Chapter 2: Metabolite extraction and fingerprinting of Terminalia arjuna extracts

Transcription

1 Chapter 2: Metabolite extraction and fingerprinting of Terminalia arjuna extracts 2.1 Introduction 2.2 Materials and Methods Plant Material Processing: Pharmacognosy analysis I. Thin layer chromatography (TLC) II. Histological studies Solvent Based Extraction I. Direct extraction II. Successive extraction Metabolic Fingerprinting of the Terminalia arjuna extracts Quantitative tests for phyto-chemicals 2.3 Results Pharmacological validation of Terminalia arjuna bark Extraction of secondary metabolites from the bark of Terminalia arjuna Metabolite profiling of different extracts from the bark of Terminalia arjuna Quantitative estimation of Terminalia arjuna bark extracts 2.4 Discussion 2.5 References 18

2 2.1 Introduction In recent years there has been considerable increase in the use of plant based medicines (Ravishankar, 2007). The renewed interest in the Indian system of medicine is due to growing recognition of these products as naturally derived, safe therapeutic interventions with less reported side effects. Infact, some of the plant based drugs such as Quinine-derived from bark of Cinchona officinalis, Taxol-dervied from bark of Taxus baccata, Colchicine from Gloriosa superba are most popular for the treatment of malaria, cancer and gout respectively (Druilhe et al., 1988; Ravishankar, 2007). In spite of ancient use of plant parts and its extracts in traditional system of medicine there have been limiting factors that have prevented the growth of this segment. Some of the major challenges faced by herbal industries include availability of large quantities of authentic plant materials, detection of adulteration with spurious plant material(s), absence of available standards for quantification of bio-actives, estimation of batch-to-batch variability during commercial productions. Composite phyto-extract is characterized by mixture of metabolites. The lack of extraction and resolution of metabolite diversity by commercially available standards makes its decoding complex. The metabolite fingerprinting methods commonly employed in the herbal industries are focused on quantitative estimation of less than 10 metabolites that occur in significant proportions (>0.1%). This approach makes it difficult to address the batch to batch variability observed in bioactivity where the minor constituents (< 0.01 %) might also be responsible for the observed bioactivity. The term Pharmacognosy is defined as the study of drugs derived from natural sources. This includes scientific and systematic study such as structural, physical, chemical and biological characters of crude drugs along with their method of cultivation, collection and preparation. 19

3 The plant parts from which the herbal drugs are derived are available in crumpled or broken pieces. It therefore becomes difficult to identify them based on their morphology. Hence microscopic characteristic of these plant materials aids in confirming the identity of the plants and thereby authenticity of the plant part in use. In addition to microscopic characters additional characters such as chemical evaluation using both qualitative chemical tests, chromatographic separation of the organic compounds of the extracts are also necessary for identification of the sample for its purity and consistency. As discussed in the previous (Chapter 1; section 1.3), Terminalia arjuna as a ingredient has been used in traditional system as therapeutic component in a range of formulations. Terminalia arjuna has been reported for mitigation of range of health related disorders due to the presence of diverse class of secondary metabolites. In effort to isolate these different classes of metabolites different extraction conditions and solvent systems have been used, which will be discussed in length in the following section (section 2.3.2). Extraction of secondary metabolites is classically performed using two different methods listed below: I. Direct extraction II. Successive extraction Both extraction procedures can be performed at room temperature under agitation or static conditions. Direct extraction is performed using single solvent system. Direct extraction is preferred in situations where complete extraction of metabolites in a particular solvent is required. Direct extraction at room temperature is done for temperature sensitive bioactive(s) by immersing the plant material in the solvent of choice under constant agitation. Once the extraction is complete, the solvent is passed through filters to remove suspended particles and then centrifuged to remove any un-dissolved fine material. 20

