Chapter 3

Preparation of the alcoholic extracts and preliminary phytochemical investigation of the selected plants Boswellia ovalifoliolata, Commiphora caudata, Saccharum spontaneum and Garcinia mangostana 3.1 Plant Collection and Authentication 3.2 Physico-chemical analysis of the selected plants 3.3 Preparation of alcoholic extracts of the selected plants 3.4 Preliminary qualitative phytochemical evaluation of the selected plant extracts 3.5 Acute oral toxicity studies of the selected plant extracts 3.6 Results and Discussion

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Chapter 3 Preparation of the alcoholic extracts and preliminary Phytochemical investigation of the selected plants Boswellia ovalifoliolata, Commiphora caudata, Saccharum spontaneum and Garcinia mangostana.
The author has selected four plant species Boswellia ovalifoliolata Bal.Henry (fam: Burseraceae), Commiphora caudata Wight & Arn (Fam: Burseraceae), Saccharum spontaneum L. (Fam: Poaceae), Garcinia mangostana L.(Fam: Clusiaceae) based on the traditional uses reported in the literature and the phytoconstituents earlier isolated.

3.1 Plant collection and authentication

Fresh plant materials were collected from the Hills of Tirumala region, Chittoor (dist).A.P, India and authenticated by Dr. Madhava chetty, Assistant Professor, Department of Botany, S.V. University, Tirupathi. A voucher specimen of all the plants was kept in our herbarium, Dept of Pharmacognosy and Phytochemistry, College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh.

3.2 Physico-chemical analysis of the selected plants

Crude drugs procured for the study were dried at room temperature and subjected to physicochemical evaluation. Physical tests, loss on drying, ash values, extractive values, fluorescence analysis, total phenolic content and total flavonoid content were performed as per Indian Pharmacopoeia. The results of physico-chemical analysis were presented in Table 3.1.

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Loss on drying About 5 g of powdered drug was accurately weighed, placed in petridish and dried in hot-air oven at 1100C for four hours. After cooling, it was placed in a desiccator, later the loss in weight was recorded and the procedure was repeated till constant weight was obtained [164].

Loss on Drying =

Loss in weight W

X 100

W = Weight of the crude drug in grams Ash value About 2g of crude drug powder was accurately weighed in a tarred and previously ignited silica crucible. It was incinerated gradually by increasing the heat, not exceeding dull red heat, until free from carbon, cooled and weighed. The percentage of ash was calculated with reference to the air-dried drug [165]. a) Acid insoluble ash The ash from the above experiment was boiled for 10 min with 25 mL of dil hydrochloric acid, and the insoluble matter was collected in a silica crucible (previously ignited and weighed). The percentage of acid-insoluble ash was calculated with reference to the air-dried drug. b) Water soluble ash The total ash was boiled for 5 min with 25 mL of water. The insoluble matter was collected in a crucible, washed with hot water, ignited and weighed. The percentage of water soluble ash was calculated with reference to air-dried drug.

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Fluorescence analysis Many crude drugs show the fluorescence when the sample is exposed to ultraviolet radiation. Evaluation of crude drugs based on fluorescence in daylight is not much used, as it is usually unreliable due to the weakness of the fluorescence effect. Fluorescence lamps are fitted with suitable filters, which eliminate visible radiation from the lamp and transmit ultraviolet radiation of definite wavelength. Several crude drugs show characteristic fluorescence useful for their evaluation. The powdered crude drugs of the selected medicinal plants were tested for fluorescence in an UV chamber [166] Determination of extractive values The extracts obtained by exhausting crude drugs with different solvents are approximate measures of their chemical constituents and hence the alcohol and water soluble extractive values of the selected plants were determined according to the following procedure, 1. About 5 g of the powdered drug was weighed in a weighing bottle and transferred to a dry 250 mL conical flask. 2. 100 mL graduated flask was filled with the solvent 90% alcohol/water. The contents of weighing bottle was transferred to a conical flask and washed with the solvent. 3. The flask was corked and set aside for 24 hr, shaking frequently. 4. The contents were filtered into a 50 mL cylinder, when sufficient filtrate was collected, then 25 mL of the filtrate was transferred to a weighed thin porcelain dish.

