Natural Remedies, Bangalore) were obtained for comparison.

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