4 Another most common method of choice for extracting phyto-chemicals using different temperature and solvent systems is the Soxhlet based extraction procedure. Soxhlet based extraction can be used for extraction of plant metabolites using single solvent (Direct extraction) or multiple solvents in series (successive extraction). Soxhlet works on the boiling temperature or vaporization temperature of the solvent. In successive extraction, the same plant material is sequentially extracted with a series of solvents (from non-polar to polar solvents). This helps in extraction and isolation of the metabolites based on its class and nature. The extended refluxes with each of this solvent system ensure extraction of components based on their polarity components minimizing the chances of carryover in the subsequent solvent. Direct/successive extracts of Terminalia arjuna bark were obtained and subjected to High Performance Liquid Chromatography (HPLC) analysis. HPLC is a technique wherein small quantity of the sample is injected into a reverse phase HPLC column (C-18 column; Waters Corporation) under high pressure and the constituents are allowed to separate based on their interaction with the column and their retention time within the column. The main purpose of HPLC analysis was to generate a metabolite fingerprint of the different plant extracts. This chapter will describe the authentication of plant material by pharmacognosy, extraction of metabolites using different solvents and extraction conditions as well as metabolite profiling of the different composite extracts of Terminalia arjuna. 21

5 2.2 Materials and Methods Plant Material Processing: Bark of Terminalia arjuna was procured from authentic vendor and from Sandur forest area which is deposited in Avesthagen Herbarium. The bark was powdered and taken forward for pharmacognosy authentication Pharmacognosy analysis I. Thin layer chromatography (TLC) Bark for Terminalia arjuna (3 grams) was extracted in 50ml methanol for hrs. The resulting methanol extract was filtered and concentrated to 1/10 th of its original volume by evaporation. 3µl of the concentrated methanol extract was spotted on the pre-coated preparative TLC plate (Silica G F 254 of 10 x 10 cm; Merck). After air drying the spotted TLC plates were placed vertically in presaturated solvent chamber (Toluene:Ethyl acetate; 8:2) such that the spotted sample does not submerge in the solvent. Due to capillary action the solvent will be absorbed on the TLC plates resulting in the mobilization and separation of the different components of the extracts. The solvent based separation is based on the difference in mobility of the different components present in the extract. After the solvent has covered 3/4 th of the TLC plate, the solvent phase is marked and the plates are air dried. The metabolite fingerprint of the extract is observed in visible range, long Ultra violet (U.V.) (366nm), short U.V light (255nm) and using detection reagent consisting of Anisaldehyde, Vanillin Sulphuric acid (H 2 SO 4 ) and 10% H 2 SO 4. The retention factor (R f ) [Formula: R f value= Distance traveled by solute/ Distance traveled by solvent] was determined for each band representing the metabolites present in the bark extract of Terminalia arjuna. 22

6 II. Histological studies Transverse section of Terminalia arjuna bark were taken and washed with water to remove debris. Sections were stained with Saffranine for 5-10 minutes and treated with increasing amount of ethanol to remove the moisture. The sections were transferred to clean slide, treated with plain clove oil for 5 minutes. Excess clove oil was drained off using tissue paper. The stained bark sections are then mounted on the glass slide using Canada balsam (Wallis, 1957) Solvent Based Extraction: I. Direct Extraction a. Soxhlet based extraction from bark of Terminalia arjuna Powdered bark of Terminalia arjuna (100 grams) was taken into the Soxhlet extractor. The top was covered with cotton. The level of plant material was kept one inch below the vapor inlet tube ml of extraction solvent was taken in to the round-bottomed flask and was placed on the mantle, temperature was set at 65 C. In order to prevent bubbling of the solvent few ceramic chips were added in to the flask. The extractor containing the bark powder was placed on the solvent containing round bottom flask. The extractor was then connected with the condenser and the cold water was circulated continuously in the condenser. On heating the vapors of the solvent from the round bottom flask passes through the inlet into the condenser and the extractor. The vapor gets condensed in the condenser and the solvent drips on top of the plant material and gets collected in the extractor (body), in this process metabolites get extracted from the plant material. When the extractor containing the plant material gets completely filled with solvent, the solvent along with the extracted metabolites gets drained in the 23