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5. The solvent was evaporated to dryness on a water-bath and dried in an oven at 1050C for 6 hrs. 6. It was cooled in a desiccator for 30 min and weighed without delay. 7. The content of extractive values was calculated in mg/gm of dried material (w/w). 8. The percentage W/W of extractive was expressed with reference to the air-dried drug. Determination of total phenolic and flavonoid content It was well-known that plant phenolics are highly effective free radical scavengers and antioxidants and the literature review revealed the presence of phenolic substances in the selected plants. Hence the total phenolic and total flavonoid content of the selected plants were determined by standard procedures available in the literature. Chemical reagents: Folin-Ciocalteaus Phenol reagent, gallic acid and catechin were perchased from Sigma Aldrich Co, St Louis, USA. All other chemicals were of analytical grade. Determination of total phenolic content The total phenolic content of the dry herbs was determined with the Folinciocalteau assay. An aliquot (1 mL) of extracts or a standard solution of gallic acid (20, 40, 60, 80 and 100 mg/L) was added to a 25 mL volumetric flask, containing 9 mL of distilled deionised water (dd water). Reagent blank using dd water was also prepared. One milliliter of the Folin-Ciocalteaus Phenol reagent was added to the mixture and shaken. After 5 min, 10 mL of 7% Sodium carbonate solution was added to the mixture. The solution was diluted to 25 mL with dd water and mixed. After incubation for 90 min at room temperature, the absorbance against the prepared reagent blank was determined

42

at 750 nm with an UV-VIS Spectrophotometer Shimadzu. The data for the total Phenolic content of the extracts were expressed as mg of gallic acid equivalents (GAE)/ 100 grams dry mass (mg GAE/100 g dw). All the samples were analysed in duplicates [167]. Gallic acid equivalents (GAE) = Absorbance (at 765 nm) / 0.0508 Determination of total flavonoid content Total flavonoid content of the extracts was estimated with an aluminium chloride colorimetric assay. An aliquot (1 mL) of extracts or a standard solution of (+) -catechin (20, 40, 60, 80 and 100 mg/L) was added to a 10 mL volumetric flask, containing 4 mL of distilled deionised water (dd water). To the flask was added 0.3 mL 5% sodium nitrite. After 5 min, 0.3 mL 10% aluminium chloride was added. At the sixth minute, 2mL 1 M Sodium hydroxide was added and the volume was made up to 10 mL with dd water. The solution was mixed well and the absorbance was measured against the prepared reagent blank at 510 nm with an UV-VIS Spectrophotometer Lamda 5. The data for the total flavonoid content of the extracts were expressed as milligrams of (+) -catechin equivalents (CE) per 100 grams dry mass (mg CE/100 g dw). All the samples were analysed in duplicates [167]. Rutin equivalents (RE) = Absorbance (at 425 nm) /13.33 The results of all the physical tests, ash values, fluorescence analysis, loss on drying, extractable matter, total phenolic and total flavonoid content of individual crude drugs are presented in the Table 3.1 and Fig. 3.1 and 3.2.

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Table 3.1: Results of Physicochemical analysis of the selected drugs S.No 1. Name of the Test Physical tests Nature Free flowing powder Colour Green Brownish black Odour Taste 2. 3. Loss on drying (%w/w) Ash values (%w/w) Total ash Acid insoluble ash Water soluble ash 4. 5. Fluorescence analysis Total phenolic content (mg/gm) 6. Total flavonoid content (mg/gm) 7. Extractive Values (%w/w) Alcohol soluble extractive Water soluble extractive 15.12 2.50 10.15 9.52 5.51 CCL: Commiphora caudata leaf, GMP: Garcinia mangostana pericarp. 12.33 9.41 8.23 8.52 17.6 9.82 16.52 1.42 1.02 7.82 2.51 1.54 8.64 5.3 1.1 1.8 Yellowish green 10.56 6.2 1.6 1.5 Dark brown 7.45 9.2 2.1 7.5 Dark yellow 18.67 6.4 1.3 4. 8 Reddish brown 5.56 4.5 1.5 2.4 Bright yellow 9.82 2.5 1.2 1.5 Yellowish brown 44.24 Characteristic Characteristic 6.6 Characteristic Characteristic 2.4 Odourless Mucilagenous 5.5 Faint odour Astringent 3.8 Odourless tasteless 9. 5 Characteristic Characteristic 7.4 Solid Free flowing powder Green Free flowing powder Cinnamon brown Free flowing powder Yellowish-Brown Free flowing powder Dark Purple BOL BOG CCL CCB SSW GMP

BOL: Boswellia ovalifoliolata leaf, CCB: Commiphora caudata bark,

BOG: Boswellia ovalifoliolata gum, SSW: Saccharum spontaneum whole plant,

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Figure 3.1: The amount of total phenols in selected plants BOL: Boswellia ovalifoliolata leaf CCL: Commiphora caudata leaf SSW: Saccharum spontaneum whole plant BOG: Boswellia ovalifoliolata gum CCB: Commiphora caudata bark GMP: Garcinia mangostana pericarp

10 8
m g/gmdr.wt.

6 4 2 0 BOL BOG CCL CCB SSW GMP

Figure 3.2: The amount of total flavonoids in selected plants BOL: Boswellia ovalifoliolata leaf CCL: Commiphora caudata leaf SSW: Saccharum spontaneum whole plant BOG: Boswellia ovalifoliolata gum CCB: Commiphora caudata bark GMP: Garcinia mangostana pericarp

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3.3 Preparation of alcoholic extracts of the selected plants B. ovalifoliolata, C. caudata, S. spontaneum and G. mangostana
Freshly collected plant material was dried under shade and coarsely powdered in a willey mill. The coarsely powdered material (1 kg) was extracted with petroleum ether to remove the fatty material and further extracted in a Soxhlet apparatus and subjected to continuous extraction with ethanol (95%). The liquid extract was collected and concentrated under reduced pressure until a waxy mass was obtained. The concentrate obtained was weighed in each case [168]. The concentrate was thoroughly air dried to remove all traces of the solvent and the percentage yield was calculated and given in Table 3.2. The extractive value of the extraction was obtained by using the relation, Weight of concentrate % Extraction = Weight of fresh material X 100

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Table 3.2: Physical tests and quantity of extracts

Name S. of the No. Extract 1. EBOL

Quantity of Extractive Odour extract value(%w/w) in gms Greenish brown Characteristic 135 13.5 Colour and nature Of the extract intractable gum

2.

EBOG

Light brown intractable gum

Characteristic 75

7.5

3.

ECCL

Dark green intractable gum

Characteristic 80

4.

ECCB

Reddish brown intractable gum

Characteristic 195

19.5

5.

ESSW

Dark green intractable gum

Odourless

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4.2

6.

EGMP

Dark Purple intractable gum

Characteristic 185

18.5

EBOL: Ethanolic extract of B.ovalifoliolata leaf ECCL: Ethanolic extract of C.caudata leaf, ESSW: Ethanolic extract of S.spontaneum whole plant

EBOG: Ethanolic extract of B.ovalifoliolata gum ECCB: Ethanolic extract of C.caudata bark EGMP: Ethanolic extract of G.mangostana pericarp