7 solvent containing round bottom flask. This completes one cycle of extraction. The extraction was continued for 8 hours, with ~4 cycles per hour. Finally the extract collected in the round bottom flask was concentrated by vacuum lyophilization. After complete extraction with the given solvent, the plant material was air dried and taken up for extraction with the next solvent and the entire procedure was repeated. The percentage yield of the extract was calculated using following formula. % Yield = weight of lyophilized extract * 100 weight of starting plant material b. Extraction from bark of Terminalia arjuna at room temperature Powered bark of Terminalia arjuna (100 grams) was taken into the conical flask. To it extraction solvent (500 ml) was added and the mouth was covered with aluminum foil to avoid solvent evaporation. The flask was then placed on an orbital shaker at 210 rpm, room temperature (25 C) for 4hours. The supernatant was centrifugation at 1000 rpm for 10 minutes and was subsequently subjected to lyophilization. The plant material was re-extraction with 500ml extraction solvent for 2 hours. Again the supernatant obtained was centrifuged at 1000 rpm for 10 minutes. The filtrate was subjected to lyophilization. Yield of the extract was calculated using the following formula: % Yield = weight of lyophilized extract * 100 weight of starting plant material 24

8 II. Successive Extractions: Successive extraction from bark of Terminalia arjuna was carried out using soxhlet extractor. The solvents used, were based on their sequential polarity starting from non-polar to polar, viz; Hexane, chloroform, ethyl acetate, acetone, ethanol, methanol and water at temperature above the boiling point of the solvents. The detailed process of successive extraction is given below: Powdered bark of Terminalia arjuna (100 grams) was taken into the Soxhlet extractor. The top was covered with cotton. The level of plant material was kept one inch below the vapor inlet tube ml of solvent (non polar to polar solvent) was taken in to the roundbottomed flask and was placed on the mantle; temperature was set above the boiling point of the solvent being used for extraction. In order to prevent bubbling of the solvent few ceramic chips were added in to the flask. The extractor containing the bark powder was placed on the solvent containing round bottom flask. The extractor was then connected with the condenser and the cold water was circulated continuously in the condenser. On heating the vapors of the solvent from the round bottom flask passes through the inlet into the condensor and the extractor. The vapor gets condensed in the condenser and the solvent drips on top of the plant material and gets collected in the extractor (body), in this process metabolites get extracted from the plant material. When the extractor containing the plant material gets completely filled with solvent, the solvent along with the extracted metabolites gets drained in the solvent containing round bottom flask. This completes one cycle of extraction. The extraction was continued for 8 hours, with ~4 cycles per hour. Finally the extract collected in the round bottom flask was concentrated by vacuum lyophilization. 25

9 After complete extraction with the given solvent, the plant material was air dried and taken up for extraction with the next solvent and the entire procedure was repeated. The percentage yield of the extract was calculated using following formula. % Yield = weight of lyophilized extract * 100 weight of starting plant material Metabolic Fingerprinting of the Terminalia arjuna extracts Metabolic fingerprinting of all the direct and successive extracts from Terminalia arjuna plant parts is done by HPLC. HPLC Fingerprinting: The samples for HPLC analysis were prepared by dissolving 10 mg of extract in 1 ml of methanol:water. These samples were filtered, collected in HPLC vials and 10 μl of extract was injected and subjected to separation by Waters 2695 HPLC instrument. Subsequently the metabolite profiles were analyzed using Waters Millennium 32 (Waters Corporation) and Metagrid TM software developed in-house (Avesthagen). The HPLC column used for separation was Atlantis dc18, 5μ, 4.6x250mm (Waters Corporation). The column temperature was maintained at 25 0 C and the solvent flow rate was set at 1.0ml per min. HPLC conditions included Gradient chromatography. Solvents used for metabolite separation were Acetonitrile (solvent A), Methanol (solvent B), Water (HPLC Grade)+ 0.1%TFA (Solvent C and D). Method standardization was done for the maximum separation of the metabolites of Terminalia arjuna direct and successive extracts. Following are the conditions that were used for separation and metabolite fingerprinting of respective extracts. 26