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3.4

Preliminary qualitative phytochemical evaluation of the selected plant extracts B. ovalifoliolata, C. caudata, S. spontaneum and G. mangostana.
Preliminary phytochemical analysis of the selected plant extracts were carried out

according to the procedures available in the standard books and the extracts gave positive tests for alkaloids, glycosides, flavonoids, saponins, carbohydrates, protein, aminoacids, lipids, sterols, and tannins [169]. The results of preliminary qualitative phytochemical screening were presented in the Table 3.3. Table 3.3: Results of Phytochemical analysis of the extracts S.No. 1. Name of the Test Test for sterols a. Test solution + Sulphur b. Libermann Reaction 2. Test for glycosides a. Keller Killaini Test b. Baljets Test c. Libermanns test 3. Test for saponins a. Haemolytic test b. Foam test 4. Tests for proteins a. Xanthoprotein test b. Millons test c. Biuret test d. Ninhydrin test 5. Test for tannins a. Gelatin test + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + EBOL EBOG ECCL ECCB ESSW EGMP

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S.No. Name of the Test b. Ferric chloride test c. Lead acetate test d. Dil HNO3 test 6. Test for alkaloids a. Dragendroffs test b. Mayers test c. Hagers test d. Wagners test 7. Test for carbohydrates a. Barfords test b. Benedicts test c. Molischs test 8. Test for Triterpenoids a. Libermann Burchardts Test b. Salkowaski Test 9. Test for flavonoids a. Shinoda test b. Alkaline reagent test c. Lead acetate test
+ - Positive, - - Negative

EBOL EBOG ECCL ECCB ESSW EGMP + + + + + + + + + + + + + + + + + +

+ + + +

+ + + +

+ + + +

+ + +

+ + +

+ + +

+ + +

+ +

+ +

+ +

+ +

+ +

+ +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

EBOL: Ethanolic extract of B.ovalifoliolata leaf ECCL: Ethanolic extract of C.caudata leaf, ESSW: Ethanolic extract of S.spontaneum whole plant

EBOG: Ethanolic extract of B.ovalifoliolata gum ECCB: Ethanolic extract of C.caudata bark EGMP: Ethanolic extract of G.mangostana pericarp

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3.5 Acute oral toxicity studies of the selected plant extracts Boswellia ovalifoliolata, Commiphora caudata, Saccharum spontaneum and Garcinia mangostana.
The alcoholic extracts of the selected plants, B. ovalifoliolata, C. caudata, S. spontaneum and G. mangostana were initially tested for acute toxicity as per the OECD guidelines set by Organization for Economic Co-operation and Development (OECD), revised draft guidelines 423, received from Committee for the purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. Principle of test The principle is based on a stepwise procedure with the use of a minimum number of animals per step; sufficient information is obtained on the acute toxicity of the test substance to enable its classification. The substance is administered orally to a group of experimental animals at one of the defined doses. The substance is tested using a stepwise procedure, each step using three animals of a single sex (normally females). Absence or presence of compound related mortality of the animals dosed at one step will determine the next step. 1. No further testing is required. 2. Dosing of three additional animals with the same dose. 3. Dosing of three animals at the next higher or the next lower dose level. The method enables a judgment with respect to classifying the test substances to one of the series of toxicity classes defined by fixed LD50 cut off values.

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2. Description of the method i) Selection of animal species Healthy young albino rats of either sex weighing between 150 to 200 gms (8 to 12 weeks old) were used for acute toxicity study to determine LD50 of extracts. Totally there were three groups, each groups consisting of three animals. ii) Housing and feeding condition The temperature in the experimental room was around 25 C. Lightning was artificial, the sequence being 12 hr dark, 12 hr light. The conventional laboratory diet was feeded, with unlimited supply of drinking water. iii) Preparation of animals The animals were randomly selected, marked to permit individual identification, and kept in their cages for five days prior to dosing to allow for acclimatization to the laboratory condition. iv) Preparation of dose All the extracts were prepared as a suspension in 1% sodium carboxy methyl cellulose (CMC). v) Administration of dose The test substances were administered in a single dose by gavage using a stomach tube. Animals were fasted prior to dosing, following fasting period the animals were weighed and test substance was administered. After the dose was administered, food was withheld for further 4 hrs. vi) Number of animals and dose levels In each step three animals were used in each group. Since there was no

information on the substance to be tested (i.e. extracts), starting dose was 300-mg/kg
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body weight up to 2000 mg/kg body weight. The procedure of dose selection and finalizing LD50 cut off values are shown in Table 3.4 . vii) Observations After dosing, animals were observed initially at least once during the first 30 minutes and periodically during the first 24 hours for any chances of toxic symptoms or mortality. Additional observations like changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention was also given to observation of tremors and convulsions. Results The results of acute toxicity studies were given in Table 3.4 and 3.5. The acute toxicity studies of the selected plant extracts showed no symptoms of toxicity or behavioral changes at the maximum dose (2000 mg/kg). Hence 1/10th of the maximum dose viz. 200 mg/kg b.w was set as high dose for further studies. Table 3.4: Evaluation of LD50 Cut-off value of the selected plant extracts B. ovalifoliolata, C. caudata, S. spontaneum and G. mangostana S. No. 1. 2. 3. 4. Extracts Boswellia ovalifoliolata -alcoholic extract LD50 Cut-Off mg/kg b.w. >2000 mg/kg, b.w. Vehicle 1 % CMC 1 % CMC 1 % CMC 1 % CMC

Commmiphora caudata -alcoholic extract >2000 mg/kg, b.w. Saccharum spontaneum -alcoholic extract Garcinia mangostana -alcoholic extract >2000 mg/kg, b.w. >2000 mg/kg, b.w.

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S. No 1 2 3 4 5

Extract EBOG ECCL ESSW EGMP Control

Table 3.5: Behavioural data of acute toxicity studies of the selected plant extracts B. ovalifoliolata, C. caudata , S. spontaneum and G. mangostana Observations Toxicity Time of death Skin & Fur Eyes RESP CNS TRE CON SALI DIAH SLEEP on set Stop x x x x X x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x

LET x x x x x

Coma x x x x x

(*TRE-Tremor, CON-Convulsions, SALI- Salivation, DIAH - Diarrhea, LET-Lethargy), x = Negative, = Positive EBOL: Ethanolic extract of B.ovalifoliolata leaf ECCL: Ethanolic extract of C.caudata leaf, ESSW: Ethanolic extract of S.spontaneum whole plant EBOG: Ethanolic extract of B.ovalifoliolata gum ECCB: Ethanolic extract of C.caudata bark EGMP: Ethanolic extract of G.mangostana pericarp

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3.6 Results and Discussion

The Physico-chemical analysis of crude drugs serve as a valuable source of information and provide suitable standards to determine the quality of the selected plant materials Boswellia ovalifoliolata, Commiphora caudata , Saccharum spontaneum and Garcinia mangostana . The moisture content of a drug will be responsible for decomposition of crude drugs either by producing chemical change or microbial growth. So the moisture content should be determined and controlled. The moisture content of the selected plants were determined and found to be 6.6 (BOL), 2.4 (BOG), 5.5 (CCL), 3.8 (CCB), 9.5 (SSW) AND 7.4 (GMP). Certain drugs produce fluorescence when the cut surface or the powder is exposed to UV radiation and is useful in the identification and also to distinguish between different varieties. The fluorescence produced by the selected plants was observed under short UV and presented in Table 3.1. The determination of ash value is useful for detecting exhausted drugs and excess of sandy or earthy matter. Total ash is useful in detecting the crude drugs that are mixed with various mineral substances like sand, soil, calcium oxalate and other inorganic contents. The total ash values of the selected plants were found to be 5.3 (BOL), 6.2 (BOG), 9.2 (CCL), 6.4 (CCB), 4.5 (SSW) and 2.5 (GMP). The calcium oxalate or carbonate, yielded by the incinerated oxalate, will be soluble in hydrochloric acid. The acid insoluble ash is used to detect the presence of excessive earthy matter which is likely to occur with roots, rhizomes, etc. The acid insoluble ash values were found to be 1.1 (BOL), 1.6 (BOG), 2.1 (CCL), 1.3 (CCB), 1.5 (SSW) and 1.2 (GMP). Water soluble ash is used to detect the presence of material exhausted by water and was found to be 1.8 (BOL), 1.5 (BOG), 7.5 (CCL), 4.8 (CCB), 2.4 (SSW) and 1.5 (GMP).
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The extracts obtained by exhausting crude drugs with different solvents are approximate measures of their chemical constituents and the selected plants appear to be rich source of secondary metabolites. Water soluble extractive is used for crude drugs containing water soluble constituents like glycosides, mucilage, etc. The water soluble extractive values of the selected plants were found to be 15.12 (BOL), 9.24 (BOG), 10.15 (CCL), 9.52 (CCB), 5.51 (SSW) and 12.33 (GMP). Alcohol soluble extractive is a measure of tannins, glycosides, resins, etc and were found to be 9.41 (BOL), 8.23 (BOG), 8.52 (CCL), 17.6 (CCB), 9.82 (SSW) and 16.52 (GMP). In this study, the contents of total phenolics were determined