10 Sample-AV016BaDi (65) 04(100) Time Program: Time Flow %A %B %C %D Curve Sample-AV016BaDi (65) 04(80) Time Program: Time Flow %A %B %C %D Curve Sample-AV016BaSu (65) 09(100) Time Program: Time Flow %A %B %C %D Curve Sample-AV016BaSu (65) 01(100) Time Program: Time Flow %A %B %C %D Curve Sample-AV016BaSu (65) 04(100) Time Program Time Flow %A %B %C %D Curve

11 Sample-AV016BaSu (65) 06(100) Time Program Time Flow %A %B %C %D Curve Sample-AV016BaSu (105) 08(100) Time Program Time Flow %A %B %C %D Curve Quantitative tests for phyto-chemicals: Determination of total phenols Weigh 10 mg of extract to falcon tube and dissolve it in 10 ml of 50% methanol. Dilute 1:10 of this solution to get 0.1 mg/ml (100 μg/ml). a. Preparation of standard Gallic acid: Weigh 10 mg of Standard Gallic acid to falcon tube and dissolve it in 10 ml of 50% methanol. Dilute 1:10 of this solution to get 0.1 mg/ml (100 μg/ml). b. Preparation of reagents: Folin-Ciocalteu (FC) reagent: 5 ml of FC reagent in 95 ml of 50% methanol 7.5% of Sodium carbonate solution: weigh 7.5 gms of sodium carbonate in 100 ml water. 28

12 c. Procedure: Pipette out the different concentration of extracts ranging 2 to 20 μg/ml into microfuge tube. Make up the volume to 200 μl with methanol. Add 1000 μl of the FC reagent to each microfuge tubes Incubate for 5 min at 37 C Add 800 μl of the 7.5% Sodium carbonate to all tubes. Incubate for 30 min at room temperature Read absorbance at 750 nm d. Calculation: OD of sample % Of Total phenol content: X 100 OD of standard 29

13 2.3 Results Pharmacognostic validation of Terminalia arjuna bark Bark of Terminalia arjuna was authenticated by Department of Forestry and Environmental Sciences, GKVK, Bangalore. Further as an in-house quality check, Pharmacognostic analysis was carried out as per Ayurvedic Pharmacopoeia of India (API) (Anonymous, 2001). As reported earlier (Anonymous, 2001), the bark of Terminalia arjuna was found to be pinkish in colour, with fibrous texture. It had a bitter and astringent taste. Further to validate the raw material TLC and histological analysis was performed. TLC based metabolite fingerprint of the Terminalia arjuna bark methanolic extract (Chapter 2; section 2.2.2) was performed to assign a reference metabolic fingerprint. As seen in Figure 2.1, metabolites with the natural pigment failed to separate using the Toulene:Ethyl acetate (8:2) solvent system (Figure 2.1, lane 1 and 2); using anisaldehyde developing reagent three distinct band at R f value 0.04, 0.31 and 0.91 were obtained (Figure 2.1, lane 3). Further in the short U.V. range (254 nm) there were no observed bands (Figure 2.1, lane 4); whereas at the longer U.V. wavelength (365nm) only one band at R f value of 0.72 was obtained (Figure 2.1, lane 5). 30

14 Figure 2.1: A B C D E E Figure 2.1: TLC based metabolic finger printing of Terminalia arjuna bark. TLC slide representing mobility of methanolic extract of Terminalia arjuna bark powder. Lane 1 [A] represents profile under visible light, lane 2 [B] represents profile after spraying with 10% Sulphuric acid, lane 3 [C] represents profile after spraying with Anisaldehyde reagent, lane 3 [D] represents profile under short UV (254 nm) and lane 5 [E] represent profile under long UV (366 nm). Further as mentioned in API (Anonymous, 2001), histological analysis of Terminalia arjuna bark was done with saffranine staining (Section 2.2.2). Transverse section of the bark shows in cortical zone medullary rays, phloem fibers and calcium oxalate crystals present in parenchyma cells alternating with fibers. Phloem fibers, which are distributed in rows and present in groups (Figure 2.2). 31

15 Figure 2.2: Medullary rays Phloem Calcium Oxalate crystal fibers Figure 2.2: Histological analysis of Terminalia arjuna bark. Transverse section of Terminalia arjuna bark stained with Saffranine. Transverse section shows medullary rays, phloem fibers, calcium oxalate crystals similar to that reported for bark of Terminalia arjuna in Ayurvedic Pharmacopoeia of India Extraction of secondary metabolites from the bark of Terminalia arjuna Metabolite extraction from bark of Terminalia arjuna was carried out using both direct extraction and successive extraction method. Direct extraction was performed at room temperature as well as in soxhlet apparatus. Successive extraction was performed in soxhlet based extraction using solvent from nonpolar to polar range. The extraction conditions and protocol are mentioned in section

16 The extracts were coded as per following nomenclature: Plant No & Avesthagen AV 016Ba Su/Di (Temp. ext) 01(20) Type of extraction: Ext. t Solvent No. & % Solvent 1. AV- first two letters represents Avesthagen. 2. Plant Name: The Plants used and in use are assigned with unique 3-digit number, 016 represents Terminalia arjuna. 3. Part of the plant /Tissue: There is a two letter ID for each plant part used. Here Ba stands for Bark. 4. Solvents: The solvents used for extraction are also assigned with two digit numbers 01 for Acetone, 02 for Benzene, 03 for Chloroform, 04 for Ethanol, 05 for Hexane, 06 for Methanol, 08 for water, 09 for ethyl acetate. Percentage of solvents used for extraction is given within bracket (20) for 20 % of that solvent. For example if 20% of Ethanol was used for extraction, 04(20). 5. Method of Extraction: Successive extraction is referred to as Su whereas direct extraction is referred to as Di, temperature for extraction is written in bracket. For example, Su(65) represents successive extraction at 65 ºC. The characteristics of extracts using direct extraction with 20% ethanol at room temperature and soxhlet based extraction using absolute ethanol are represented in Table 2.1. Similarly, Table 2.2 lists the characteristics of soxhlet based successive extraction with non-polar to polar solvents. 33

17 Table 2.1: Characteristics of direct extracts using 100% and 20% ethanol Solvent Extract Extract Sl. No Extract ID used color Texture 1 AV016BaDi(65)04(100) Ethanol Dark Brown powder 2 AV016BaDi(25)04(20) Hydroalcohol Brownish powder Table 2.2: Characteristics of successive extracts using solvents ranging from non-polar to polar solvents Sl. No Extract ID Solvent used Extract color Extract Texture 1 AV016BaSu(65)05(100) Hexane Yellowish Sticky 2 AV016BaSu(65)03(100) Chloroform Greenish Sticky 3 AV016BaSu(65)09(100) Ethyl acetate Orangeish brown powder 4 AV016BaSu(65)01(100) Acetone Reddish brown powder 5 AV016BaSu(65)06(100) Methanol Brownish powder 6 AV016BaSu(65)04(100) Ethanol Reddish brown powder 7 AV016BaSu(65)08(100) Water Brownish powder 34