spectrophotometrically according to the Folin-Ciocalteau method and calculated as gallic acid equivalents (GAE). Quantification of the phenolic compounds showed that Garcinia mangostana pericarp (44.24 mg/g) and Commiphora caudata leaves (18.67 mg/g) contain higher phenolic content and the phenolic contents of the selected plants were found to be in the order GMP (44.24) > CCL (18.67) > BOL(10.56)> SSW (9.82)> BOG (7.45)> CCB ( 5.56). The total flavonoid content was estimated with an Aluminium chloride colorimetric assay and calculated as Rutin equivalents. The flavonoid content was found to be higher in GMP (8.64 mg/g) followed by CCL (7.82), CCB (2.51), SSW (1.54), BOL (1.42) and BOG (1.02). It was well-known that plant phenolics are highly effective free radical scavengers and antioxidants. G. mangostana contained high amounts of xanthones, a class of phenolic compounds (Table 2.7 ). The physico-chemical analysis of the selected plant extracts was followed by continuous extraction with alcohol in a soxhlet apparatus. The alcoholic extracts were obtained as intractable gum and possessed characteristic odour. The percentage yield of

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the extracts obtained were found to be 13.5 (BOL), 7.5 (BOG), 8 (CCL), 19.5 (CCB), 4.2 (SSW) and 18.5 (GMP). The preliminary phytochemical investigations were carried out using standard procedures available in the literature. The alcoholic extracts of the selected plants B. ovalifoliolata, C. caudata, S. spontaneum and G. mangostana showed in common the presence of phytoconstituents like the steroids, terpenoids, phenols, flavonoids, glycosides and tannins. The alcoholic extracts of the selected plants, B. ovalifoliolata, C. caudata, S. spontaneum and G. mangostana were initially tested for acute oral toxicity as per the OECD guidelines. Healthy young albino rats of either sex weighing between 150 to 200 gms were used for acute toxicity study and tested upto 2000 mg/kg body weight. After dosing, animals were observed initially at least once during the first 30 minutes and periodically during the first 24 hours for any chances of toxic symptoms or mortality. . The acute toxicity studies of the selected plant extracts showed no symptoms of toxicity or behavioral changes at the maximum dose (2000 mg/kg). Hence 1/10th of the maximum dose viz. 200 mg/kg b.w was set as high dose for further studies. In view of the important phytoconstituents such as phenols and flavonoids present in the selected plants, and taking into account the free radical scavenging activity of the phenolic compounds and their possible role in the treatment of oxidative stress related diseases such as atherosclerosis, the author has further tested the in-vitro free radical scavenging activity of the selected plant extracts, B.ovalifoliolata , C. caudata, S. spontaneum and G. mangostana.

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