18 2.3.3 Metabolite profiling of different extracts from the bark of Terminalia arjuna Metabolomics is defined as comprehensive metabolite profiling of a given biological system. Metabolite profiling of Terminalia arjuna was a technically challenging task due to diversity of the metabolites along with its varying concentrations. Absence of single analytical method that is capable of extracting and detecting all metabolites at once makes the task even more challenging. Currently, the most widely used technique involves reverse phase - HPLC coupled with photodiode array (PDA), with or without mass spectrometry. Till date this is the most sensitive method enabling the detection of hundreds of compounds from a given extract. To decipher metabolite fingerprints of different Terminalia arjuna bark extracts, I have used MetaGrid TM. MetaGrid TM is a reproducible analytical technique for metabolite fingerprinting, developed at Avesthagen Limited, Bangalore. This technique consists of reverse phase liquid chromatography coupled with photodiode array (PDA). The outputs of these results were analyzed using an inhouse developed software; where the results are depicted in the form a metabolite signature. To obtain the metabolite fingerprint of different Terminalia arjuna extracts, individual extracts were subjected to reverse phase HPLC based separation. Each of the extract was standardized with respect to the mobile solvent system and the run conditions for maximum separation of the metabolites in composite extracts (section 2.2.4). Three HPLC runs are performed per extract to generate a metabolite profile. HPLC profiles of different extracts are represented in form of PDA chromatogram. Here, the absorption maxima of the different metabolites present in respective composite extracts are recorded from 200 to 700nm at an interval of 10nm. Millenium 32 software (Waters Corporation) is used to generate this PDA chromatogram. PDA chromatogram is a 3- dimensional chromatogram, where the retention times of the data points are represented by x-axis, the wave-length (nm) of analysis on z-axis and the intensity of nm absorption on y-axis. 35

19 Further, the PDA chromatogram is used as an input data for MetaGrid TM software, which in turn represents the data in form of metabolite fingerprint. Here, the x-axis represents the absorption range from nm and y-axis represents the retention time. Additionally, the MetaGrid TM software also gives percentage conservation of metabolites between three individual HPLC runs for each extract. Direct and successive extracts from bark of Terminalia arjuna were subjected to above mentioned HPLC based fingerprinting and MetaGrid TM analysis. A representative PDA chromatogram of each Terminalia arjuna extract at 254nm is shown in Figure 2.3A-2.9A. Metabolite profile of Terminalia arjuna direct extracts (Figure 2.3B-2.4B) and successive extracts (Figure 2.5B-2.9B) at 254nm gives a representative metabolite signature of each extract. 36

20 Figure 2.3: A AU Minutes B. Figure 2.3: HPLC profile and Metabolite finger printing of Direct 100% ethanol extract (AV016BaDi(65)04(100)). (A) Representative HPLC profile of the extract at 254 nm. (B) Profile of metabolite fingerprint for extract. 37

21 Figure 2.4: A. 700 mau 254nm4nm (1.00) min B. Figure 2.4: HPLC profile and Metabolite finger printing of Direct 20% ethanol extract (AV016BaDi(65)04(20)). (A) Representative HPLC profile of the extract at 254 nm. (B) Profile of metabolite fingerprint for extract. 38

22 Figure 2.5: A. Metabolite profile of the extract at 254 nm AU Minutes B. Figure 2.5: HPLC profile and metabolite finger printing of Successive 100% ethyl acetate extract (AV016BaSu(65)09(100)). (A) Representative HPLC profile of the extract at 254 nm. (B) Profile of metabolite fingerprint for extract. 39

23 Figure 2.6: A AU Minutes B. Figure 2.6: HPLC profile and metabolite finger printing of Successive 100% acetone extract (AV016BaSu(65)01(100)). (A) Representative HPLC profile of the extract at 254 nm. (B) Profile of metabolite fingerprint for extract. 40

24 Figure 2.7: A AU Minutes B. Figure 2.7: HPLC profile and metabolite finger printing of Successive 100% ethanol extract (AV016BaSu(65)04(100)). (A) Representative HPLC profile of the extract at 254 nm. (B) Profile of metabolite fingerprint for extract. 41

25 Figure 2.8: A AU Minutes B. Figure 2.8: HPLC profile and metabolite finger printing of Successive 100% methanol extract (AV016BaSu(65)06(100)). (A) Representative HPLC profile of the extract at 254 nm. (B) Profile of metabolite fingerprint for extract. 42

26 Figure 2.9: A AU Minutes B. Figure 2.9: HPLC profile and metabolite finger printing of Successive 100% water extract (AV016BaSu(105)08(100)). (A) Representative HPLC profile of the extract at 254 nm. (B) Profile of metabolite fingerprint for extract. 43

27 2.3.4 Quantitative estimation of Terminalia arjuna bark extracts In absence of available standards, concentration of the class of metabolites is used as parameter for representing reproducibility of extracts in commercial batches. Standardized commercial phyto-extracts from green tea, grape seeds, and other plant extracts are represented by presence of minimum concentration of class of metabolites like total phenols. With the subsequent fractionations and identifications of active molecule(s), using standards, the extracts are then represented as minimum concentration of bioactive, eg catechins in green tea extracts (Khokhar and Magnusdottir, 2002). Additionally, there as been positive relationship between the total phenol content and free radical scavenging potential. In addition to metabolic fingerprinting, Terminalia arjuna direct and successive extracts were also estimated for total phenol content to account for batch to batch variability. Gallic acid was used as standard and total phenol content of Terminalia arjuna bark successive and direct extracts were represented as gallic acid equivalent. Standard graph of gallic acid (Figure 2.10) was made with concentrations ranging from 2 to 20 μg/ml following procedure mentioned in section Figure 2.10: Absorbance at 750 nm Concentration (μg/ml) Figure 2.10: Standard curve with Gallic acid for determination of total phenol. Graph representing Gallic acid standard curve. X-axis represents concentration of gallic acid represented in micrograms per milliliter. Y-axis represents absorbance at 750 nanometers. 44

28 It was found that the different Terminalia arjuna extracts showed total phenol concentration on the range from 33 to 75% (Table 2.3). Table 2.3: Total Phenol content of direct and successive extracts from bark of Terminalia arjuna Sr. No. Phyto-extract codes Name Of The Phyto-extract Total Phenol ( % Equivalent To Gallic Acid) 1 Successive AV016BaSu(65)09(100) Ethyl acetate AV016BaSu(65)04(100) Ethanol AV016BaSu(65)07(100) Methanol AV016BaSu(105)08(100) Water Direct 100% AV016BaDi(65)04(100) ethanol Direct 20% AV016BaDi(65)04(20) ethanol

29 2.4 Discussion One of the challenges in herbal industry is to have quality control checks leading to standardized methods for validation of the raw materials and the metabolites extracted from plants. In plants without know standards this becomes even more challenging. In this study Terminalia arjuna bark under study was analyzed for its authenticity by morphological, organoleptic, histological and chromatographic studies. Result obtained reported desired characteristic of Terminalia arjuna bark (section 2.3.1; Figure 2.1 & Figure 2.2). The bark was then taken for further solvent based extraction. Direct and Successive extracts of Terminalia arjuna bark were profiled by HPLC and Metagrid software. The Metagrid TM generated metabolite profiles for all the composite extracts, which became a reference fingerprint for quality control and for checking variability between samples and extraction procedures. The extracts under study showed metabolite conservation across HPLC runs (section 2.3.3; Figure ). The composite successive and direct extracts from bark of Terminalia arjuna were further taken forward for activity validation using cell free and cell based assay systems is discussed in Chapter 3 46

30 2.5 References : Anonymous (2001). The Ayurvedic Pharmacopoeia of India Vol 1-4 (Delhi: The Controller of Publications ). Druilhe, P., Brandicourt, O., Chongsuphajaisiddhi, T., and Berthe, J. (1988). Activity of a combination of three cinchona bark alkaloids against Plasmodium falciparum in vitro. Antimicrob Agents Chemother 32, Khokhar, S. and Magnusdottir, S. G. M. (2002). Total Phenol, Catechin, and Caffeine Contents of Teas Commonly Consumed in the United Kingdom. J. Agric. Food Chem, 50 (3), Ravishankar, B. a. S., V.J. (2007). INDIAN SYSTEMS OF MEDICINE: A BRIEF PROFILE. African Journal of Traditional, Complimentary and Alternative Medicines 4, Wallis, T. E. (1957). Crystals in the leaf of Lobelia inflata Linn. J Pharm Pharmacol 9,